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Sample records for ras induces p19arf

  1. C/EBPβ represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19Arf

    Science.gov (United States)

    Ewing, SJ; Zhu, S; Zhu, F; House, JS; Smart, RC

    2013-01-01

    CCAAT/enhancer-binding protein-β (C/EBPβ) is a mediator of cell survival and tumorigenesis. When C/EBPβ−/− mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19Arf and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19Arf is dramatically elevated in C/EBPβ−/− epidermis and that C/EBPβ represses a p19Arf promoter reporter. To determine whether p19Arf is responsible for the proapoptotic phenotype in C/EBPβ−/− mice, C/EBPβ−/−;p19Arf−/− mice were generated. C/EBPβ−/−;p19Arf−/− mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19Arf is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPβ−/− epidermis, we generated K14-ER:Ras; C/EBPβ−/− mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPβ−/− mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPβ represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPβ may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents. PMID:18636078

  2. Mutated N-ras does not induce p19 arf in CO25 cell line | Saleh ...

    African Journals Online (AJOL)

    The mouse cell line (CO25) used in this study was transfected with a glucocorticoid inducible mutated human N-ras oncogene under transcriptional control of the steroid-sensitive promoter of the mouse mammary tumors virus long terminal repeat MMTV-LTR. This study was aimed to investigate the expression of p19arf and ...

  3. Induction of nonapoptotic cell death by activated Ras requires inverse regulation of Rac1 and Arf6.

    Science.gov (United States)

    Bhanot, Haymanti; Young, Ashley M; Overmeyer, Jean H; Maltese, William A

    2010-10-01

    Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together, the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. The latter seems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death.

  4. Induction of Non-Apoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

    Science.gov (United States)

    Bhanot, Haymanti; Young, Ashley M.; Overmeyer, Jean H.; Maltese, William A.

    2010-01-01

    Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating-protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or co-expression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by shRNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1, combined with reciprocal inactivation of Arf6. The latter appears to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for induction of methuosis in cancers that are resistant to apoptotic cell death. PMID:20713492

  5. Nucleophosmin is required for DNA integrity and p19Arf protein stability

    DEFF Research Database (Denmark)

    Colombo, Emanuela; Bonetti, Paola; Lazzerini Denchi, Eros

    2005-01-01

    , such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM....

  6. Induction of Non-Apoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

    OpenAIRE

    Bhanot, Haymanti; Young, Ashley M.; Overmeyer, Jean H.; Maltese, William A.

    2010-01-01

    Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating-protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to...

  7. Dysregulated ΔNp63α inhibits expression of Ink4a/arf, blocks senescence, and promotes malignant conversion of keratinocytes.

    Directory of Open Access Journals (Sweden)

    Linan Ha

    Full Text Available p63 is critical for squamous epithelial development, and elevated levels of the ΔNp63α isoform are seen in squamous cell cancers of various organ sites. However, significant controversy exists regarding the role of p63 isoforms as oncoproteins or tumor suppressors. Here, lentiviruses were developed to drive long-term overexpression of ΔNp63α in primary keratinocytes. Elevated levels of ΔNp63α in vitro promote long-term survival and block both replicative and oncogene-induced senescence in primary keratinocytes, as evidenced by the expression of SA-β-gal and the presence of nuclear foci of heterochromatin protein 1γ. The contribution of ΔNp63α to cancer development was assessed using an in vivo grafting model of experimental skin tumorigenesis that allows distinction between benign and malignant tumors. Grafted lenti-ΔNp63α keratinocytes do not form tumors, whereas lenti-GFP/v-ras(Ha keratinocytes develop well-differentiated papillomas. Lenti-ΔNp63α/v-ras(Ha keratinocytes form undifferentiated carcinomas. The average volume of lenti-ΔNp63α/v-ras(Ha tumors was significantly higher than those in the lenti-GFP/v-ras(Ha group, consistent with increased BrdU incorporation detected by immunohistochemistry. The block in oncogene-induced senescence corresponds to sustained levels of E2F1 and phosphorylated AKT, and is associated with loss of induction of p16(ink4a/p19(arf. The relevance of p16(ink4a/p19(arf loss was demonstrated in grafting studies of p19(arf-null keratinocytes, which develop malignant carcinomas in the presence of v-ras(Ha similar to those arising in wildtype keratinocytes that express lenti-ΔNp63α and v-ras(Ha. Our findings establish that ΔNp63α has oncogenic activity and its overexpression in human squamous cell carcinomas contributes to the malignant phenotype, and implicate its ability to regulate p16(ink4a/p19(arf in the process.

  8. The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas.

    Science.gov (United States)

    Kannan, K; Munirajan, A K; Krishnamurthy, J; Bhuvarahamurthy, V; Mohanprasad, B K; Panishankar, K H; Tsuchida, N; Shanmugam, G

    2000-03-01

    Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.

  9. p53-Induced Apoptosis Occurs in the Absence of p14ARF in Malignant Pleural Mesothelioma

    Directory of Open Access Journals (Sweden)

    Sally Hopkins-Donaldson

    2006-07-01

    Full Text Available Malignant pleural mesotheliomas (MPMs are usually wild type for the p53 gene but contain homozygous deletions in the INK4A locus that encodes p14ARF, an inhibitor of p53-MDM2 interaction. Previous findings suggest that lack of p14ARF expression and the presence of SV40 large T antigen (L-Tag result in p53 inactivation in MPM. We did not detect SV40 L-Tag mRNA in either MPM cell lines or primary cultures, treatment of p14ARF-deficient cells with cisplatin (CDDP increased both total and phosphorylated p53 and enhanced p53 DNA-binding activity. On incubation with CDDP, levels of positively regulated p53 transcriptional targets p21WAF, PIG3, MDM2, Bax, PUMA increased in p14ARF-deficient cells, whereas negatively regulated survivin decreased. Significantly, p53-induced apoptosis was activated by CDDP in p14ARF-deficient cells, treatment with p53-specific siRNA rendered them more CDDP-resistant. p53 was also activated by: 1 inhibition of MDM2 (using nutlin-3; 2 transient overexpression of p14ARF; and 3 targeting of survivin using antisense oligonucleotides. However, it is noteworthy that only survivin downregulation sensitized cells to CDDP-induced apoptosis. These results suggest that p53 is functional in the absence of p14ARF in MPM and that targeting of the downstream apoptosis inhibitor survivin can sensitize to CDDP-induced apoptosis.

  10. Intratumoral Immunization by p19Arf and Interferon-β Gene Transfer in a Heterotopic Mouse Model of Lung Carcinoma

    Directory of Open Access Journals (Sweden)

    João Paulo Portela Catani

    2016-12-01

    Full Text Available Therapeutic strategies that act by eliciting and enhancing antitumor immunity have been clinically validated as an effective treatment modality but may benefit from the induction of both cell death and immune activation as primary stimuli. Using our AdRGD-PG adenovector platform, we show here for the first time that in situ gene transfer of p19Arf and interferon-β (IFNβ in the LLC1 mouse model of lung carcinoma acts as an immunotherapy. Although p19Arf is sufficient to induce cell death, only its pairing with IFNβ significantly induced markers of immunogenic cell death. In situ gene therapy with IFNβ, either alone or in combination with p19Arf, could retard tumor progression, but only the combined treatment was associated with a protective immune response. Specifically in the case of combined intratumoral gene transfer, we identified 167 differentially expressed genes when using microarray to evaluate tumors that were treated in vivo and confirmed the activation of CCL3, CXCL3, IL1α, IL1β, CD274, and OSM, involved in immune response and chemotaxis. Histologic evaluation revealed significant tumor infiltration by neutrophils, whereas functional depletion of granulocytes ablated the antitumor effect of our approach. The association of in situ gene therapy with cisplatin resulted in synergistic elimination of tumor progression. In all, in situ gene transfer with p19Arf and IFNβ acts as an immunotherapy involving recruitment of neutrophils, a desirable but previously untested outcome, and this approach may be allied with chemotherapy, thus providing significant antitumor activity and warranting further development for the treatment of lung carcinoma.

  11. Azidothymidine and cisplatin increase p14ARF expression in OVCAR-3 ovarian cancer cell line

    International Nuclear Information System (INIS)

    Vaskivuo, Liisa; Rysae, Jaana; Koivuperae, Johanna; Myllynen, Paeivi; Vaskivuo, Tommi; Chvalova, Katerina; Serpi, Raisa; Savolainen, Eeva-Riitta; Puistola, Ulla; Vaehaekangas, Kirsi

    2006-01-01

    p14 ARF tumor suppressor protein regulates p53 by interfering with mdm2-p53 interaction. p14 ARF is activated in response to oncogenic stimuli but little is known of the responses of endogenous p14 ARF to different types of cellular stress or DNA damage. Azidothymidine (AZT) is being tested in several clinical trials as an enhancer of anticancer chemotherapy. However, the knowledge of the relationship between AZT and cellular pathways, e.g. p53 pathway, is very limited. In this study, we show that AZT, cisplatin (CDDP) and docetaxel (DTX) all induce unique molecular responses in OVCAR-3 ovarian carcinoma cells carrying a mutated p53, while in A2780, ovarian carcinoma and MCF-7 breast carcinoma cells with wild type p53, all of these drugs cause similar p53 responses. We found that endogenous p14 ARF protein in OVCAR-3 cells is down-regulated by DTX but induced by AZT and a short CDDP pulse treatment. In HT-29 colon carcinoma cells with a mutated p53, all treatments down-regulated p14 ARF protein. Both CDDP and AZT increased the expression of p14ARF mRNA in OVCAR-3 cells. Differences in cell death induced by these drugs did not explain the differences in protein and mRNA expressions. No increase in the level of either c-Myc or H-ras oncoproteins was seen in OVCAR-3 cells after AZT or CDDP-treatment. These results suggest that p14 ARF can respond to DNA damage without oncogene activation in cell lines without functional p53

  12. Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

    Science.gov (United States)

    Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

    2016-06-07

    Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

  13. Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

    Directory of Open Access Journals (Sweden)

    Zanatta Daniela B

    2010-06-01

    Full Text Available Abstract Background Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods B16 (mouse melanoma and C6 (rat glioma cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet

  14. Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

    International Nuclear Information System (INIS)

    Merkel, Christian A; Silva Soares, Rafael B da; Carvalho, Anna Carolina V de; Zanatta, Daniela B; Bajgelman, Marcio C; Fratini, Paula; Costanzi-Strauss, Eugenia; Strauss, Bryan E

    2010-01-01

    Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19

  15. K-Ras(V14I) -induced Noonan syndrome predisposes to tumour development in mice.

    Science.gov (United States)

    Hernández-Porras, Isabel; Schuhmacher, Alberto J; Garcia-Medina, Raquel; Jiménez, Beatriz; Cañamero, Marta; de Martino, Alba; Guerra, Carmen

    2016-06-01

    The Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. A significant proportion of NS patients may also develop myeloproliferative disorders (MPDs), including juvenile myelomonocytic leukaemia (JMML). Surprisingly, scarce information is available in relation to other tumour types in these patients. We have previously developed and characterized a knock-in mouse model that carries one of the most frequent KRAS-NS-related mutations, the K-Ras(V14I) substitution, which recapitulates most of the alterations described in NS patients, including MPDs. The K-Ras(V14I) mutation is a mild activating K-Ras protein; thus, we have used this model to study tumour susceptibility in comparison with mice expressing the classical K-Ras(G12V) oncogene. Interestingly, our studies have shown that these mice display a generalized tumour predisposition and not just MPDs. In fact, we have observed that the K-Ras(V14I) mutation is capable of cooperating with the p16Ink4a/p19Arf and Trp53 tumour suppressors, as well as with other risk factors such as pancreatitis, thereby leading to a higher cancer incidence. In conclusion, our results illustrate that the K-Ras(V14I) activating protein is able to induce cancer, although at a much lower level than the classical K-Ras(G12V) oncogene, and that it can be significantly modulated by both genetic and non-genetic events. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  16. Potential Therapeutic Uses of p19ARF Mimics in Mammary Tumorigenesis

    National Research Council Canada - National Science Library

    Hann, Stephen R

    2005-01-01

    Since many breast tumors have deregulated c-Myc we hypothesize that an ARF mimic would be a valuable therapeutic agent for breast cancer to inhibit c-Myc-induced transformation/tumorigenesis without...

  17. Losses of both products of the Cdkn2a/Arf locus contribute to asbestos-induced mesothelioma development and cooperate to accelerate tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Deborah A Altomare

    2011-04-01

    Full Text Available The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A and p14(ARF, which are frequently co-deleted in human malignant mesothelioma (MM. The importance of p16(INK4A loss in human cancer is well established, but the relative significance of p14(ARF loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1β, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/- and Arf(+/- mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/- and Arf(+/- mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/- mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b. In contrast, MMs from Arf(+/- mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a and p19(Arf cooperate to accelerate asbestos-induced tumorigenesis.

  18. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

    International Nuclear Information System (INIS)

    Tsujita, Maristela; Batista, Wagner L.; Ogata, Fernando T.; Stern, Arnold; Monteiro, Hugo P.; Arai, Roberto J.

    2008-01-01

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras C118S ) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG

  19. Somatotropinomas, but not nonfunctioning pituitary adenomas, maintain a functional apoptotic RET/Pit1/ARF/p53 pathway that is blocked by excess GDNF.

    Science.gov (United States)

    Diaz-Rodriguez, Esther; Garcia-Rendueles, Angela R; Ibáñez-Costa, Alejandro; Gutierrez-Pascual, Ester; Garcia-Lavandeira, Montserrat; Leal, Alfonso; Japon, Miguel A; Soto, Alfonso; Venegas, Eva; Tinahones, Francisco J; Garcia-Arnes, Juan A; Benito, Pedro; Angeles Galvez, Maria; Jimenez-Reina, Luis; Bernabeu, Ignacio; Dieguez, Carlos; Luque, Raul M; Castaño, Justo P; Alvarez, Clara V

    2014-11-01

    Acromegaly is caused by somatotroph cell adenomas (somatotropinomas [ACROs]), which secrete GH. Human and rodent somatotroph cells express the RET receptor. In rodents, when normal somatotrophs are deprived of the RET ligand, GDNF (Glial Cell Derived Neurotrophic Factor), RET is processed intracellularly to induce overexpression of Pit1 [Transcription factor (gene : POUF1) essential for transcription of Pituitary hormones GH, PRL and TSHb], which in turn leads to p19Arf/p53-dependent apoptosis. Our purpose was to ascertain whether human ACROs maintain the RET/Pit1/p14ARF/p53/apoptosis pathway, relative to nonfunctioning pituitary adenomas (NFPAs). Apoptosis in the absence and presence of GDNF was studied in primary cultures of 8 ACROs and 3 NFPAs. Parallel protein extracts were analyzed for expression of RET, Pit1, p19Arf, p53, and phospho-Akt. When GDNF deprived, ACRO cells, but not NFPAs, presented marked level of apoptosis that was prevented in the presence of GDNF. Apoptosis was accompanied by RET processing, Pit1 accumulation, and p14ARF and p53 induction. GDNF prevented all these effects via activation of phospho-AKT. Overexpression of human Pit1 (hPit1) directly induced p19Arf/p53 and apoptosis in a pituitary cell line. Using in silico studies, 2 CCAAT/enhancer binding protein alpha (cEBPα) consensus-binding sites were found to be 100% conserved in mouse, rat, and hPit1 promoters. Deletion of 1 cEBPα site prevented the RET-induced increase in hPit1 promoter expression. TaqMan qRT-PCR (real time RT-PCR) for RET, Pit1, Arf, TP53, GDNF, steroidogenic factor 1, and GH was performed in RNA from whole ACRO and NFPA tumors. ACRO but not NFPA adenomas express RET and Pit1. GDNF expression in the tumors was positively correlated with RET and negatively correlated with p53. In conclusion, ACROs maintain an active RET/Pit1/p14Arf/p53/apoptosis pathway that is inhibited by GDNF. Disruption of GDNF's survival function might constitute a new therapeutic route in

  20. Oncogenic c-Myc-induced lymphomagenesis is inhibited non-redundantly by the p19Arf–Mdm2–p53 and RP–Mdm2–p53 pathways

    OpenAIRE

    Meng, X; Carlson, NR; Dong, J; Zhang, Y

    2015-01-01

    The multifaceted oncogene c-Myc plays important roles in the development and progression of human cancer. Recent in vitro and in vivo studies have shown that the p19Arf–Mdm2–p53 and the ribosomal protein (RP)–Mdm2–p53 pathways are both essential in preventing oncogenic c-Myc-induced tumorigenesis. Disruption of each pathway individually by p19Arf deletion or by Mdm2C305F mutation, which disrupts RP-Mdm2 binding, accelerates Eμ-myc transgene-induced pre-B/B-cell lymphoma in mice at seemingly s...

  1. Increased Arf/p53 activity in stem cells, aging and cancer.

    Science.gov (United States)

    Carrasco-Garcia, Estefania; Moreno, Manuel; Moreno-Cugnon, Leire; Matheu, Ander

    2017-04-01

    Arf/p53 pathway protects the cells against DNA damage induced by acute stress. This characteristic is the responsible for its tumor suppressor activity. Moreover, it regulates the chronic type of stress associated with aging. This is the basis of its anti-aging activity. Indeed, increased gene dosage of Arf/p53 displays elongated longevity and delayed aging. At a cellular level, it has been recently shown that increased dosage of Arf/p53 delays age-associated stem cell exhaustion and the subsequent decline in tissue homeostasis and regeneration. However, p53 can also promote aging if constitutively activated. In this context, p53 reduces tissue regeneration, which correlates with premature exhaustion of stem cells. We discuss here the current evidence linking the Arf/p53 pathway to the processes of aging and cancer through stem cell regulation. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  2. A novel proapoptotic gene PANO encodes a post-translational modulator of the tumor suppressor p14ARF

    Energy Technology Data Exchange (ETDEWEB)

    Watari, Akihiro; Li, Yang; Higashiyama, Shinji; Yutsudo, Masuo, E-mail: yutsudo@biken.osaka-u.ac.jp

    2012-02-01

    The protein p14ARF is a known tumor suppressor protein controlling cell proliferation and survival, which mainly localizes in nucleoli. However, the regulatory mechanisms that govern its activity or expression remain unclear. Here, we report that a novel proapoptotic nucleolar protein, PANO, modulates the expression and activity of p14ARF in HeLa cells. Overexpression of PANO enhances the stability of p14ARF protein by protecting it from degradation, resulting in an increase in p14ARF expression levels. Overexpression of PANO also induces apoptosis under low serum conditions. This effect is dependent on the nucleolar localization of PANO and inhibited by knocking-down p14ARF. Alternatively, PANO siRNA treated cells exhibit a reduction in p14ARF protein levels. In addition, ectopic expression of PANO suppresses the tumorigenicity of HeLa cells in nude mice. These results indicate that PANO is a new apoptosis-inducing gene by modulating the tumor suppressor protein, p14ARF, and may itself be a new candidate tumor suppressor gene.

  3. Expression of arf tumor suppressor in spermatogonia facilitates meiotic progression in male germ cells.

    Directory of Open Access Journals (Sweden)

    Michelle L Churchman

    2011-07-01

    Full Text Available The mammalian Cdkn2a (Ink4a-Arf locus encodes two tumor suppressor proteins (p16(Ink4a and p19(Arf that respectively enforce the anti-proliferative functions of the retinoblastoma protein (Rb and the p53 transcription factor in response to oncogenic stress. Although p19(Arf is not normally detected in tissues of young adult mice, a notable exception occurs in the male germ line, where Arf is expressed in spermatogonia, but not in meiotic spermatocytes arising from them. Unlike other contexts in which the induction of Arf potently inhibits cell proliferation, expression of p19(Arf in spermatogonia does not interfere with mitotic cell division. Instead, inactivation of Arf triggers germ cell-autonomous, p53-dependent apoptosis of primary spermatocytes in late meiotic prophase, resulting in reduced sperm production. Arf deficiency also causes premature, elevated, and persistent accumulation of the phosphorylated histone variant H2AX, reduces numbers of chromosome-associated complexes of Rad51 and Dmc1 recombinases during meiotic prophase, and yields incompletely synapsed autosomes during pachynema. Inactivation of Ink4a increases the fraction of spermatogonia in S-phase and restores sperm numbers in Ink4a-Arf doubly deficient mice but does not abrogate γ-H2AX accumulation in spermatocytes or p53-dependent apoptosis resulting from Arf inactivation. Thus, as opposed to its canonical role as a tumor suppressor in inducing p53-dependent senescence or apoptosis, Arf expression in spermatogonia instead initiates a salutary feed-forward program that prevents p53-dependent apoptosis, contributing to the survival of meiotic male germ cells.

  4. E2F-dependent induction of p14ARF during cell cycle re-entry in human T cells

    DEFF Research Database (Denmark)

    del Arroyo, Ana Gutierrez; El Messaoudi, Selma; Clark, Paula A

    2007-01-01

    The ARF protein, encoded by alternate exon usage within the CDKN2A locus, provides a link between the retinoblastoma (pRb) and p53 tumor suppressor pathways. Agents that disable pRb or otherwise impinge on the E2F family of transcription factors induce expression of ARF, resulting in stabilization...... of p53 and activation of p53-regulated genes. However, in some cell types ARF is not induced upon cell cycle re-entry, as expected of a conventional E2F target gene, leading to the suggestion that the ARF promoter only responds to supra-physiological or aberrant levels of E2F. These properties have...

  5. Nitric oxide induces thioredoxin-1 nuclear translocation: Possible association with the p21Ras survival pathway

    International Nuclear Information System (INIS)

    Arai, Roberto J.; Masutani, H.; Yodoi, J.; Debbas, V.; Laurindo, Francisco R.; Stern, A.; Monteiro, Hugo P.

    2006-01-01

    One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21 RasC118S ). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway

  6. Opposite effects of Ha-Ras and Ki-Ras on radiation-induced apoptosis via differential activation of PI3K/Akt and Rac/p38 mitogen-activated protein kinase signaling

    International Nuclear Information System (INIS)

    Choi, J.-A.; Kang, C.-M.; Lee, Y.-S.; Lee, S.-J.; Bae, S.-W.; Cho, C.-K.

    2003-01-01

    It has been well known that Ras signaling is involved in various cellular processes, including proliferation, differentiation, and apoptosis. However, distinct cellular functions of Ras isozymes are not fully understood. Here we show the opposing roles of Ha-Ras and Ki-Ras genes in the modulation of cell sensitivity to ionizing radiation. Overexpression of active isoform of Ha-Ras (12V-Ha- Ras) in Rat2 cells increases resistance to the ionizing radiation. Constitutive activation of phosphoinositide-3-kinase (PI3K) and Akt is detected specifically in 12V-Ha-Ras-overexpressing cells. The specific PI3K inhibitor LY294002 inhibits PI3K/Akt signaling and potentiates the radiation-induced apoptosis, suggesting that activation of PI3K/Akt signaling pathway is involved in the increased radio-resistance in cells overexpressing 12V-Ha-Ras. Overexpression of activated Ki-Ras (12V-Ki-Ras), on the other hand, markedly increases radiation sensitivity. The p38 mitogen-activated protein (MAP) kinase activity is selectively enhanced by ionizing radiation in cells overexpressing 12V-Ki-Ras. The specific p38 MAP kinase inhibitor, PD169316, or dominant-negative p38 MAP kinase decreases radiation-induced cell death. We further show that the mechanism that underlies potentiation of cell death in cells overexpressing 12V-Ki-Ras involves Bax translocation to the mitochondrial membrane. Elevated Bax translocation following ionizing irradiation in 12V-Ki-Ras-overexpressing cells is completely inhibited by PD169316 or dominant-negative p38 MAP kinase. In addition, introduction of cells with RacN17, a dominant negative mutant of Rac, resulted in a marked inhibition of radiation-induced Bax translocation and apoptotic cell death as well as p38 MAP kinase activation. Taken together, these findings explain the opposite effects of Ha-Ras and Ki-Ras on modulation of radio-sensitivity, and suggest that differential activation of PI3K/Akt and Rac/p38 MAP kinase signaling by Ha-Ras and Ki-Ras may

  7. The ionizing radiation inducible gene PARX/ARAP2 participates in Rho and ARF signaling

    International Nuclear Information System (INIS)

    Wong, J.A.; Chen, Z.; Zhao, Y.; Vallis, K.A.; Marignani, P.A.; Randazzo, P.A.

    2003-01-01

    Full text: PARX/ARAP2 is a novel protein that we identified in a gene trap screen for ionizing radiation (IR)-regulated genes. It belongs to a recently described family of proteins that link Rho, ADP-ribosilation factor (ARF) and phosphoinositide 3-kinase (PI3-K) signaling. We have cloned the full length human PARX. Domain analysis of the predicted protein revealed a sterile-alpha motif, five pleckstrin homology domains, a RhoGTPase activating domain (RhoGAP) and an ARF activating domain (ARFGAP). PARX is early inducible by IR in a dose-dependent manner in murine ES cells and in several human B-cell lymphoma lines with up to six-fold induction at the mRNA level at 2 hours (10 Gy). Thus, the kinetics of PARX induction follows the pattern of the rapid response typical of many stress-induced immediate-early genes. PARX expression is also induced in response to other cellular stressors including sorbitol and bleomycin. PARX induction is dependent on PI3-K activity and can be suppressed by the PI3-K inhibitor LY294002. Induction of PARX in response to IR has been observed in cell lines that are p53 mutant indicating up-regulation independent of normal p53 function. The role of p53 in PARX induction is currently being studied using cell lines expressing temperature sensitive p53. Biochemical studies reveal that human PARX has in vivo RhoGAP activity for Rac1 and phosphatidylinositol 3,4,5-trisphosphate dependent ARFGAP activity for ARF1, ARF5 and ARF6. Also, temporal changes in PARX cellular localization following IR are currently being investigated using confocal microscopy. PARX is a gene with a potential role in the cellular response to genotoxic stress, and may illuminate the currently unclear role the small GTPases Rho and ARF play in the radiation response

  8. ARF and ATM/ATR cooperate in p53-mediated apoptosis upon oncogenic stress

    International Nuclear Information System (INIS)

    Pauklin, Siim; Kristjuhan, Arnold; Maimets, Toivo; Jaks, Viljar

    2005-01-01

    Induction of apoptosis is pivotal for eliminating cells with damaged DNA or deregulated proliferation. We show that tumor suppressor ARF and ATM/ATR kinase pathways cooperate in the induction of apoptosis in response to elevated expression of c-myc, β-catenin or human papilloma virus E7 oncogenes. Overexpression of oncogenes leads to the formation of phosphorylated H2AX foci, induction of Rad51 protein levels and ATM/ATR-dependent phosphorylation of p53. Inhibition of ATM/ATR kinases abolishes both induction of Rad51 and phosphorylation of p53, and remarkably reduces the level of apoptosis induced by co-expression of oncogenes and ARF. However, the induction of apoptosis is downregulated in p53-/- cells and does not depend on activities of ATM/ATR kinases, indicating that efficient induction of apoptosis by oncogene activation depends on coordinated action of ARF and ATM/ATR pathways in the regulation of p53

  9. Pokemon enhances proliferation, cell cycle progression and anti-apoptosis activity of colorectal cancer independently of p14ARF-MDM2-p53 pathway.

    Science.gov (United States)

    Zhao, Yi; Yao, Yun-hong; Li, Li; An, Wei-fang; Chen, Hong-zen; Sun, Li-ping; Kang, Hai-xian; Wang, Sen; Hu, Xin-rong

    2014-12-01

    Pokemon has been showed to directly suppress p14(ARF) expression and also to overexpress in multiple cancers. However, p14(ARF)-MDM2-p53 pathway is usually aberrant in colorectal cancer (CRC). The aim is to confirm whether Pokemon plays a role in CRC and explore whether Pokemon works through p14(ARF)-MDM2-p53 pathway in CRC. Immunohistochemistry for Pokemon, p14(ARF) and Mtp53 protein was applied to 45 colorectal epitheliums (CREs), 42 colorectal adenomas (CRAs) and 66 CRCs. Pokemon was knocked down with RNAi technique in CRC cell line Lovo to detect mRNA expression of p14(ARF) with qRT-PCR, cell proliferation with CCK8 assay, and cell cycle and apoptosis with flowcytometry analysis. The protein expression rates were significantly higher in CRC (75.8%) than in CRE (22.2 %) or CRA (38.1%) for Pokemon and higher in CRC (53.0%) than in CRE (0) or CRA (4.8%) for Mtp53, but not significantly different in CRC (86.4 %) versus CRE (93.3%) or CRA (90.5 %) for p14(ARF). Higher expression rate of Pokemon was associated with lymph node metastasis and higher Duke's stage. After knockdown of Pokemon in Lovo cells, the mRNA level of p14(ARF) was not significantly changed, the cell proliferation ability was decreased by 20.6%, cell cycle was arrested by 55.7% in G0/G1 phase, and apoptosis rate was increased by 19.0%. Pokemon enhanced the oncogenesis of CRC by promoting proliferation, cell cycle progression and anti-apoptosis activity of CRC cells independently of p14(ARF)-MDM2-p53 pathway. This finding provided a novel idea for understanding and further studying the molecular mechanism of Pokemon on carcinogenesis of CRC.

  10. Molecular analysis of p53 and K-ras in lung carcinomas of coal miners

    Energy Technology Data Exchange (ETDEWEB)

    Sarkar, F.H.; Li, Y.W.; Vallyathan, V. [Wayne State University, Detroit, MI (United States). School of Medicine, Dept. of Pathology

    2001-10-01

    Thirty-three cases of non-small cell lung cancers (NSCLC) from the archives of National Coal Workers' Autopsy Study were studied for mutational alterations in p53 and K-ras using PCR-SSCP, DNA sequencing and PCR-oligonucleotide probe hybridization techniques. Mutations of the p53 were observed in 4 smokers (19%) and one in a never smoker (8%). Two polymorphisms in smokers were detected at codon 213, a common site for sequence variation. Among the smokers the p53 mutations were in the heavy smokers. In never smokers there was only a single p53 mutation and two K-ras mutations. In never smokers the frequency of K-ras mutations was similar (17%) in smokers, but one never smoker had two K-ras mutations. Mutations of p53 were more frequent in adenocarcinomas (27%) and they were AT-GC transitions. There were two large cell undifferentiated carcinomas with p53 mutation and one with a K-ras mutation. Two of the 16 squamous cell carcinomas were positive for p53 mutation, while no K-ras mutations were found in this group. The results of these preliminary studies indicate a moderately different mutational spectrum of p53 and K-ras in coal miners independent of cigarette smoking. The mutational spectrum observed in this study of coal miners with heavy cigarette smoking history suggest a protective effect of coal mine dust in preventing abnormal mutations induced by chemical carcinogens in cigarette smoke or reactive oxygen species.

  11. Molecular dynamics simulations of membrane deformation induced by amphiphilic helices of Epsin, Sar1p, and Arf1

    Science.gov (United States)

    Li, Zhen-Lu

    2018-03-01

    The N-terminal amphiphilic helices of proteins Epsin, Sar1p, and Arf1 play a critical role in initiating membrane deformation. The interactions of these amphiphilic helices with the lipid membranes are investigated in this study by combining the all-atom and coarse-grained simulations. In the all-atom simulations, the amphiphilic helices of Epsin and Sar1p are found to have a shallower insertion depth into the membrane than the amphiphilic helix of Arf1, but remarkably, the amphiphilic helices of Epsin and Sar1p induce higher asymmetry in the lipid packing between the two monolayers of the membrane. The insertion depth of amphiphilic helix into the membrane is determined not only by the overall hydrophobicity but also by the specific distributions of polar and non-polar residues along the helix. To directly compare their ability to deform the membrane, the coarse-grained simulations are performed to investigate the membrane deformation under the insertion of multiple helices. Project supported by the National Natural Science Foundation of China (Grant Nos. 91427302 and 11474155).

  12. Loss of p19(Arf facilitates the angiogenic switch and tumor initiation in a multi-stage cancer model via p53-dependent and independent mechanisms.

    Directory of Open Access Journals (Sweden)

    Danielle B Ulanet

    2010-08-01

    Full Text Available The Arf tumor suppressor acts as a sensor of oncogenic signals, countering aberrant proliferation in large part via activation of the p53 transcriptional program, though a number of p53-independent functions have been described. Mounting evidence suggests that, in addition to promoting tumorigenesis via disruptions in the homeostatic balance between cell proliferation and apoptosis of overt cancer cells, genetic alterations leading to tumor suppressor loss of function or oncogene gain of function can also incite tumor development via effects on the tumor microenvironment. In a transgenic mouse model of multi-stage pancreatic neuroendocrine carcinogenesis (PNET driven by inhibition of the canonical p53 and Rb tumor suppressors with SV40 large T-antigen (Tag, stochastic progression to tumors is limited in part by a requirement for initiation of an angiogenic switch. Despite inhibition of p53 by Tag in this mouse PNET model, concomitant disruption of Arf via genetic knockout resulted in a significantly accelerated pathway to tumor formation that was surprisingly not driven by alterations in tumor cell proliferation or apoptosis, but rather via earlier activation of the angiogenic switch. In the setting of a constitutional p53 gene knockout, loss of Arf also accelerated tumor development, albeit to a lesser degree. These findings demonstrate that Arf loss of function can promote tumorigenesis via facilitating angiogenesis, at least in part, through p53-independent mechanisms.

  13. Regulation of p21ras activity

    DEFF Research Database (Denmark)

    Lowy, D R; Zhang, K; DeClue, J E

    1992-01-01

    The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control...... mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange....

  14. Analysis of the K-ras and p53 pathways in x-ray-induced lung tumors in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Belinsky, S.A.; Middleton, S.K.; Hahn, F.F.; Nikula, K.J. [Inhalation Toxicology Research Inst., Albuquerque, NM (United States); Picksley, S.M. [Medical Sciences Inst., Dundee (United Kingdom)

    1996-04-01

    The risk from exposure to low-dose radiation in conjunction with cigarette smoking has not been estimated due in part to lmited knowledge surrounding the molecular mechanisms underlying radiation-induced cancers. The purpose of this investigation was to determine the frequency for alterations in genes within the K-ras and p53 signal and cell cycle regulatory pathways, respectively, in X-ray-induced lung tumors in the F344/N rat. These tumors were examined for genetic alterations in the K-ras, c-raf-1, p53, mdm2 and cip1 genes. No K-ras mutations were detected by sequencing in 18 squamous cell carcinomas (SCCs) or 17 adenocarcinomas. However, using a K-ras codon 12 mutation selection assay, a codon 12 GGT {r_arrow} GAT mutation was detected in one SCC, suggesting that activation of the K-ras proto-oncogene is both a rare and late event. Single-strand conformation polymorphism (SSCP) analysis of the kinase-binding domain of the c-raf-1 gene did not detect any polymorphisms. Three of 18 SCCs but none of the adenocarcinomas showed p53 nuclear immunoreactivity. Single-strand conformation polymorphism analysis of exons 4-9 of the p53 gene detected only an exon 9 mutation in one SCC. Mutations were not detected in the three SCCs with immunoreactive p53 protein. No amplification of the mdm2 gene was detected; however, nuclear mdm2 immunoreactivity was present in one of the three SCCs that stained positive for the p53 protein. The complete cDNA of the rat cip1 gene comprising 810 bases was cloned and sequenced. The frequency of somatic mutations in exon 2 of the cip1 gene was determined by SSCP analysis. No alterations in electrophoretic mobility were detected. The results of this investigation indicate that alterations in the K-ras and p53 pathways do not play a major role in the genesis of X-ray-induced lung tumors in the rat. 49 refs., 5 figs.

  15. Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells.

    Science.gov (United States)

    Xu, Rui; Zhang, Yujie; Gu, Luo; Zheng, Jianchao; Cui, Jie; Dong, Jing; Du, Jun

    2015-01-01

    E-cadherin internalization facilitates dissolution of adherens junctions and promotes tumor cell epithelial-mesenchymal transition (EMT) and migration. Our previous results have shown that Arf6 exerts pro-migratory action in breast cancer cells after EGF stimulation. Despite the fact that EGF signaling stimulates EMT of breast cancer cells, the effect of Arf6 on internalization of E-cadherin of breast cancer cells under EGF treatment remains to be determined. Here, we showed that EGF dose-dependently stimulated E-cadherin internalization by MCF-7 cells with the maximal effect at 50 ng/ml. Meanwhile, EGF treatment markedly increased Arf6 activation. Arf6 was involved in complexes of E-cadherin, and more E-cadherin was pulled down with Arf6 when the activity of the latter was increased. Immunoblotting and immunofluorescence assays showed that transfection breast cancer cells with Arf6-T27N or Arf6 siRNA suppressed EGF-induced E-cadherin internalization. Taken together, our study demonstrated that Arf6 activation plays a potential role in EGF-induced E-cadherin internalization, providing new mechanism underlying the effect of Arf6 on promoting breast cancer cell metastasis.

  16. The AP2/EREBP gene PUCHI Co-Acts with LBD16/ASL18 and LBD18/ASL20 downstream of ARF7 and ARF19 to regulate lateral root development in Arabidopsis.

    Science.gov (United States)

    Kang, Na Young; Lee, Han Woo; Kim, Jungmook

    2013-08-01

    The developmental process of lateral root formation consists of priming, initiation, primordium development and the emergence of lateral roots from the primary root. Molecular genetic studies with Arabidopsis have revealed several key transcriptional regulators involved in lateral root development. However, their functional interaction has not been fully characterized yet. Here we utilized a genetic approach to understand some of these interactions, revealing that PUCHI functioning in morphogenesis of early lateral root primordium is regulated downstream of ARF7/ARF19 and acts with LBD16(ASL18)/LBD18(ASL20) to regulate lateral root development. We showed that auxin-responsive expression of PUCHI was significantly reduced in arf7 or arf19 single mutants and completely abolished in arf7 arf19 double mutants. Consistent with this, β-glucuronidase (GUS) expression under the PUCHI promoter in arf7 arf19 was greatly reduced in the lateral root primordium compared with that in the wild type and did not respond to exogenous auxin. Results of GUS expression analyses under the PUCHI, LBD16 or LBD18 promoter in lbd16, lbd18 single and double mutants or puchi demonstrated that PUCHI and LBD16 or LBD18 do not regulate each other's expression. Lateral root phenotypes of double and triple mutants of lbd16, lbd18 and puchi showed that the puchi mutation in lbd16 and lbd18 mutants synergistically decreased the number of emerged lateral roots. These analyses also showed that puchi affected lateral root primordium development of lbd16 or lbd18 additively but differentially. Taken together, these results suggest that PUCHI co-acts with LBD16 and LBD18 to control lateral root primordium development and lateral root emergence.

  17. Concurrent disruption of p16INK4a and the ARF-p53 pathway predicts poor prognosis in aggressive non-Hodgkin's lymphoma

    DEFF Research Database (Denmark)

    Grønbaek, K; de Nully Brown, P; Møller, Michael Boe

    2000-01-01

    . By using a panel of PCR-based methods, we have examined the status of the p16INK4a, ARF and p53 genes in 123 cases of non-Hodgkin's lymphoma (NHL) at diagnosis. Alterations of one or more of these genes were detected in seven of 36 (19%) cases with low- to intermediate-grade histology, and in 35 of 87 (40...

  18. p53, erbB-2 and K-ras gene alterations are rare in spontaneous and plutonium-239-induced canine lung neoplasia

    International Nuclear Information System (INIS)

    Tierney, L.A.; Hahn, F.F.; Lechner, J.F.

    1996-01-01

    Inhalation of high-linear energy transfer radiation in the form of radon progeny is a suspected cause of human lung cancer. To gain insight into the types of genetic derangements caused by this type of radiation, lung tumors from beagle dogs exposed to 239 PuO 2 and those arising in animals with no known carcinogen exposure were examined for evidence of aberrations in genes known to be altered in lung tumors. Altered expression of the p53 tumor suppressor gene and proto-oncogene erbB-2 proteins (p185 erbB2 ) was evaluated by immunohistochemical analysis of 117 tumors representing different histological types in exposed (n = 80) and unexposed (n = 37) animals. Twenty-eight tumors were analyzed for K-ras proto-oncogene mutations by polymerase chain reaction amplification and direct sequencing. Fourteen percent (16/116) of all lung neoplasms showed elevated nuclear accumulation of p53 protein. Regardless of exposure history, adenosquamous and squamous cell cancers comprised 94% of all tumors with p53 abnormalities. Eighteen percent (21/117) of all tumors had evidence of erbB-2 protein overexpression. K-ras mutations were not detected in codons 12, 13 or 61 of tumors from unexposed (n = 9) or plutonium-exposed dogs (n = 19). These data indicate that p53 and K-ras gene abnormalities as a result of missense mutation are infrequent events in spontaneous and 239 PuO 2 -induced lung neoplasia in this colony of beagle dogs. Alternative mechanisms of gene alteration may be involved in canine pulmonary carcinogenesis. 45 refs., 3 figs., 2 tabs

  19. The H3K27me3 demethylase JMJD3 contributes to the activation of the INK4A-ARF locus in response to oncogene- and stress-induced senescence

    DEFF Research Database (Denmark)

    Agger, Karl; Cloos, Paul A C; Rudkjaer, Lise

    2009-01-01

    The tumor suppressor proteins p16INK4A and p14ARF, encoded by the INK4A-ARF locus, are key regulators of cellular senescence. The locus is epigenetically silenced by the repressive H3K27me3 mark in normally growing cells, but becomes activated in response to oncogenic stress. Here, we show that e...... in mouse embryonic fibroblasts results in suppression of p16Ink4a and p19Arf expression and in their immortalization....

  20. H-Ras and K-Ras Oncoproteins Induce Different Tumor Spectra When Driven by the Same Regulatory Sequences.

    Science.gov (United States)

    Drosten, Matthias; Simón-Carrasco, Lucía; Hernández-Porras, Isabel; Lechuga, Carmen G; Blasco, María T; Jacob, Harrys K C; Fabbiano, Salvatore; Potenza, Nicoletta; Bustelo, Xosé R; Guerra, Carmen; Barbacid, Mariano

    2017-02-01

    Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-Ras G12V oncogene sequences. Germline expression of H-Ras G12V or K-Ras G12V from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-Ras G12V elicited papillomas and hematopoietic tumors, K-Ras G12V induced lung tumors and gastric lesions. Pulmonary expression of H-Ras G12V created a senescence-like state caused by excessive MAPK signaling. Likewise, H-Ras G12V but not K-Ras G12V induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-Ras G12V expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins. Cancer Res; 77(3); 707-18. ©2016 AACR. ©2016 American Association for Cancer Research.

  1. High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma.

    Science.gov (United States)

    Inda, M M; Muñoz, J; Coullin, P; Fauvet, D; Danglot, G; Tuñón, T; Bernheim, A; Castresana, J S

    2006-04-01

    Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.

  2. ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

    Science.gov (United States)

    Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

    2012-01-01

    Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

  3. ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

    Directory of Open Access Journals (Sweden)

    Venkateswarlu Kanamarlapudi

    Full Text Available Adenosine diphosphate (ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1 and P2Y(12 purinoceptors. Recently, we demonstrated that P2Y(1 and P2Y(12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6 in the internalization and function of P2Y(1 and P2Y(12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1 or P2Y(12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

  4. Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk signaling pathway

    Science.gov (United States)

    Drosten, Matthias; Sum, Eleanor Y. M.; Lechuga, Carmen G.; Simón-Carrasco, Lucía; Jacob, Harrys K. C.; García-Medina, Raquel; Huang, Sidong; Beijersbergen, Roderick L.; Bernards, Rene; Barbacid, Mariano

    2014-01-01

    The Ras family of small GTPases constitutes a central node in the transmission of mitogenic stimuli to the cell cycle machinery. The ultimate receptor of these mitogenic signals is the retinoblastoma (Rb) family of pocket proteins, whose inactivation is a required step to license cell proliferation. However, little is known regarding the molecular events that connect Ras signaling with the cell cycle. Here, we provide genetic evidence to illustrate that the p53/p21 Cdk-interacting protein 1 (Cip1)/Rb axis is an essential component of the Ras signaling pathway. Indeed, knockdown of p53, p21Cip1, or Rb restores proliferative properties in cells arrested by ablation of the three Ras loci, H-, N- and K-Ras. Ras signaling selectively inactivates p53-mediated induction of p21Cip1 expression by inhibiting acetylation of specific lysine residues in the p53 DNA binding domain. Proliferation of cells lacking both Ras proteins and p53 can be prevented by reexpression of the human p53 ortholog, provided that it retains an active DNA binding domain and an intact lysine residue at position 164. These results unveil a previously unidentified role for p53 in preventing cell proliferation under unfavorable mitogenic conditions. Moreover, we provide evidence that cells lacking Ras and p53 proteins owe their proliferative properties to the unexpected retroactivation of the Raf/Mek/Erk cascade by a Ras-independent mechanism. PMID:25288756

  5. Stat1 phosphorylation determines Ras oncogenicity by regulating p27 kip1.

    Directory of Open Access Journals (Sweden)

    Shuo Wang

    Full Text Available Inactivation of p27 Kip1 is implicated in tumorigenesis and has both prognostic and treatment-predictive values for many types of human cancer. The transcription factor Stat1 is essential for innate immunity and tumor immunosurveillance through its ability to act downstream of interferons. Herein, we demonstrate that Stat1 functions as a suppressor of Ras transformation independently of an interferon response. Inhibition of Ras transformation and tumorigenesis requires the phosphorylation of Stat1 at tyrosine 701 but is independent of Stat1 phosphorylation at serine 727. Stat1 induces p27 Kip1 expression in Ras transformed cells at the transcriptional level through mechanisms that depend on Stat1 phosphorylation at tyrosine 701 and activation of Stat3. The tumor suppressor properties of Stat1 in Ras transformation are reversed by the inactivation of p27 Kip1. Our work reveals a novel functional link between Stat1 and p27 Kip1, which act in coordination to suppress the oncogenic properties of activated Ras. It also supports the notion that evaluation of Stat1 phosphorylation in human tumors may prove a reliable prognostic factor for patient outcome and a predictor of treatment response to anticancer therapies aimed at activating Stat1 and its downstream effectors.

  6. The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4.

    Science.gov (United States)

    Isabet, Tatiana; Montagnac, Guillaume; Regazzoni, Karine; Raynal, Bertrand; El Khadali, Fatima; England, Patrick; Franco, Michel; Chavrier, Philippe; Houdusse, Anne; Ménétrey, Julie

    2009-09-16

    The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 A resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6-JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6-mediated motor switch regulatory function.

  7. Analysis of the ARF/p53 Pathway During Oncogenic Stimulation

    National Research Council Canada - National Science Library

    Nahle, Zaher

    2003-01-01

    ... or deficient for the ARF and/or p53 genes. We found that the ElA oncoprotein regulates the expression of a myriad of targets involved in a diversity of functions such as apoptosis, cell cycle progression, checkpoint control, DNA replication...

  8. K-RAS and N-RAS mutations in testicular germ cell tumors

    Directory of Open Access Journals (Sweden)

    Bekir Muhammet Hacioglu

    2017-05-01

    Full Text Available Testicular cancer is a relatively rare tumor type, accounting for approximately 1% of all cancers in men. However, among men aged between 15 and 40 years, testicular cancer is the most commonly diagnosed malignancy. Testicular germ cell tumors (TGCTs are classified as seminoma and non-seminoma. The RAS oncogene controls several cellular functions, including cell proliferation, apoptosis, migration, and differentiation. Thus, RAS signaling is important for normal germ cell development. Mutations of the Kirsten RAS (K-RAS gene are present in over 20% of all cancers. RAS gene mutations have also been reported in TGCTs. We investigated K-RAS and N-RAS mutations in seminoma and non-seminoma TGCT patients. A total of 24 (55% pure seminoma cases and 19 (45% non-seminoma cases were included in the study. K-RAS and N-RAS analyses were performed in our molecular pathology laboratory, using K-RAS and N-RAS Pyro Kit 24 V1 (Qiagen. In total, a RAS mutation was present in 12 patients (27%: 7 seminoma (29% and 5 non-seminoma cases (26% [p = 0.55]. A K-RAS mutation was present in 4 pure seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63], and an N-RAS mutation was observed in 4 seminoma tumors (16% and 3 non-seminoma tumors (15% [p = 0.63]. Both, K-RAS and N-RAS mutations were present in two patients: one with seminoma tumor and the other with non-seminoma tumor. To date, no approved targeted therapy is available for the treatment of TGCTs. The analysis of K-RAS and N-RAS mutations in these tumors may provide more treatment options, especially in platinum-resistant tumors.

  9. The human ARF tumor suppressor senses blastema activity and suppresses epimorphic tissue regeneration

    Science.gov (United States)

    Hesse, Robert G; Kouklis, Gayle K; Ahituv, Nadav; Pomerantz, Jason H

    2015-01-01

    The control of proliferation and differentiation by tumor suppressor genes suggests that evolution of divergent tumor suppressor repertoires could influence species’ regenerative capacity. To directly test that premise, we humanized the zebrafish p53 pathway by introducing regulatory and coding sequences of the human tumor suppressor ARF into the zebrafish genome. ARF was dormant during development, in uninjured adult fins, and during wound healing, but was highly expressed in the blastema during epimorphic fin regeneration after amputation. Regenerative, but not developmental signals resulted in binding of zebrafish E2f to the human ARF promoter and activated conserved ARF-dependent Tp53 functions. The context-dependent activation of ARF did not affect growth and development but inhibited regeneration, an unexpected distinct tumor suppressor response to regenerative versus developmental environments. The antagonistic pleiotropic characteristics of ARF as both tumor and regeneration suppressor imply that inducing epimorphic regeneration clinically would require modulation of ARFp53 axis activation. DOI: http://dx.doi.org/10.7554/eLife.07702.001 PMID:26575287

  10. BIM is the primary mediator of MYC-induced apoptosis in multiple solid tissues.

    Science.gov (United States)

    Muthalagu, Nathiya; Junttila, Melissa R; Wiese, Katrin E; Wolf, Elmar; Morton, Jennifer; Bauer, Barbara; Evan, Gerard I; Eilers, Martin; Murphy, Daniel J

    2014-09-11

    MYC is one of the most frequently overexpressed oncogenes in human cancer, and even modestly deregulated MYC can initiate ectopic proliferation in many postmitotic cell types in vivo. Sensitization of cells to apoptosis limits MYC's oncogenic potential. However, the mechanism through which MYC induces apoptosis is controversial. Some studies implicate p19ARF-mediated stabilization of p53, followed by induction of proapoptotic BH3 proteins NOXA and PUMA, whereas others argue for direct regulation of BH3 proteins, especially BIM. Here, we use a single experimental system to systematically evaluate the roles of p19ARF and BIM during MYC-induced apoptosis, in vitro, in vivo, and in combination with a widely used chemotherapeutic, doxorubicin. We find a common specific requirement for BIM during MYC-induced apoptosis in multiple settings, which does not extend to the p53-responsive BH3 family member PUMA, and find no evidence of a role for p19ARF during MYC-induced apoptosis in the tissues examined. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  11. ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

    International Nuclear Information System (INIS)

    Fujita, Atsushi

    2008-01-01

    Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6 + , encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6Δ cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p, for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast

  12. Alterations in the K-ras and p53 genes in rat lung tumors

    Energy Technology Data Exchange (ETDEWEB)

    Belinsky, S.A.; Swafford, D.S.; Finch, G.L.; Mitchell, C.E. [Inhalation Toxicology Research Institute, Albuquerque, NM (United States)] [and others

    1997-06-01

    Activation of the K-ras protooncogene and inactivation of the p53 tumor suppressor gene are events common to many types of human cancers. Molecular epidemiology studies have associated mutational profiles in these genes with specific exposures. The purpose of this paper is to review investigations that have examined the role of the K-ras and p53 genes in lung tumors induced in the F344 rat by mutagenic and nonmutagenic exposures. Mutation profiles within the K-ras and p53 genes, if present in rat lung tumors, would help to define some of the molecular mechanisms underlying cancer induction by various environmental agents. Pulmonary adenocarcinomas or squamous cell carcinomas were induced by tetranitromethane (TNM), 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), beryllium metal, plutonium-239, X-ray, diesel exhaust, or carbon black. These agents were chosen because the tumors they produced could arise via different types of DNA damage. Mutation of the K-ras gene was determined by approaches that included DNA transfection, direct sequencing, mismatch hybridization, and restriction fragment length polymorphism analysis. The frequency for mutation of the K-ras gene was exposure dependent. The transition mutations formed could have been derived from deamination of cytosine. Alteration in the p53 gene was assessed by immunohistochemical analysis for p53 protein and single-strand conformation polymorphism (SSCP) analysis of exons 4 to 9. None of the 93 adenocarinomas examined was immunoreactive toward the anti-p53 antibody CM1. In contrast, 14 of 71 squamous cell carcinomas exhibited nuclear p53 immunoreactivity with no correlation to type of exposure. However, SSCP analysis only detected mutations in 2 of 14 squamous cell tumors that were immunoreactive, suggesting that protein stabilization did not stem from mutations within the p53 gene. Thus, the p53 gene does not appear to be involved in the genesis of most rat lung tumors. 2 figs., 2 tabs., 48 refs.

  13. Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras

    Science.gov (United States)

    Acin, Sergio; Li, Zhongyou; Mejia, Olga; Roop, Dennis R; El-Naggar, Adel K; Caulin, Carlos

    2015-01-01

    Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53R172H, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53R172H. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53R172H. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53. PMID:21952947

  14. Genetic analysis of radiation-induced mouse thymic lymphomas

    International Nuclear Information System (INIS)

    Kominami, R.; Wakabayashi, Y.; Niwa, O.

    2003-01-01

    Mouse thymic lymphomas are one of the classic models of radiation-induced malignancies, and the model has been used for the study of genes involved in carcinogenesis. ras oncogenes are the first isolate which undergoes mutations in 10 to 30 % of lymphomas, and p16INK4a and p19ARF in the INK4a-ARF locus are also frequently inactivated. In our previous study, the inactivation of Ikaros, a key regurator of lymphoid system, was found in those lymphomas, and it was suggested that there are other responsible genes yet to be discovered. On the other hand, genetic predisposition to radiation-induced lymphoma often differs in different strains, and this reflects the presence of low penetrance genes that can modify the impact of a given mutation. Little study of such modifiers or susceptibility genes has been performed, either. Recent availability of databases on mouse genome information and the power of mouse genetic system underline usefulness of the lymphoma model in search for novel genes involved, which may provide clues to molecular mechanisms of development of the radiogenic lymphoma and also genes involved in human lymphomas and other malignancies. Accordingly, we have carried out positional cloning for the two different types of tumor-related genes. In this symposium, our current progress is presented that includes genetic mapping of susceptibility/ resistance loci on mouse chromosomes 4, 5 and 19, and also functional analysis of a novel tumor suppressor gene, Rit1/Bcl11b, that has been isolated from allelic loss (LOH) mapping and sequence analysis for γ -ray induced mouse thymic lymphomas

  15. Overexpression of K-p21Ras play a prominent role in lung cancer

    Science.gov (United States)

    Zhang, Peng-bo; Zhou, Xin-liang; Yang, Ju-lun

    2018-06-01

    The proto-oncogene ras product, p21Ras, has been found overexpression in many human tumors. However, the subtypes of overexpressed p21Ras still remain unclear. The purpose of this study was to investigate overexpressed isoforms of p21Ras and their roles in the progress of lung cancer. Method: The expression of total p21Ras in normal lung tissues and lung cancers was determined by immunohistochemically staining with monoclonal antibody (Mab) KGHR-1 which could recognize and broad spectrum reaction with the (K/H/N) ras protein. Then, the isoforms of p21Ras was examined by specific Mab for each p21Ras subtypes. Results: Low expression of total p21Ras was found in 26.67% (8/30) of normal lung tissues, and 81.31% (87/107) of adenocarcinoma harbored overexpressed total p21Ras. Besides, 70.00% (35/50) of squamous cell carcinoma were detected overexpressed total p21Ras. In addition, 122 lung cancer tissues from overexpression of total p21Ras protein were selected to detect the expression of each subtype. And all the 122 lung cancer tissues were K-p21Ras overexpression. Moreover, there was a statistical significance difference between the expression level of total p21Ras and differentiation, and the same results were observed between the expression level of total p21Ras and lymph node metastasis (P0.05). Conclusions: Overexpression of K-p21Ras plays a prominent role in the progress of lung cancer and it is suggested that the p21Ras could serve as a promising treatment target in lung cancer.

  16. A Histidine pH sensor regulates activation of the Ras-specific guanine nucleotide exchange factor RasGRP1.

    Science.gov (United States)

    Vercoulen, Yvonne; Kondo, Yasushi; Iwig, Jeffrey S; Janssen, Axel B; White, Katharine A; Amini, Mojtaba; Barber, Diane L; Kuriyan, John; Roose, Jeroen P

    2017-09-27

    RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

  17. H-RAS, K-RAS, and N-RAS gene activation in human bladder cancers.

    Science.gov (United States)

    Przybojewska, B; Jagiello, A; Jalmuzna, P

    2000-08-01

    Bladder cancer is one of the leading causes of cancer death in most developed countries. In this work, 19 bladder cancer specimens, along with their infiltrations of the urinary bladder wall from the same patients, were examined for the presence of H-RAS, K-RAS, and N-RAS activation using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The H-RAS activation was found in 15 (about 84%) of the 19 bladder cancers studied. The same results were obtained in the infiltrating urinary bladder wall samples. N-RAS gene mutations were observed in all cases (except 1) in which H-RAS gene mutations were detected. The results suggest a strong relationship between H-RAS and N-RAS gene activation in bladder cancer. Changes in the K-RAS gene in bladder cancers seem to be a rare event; this is in agreement with findings of other authors. We found activation of the gene in one specimen of bladder cancer and its infiltration of the urinary bladder wall in the same patient.

  18. P53- and mevalonate pathway–driven malignancies require Arf6 for metastasis and drug resistance

    Science.gov (United States)

    Hashimoto, Ari; Oikawa, Tsukasa; Hashimoto, Shigeru; Sugino, Hirokazu; Yoshikawa, Ayumu; Otsuka, Yutaro; Handa, Haruka; Onodera, Yasuhito; Nam, Jin-Min; Oneyama, Chitose; Okada, Masato; Fukuda, Mitsunori

    2016-01-01

    Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial–mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program. The MVP enzyme geranylgeranyl transferase II (GGT-II) and its substrate Rab11b are critical for Arf6 trafficking to the plasma membrane, where it is activated by receptor tyrosine kinases. Consistently, mutant p53, which is known to support tumorigenesis via MVP, promotes Arf6 activation via GGT-II and Rab11b. Inhibition of MVP and GGT-II blocked invasion and metastasis and reduced cancer cell resistance against chemotherapy agents, but only in cells overexpressing Arf6 and components of the mesenchymal program. Overexpression of Arf6 and mesenchymal proteins as well as enhanced MVP activity correlated with poor patient survival. These results provide insights into the molecular basis of MVP-driven malignancy. PMID:27044891

  19. p53 and ARF: Unexpected players in autophagy

    OpenAIRE

    Balaburski, Gregor M.; Hontz, Robert D.; Murphy, Maureen E.

    2010-01-01

    p53 and ARF are well-established tumor suppressor proteins that function together in the negative regulation of cancer. Recently, both of these proteins were found to play surprising roles in autophagy. Autophagy (“self-eating”) is a critical response of eukaryotic cells to metabolic and other stress. During this process, portions of the cytosol are sequestered into characteristic double membrane vesicles that are delivered to the lysosome for degradation, leading to the release of free amino...

  20. Molecular characterization of radon-induced rat lung tumors

    International Nuclear Information System (INIS)

    Guillet Bastide, K.

    2008-11-01

    The radon gas is a well known lung carcinogenic factor in human at high doses but the cancer risk at low doses is not established. Indeed, epidemiological studies at low doses are difficult to conduct because of the human exposure to other lung carcinogenic factors. These data underlined the necessity to conduct experiments on lung tumors developed on animal model. The aim of this work was to characterize rat lung tumors by working on a series of radon-induced tumors that included adenocarcinomas (A.C.), squamous cell carcinomas (S.C.C.) and adeno-squamous carcinomas (A.S.C.), that are mixed tumors with both A.C. and S.C.C. cellular components. A C.G.H. analysis of the three types of tumors allowed us to define chromosomal recurrent unbalances and to target candidate genes potentially implicated in lung carcinogenesis, as p16Ink4a, p19Arf, Rb1, K-Ras or c-Myc. A more precise analysis of the p16Ink4a/Cdk4/Rb1 and p19Arf/Mdm2/Tp53 pathways was performed and indicated that the Rb1 pathway was frequently inactivated through an absence of p16 Ink4a protein expression, indicating that it has a major role in rat lung carcinogenesis. Finally, a comparative transcriptomic analysis of the three types of tumors allowed us to show for the first time that the complex tumors A.S.C. have a transcriptomic profile in accordance with their mixed nature but that they also display their own expression profiles specificities. This work allowed us to find molecular characteristics common to murine and human lung tumors, indicating that the model of lung tumors in rat is pertinent to search for radiation-induced lung tumors specificities and to help for a better molecular identification of this type of tumors in human. (author)

  1. ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation.

    Science.gov (United States)

    Koubek, Emily J; Santy, Lorraine C

    2018-05-04

    Hepatocyte growth factor (HGF) is a potent signaling factor that acts on epithelial cells, causing them to dissociate and scatter. This migration is coordinated by a number of small GTPases, such as ARF6 and Rac1. Active ARF6 is required for HGF-stimulated migration and intracellular levels of ARF6-GTP and Rac1-GTP increase following HGF treatment. During migration, cross talk between ARF6 and Rac1 occurs through formation of a multi-protein complex containing the ARF-GEF cytohesin-2, the scaffolding protein GRASP/Tamalin, and the Rac1-GEF Dock180. Previously, the role of ARF6 in this process was unclear. We have now found that ARF6 and ARF1 regulate trafficking of GRASP and Dock180 to the plasma membrane following HGF treatment. Trafficking of GRASP and Dock180 is impaired by blocking ARF6-mediated recycling pathways and is required for HGF-stimulated Rac1 activation. Finally, HGF treatment stimulates association of GRASP and Dock180. Inhibition of ARF6 trafficking pathways traps GRASP and Dock180 as a complex in the cell.

  2. Survey of familial glioma and role of germline p16INK4A/p14ARF and p53 mutation

    DEFF Research Database (Denmark)

    Robertson, Lindsay B; Armstrong, Georgina N; Olver, Bianca D

    2010-01-01

    There is increasing recognition of familial propensity to glioma as a distinct clinical entity beyond a few rare syndromes; however its genetic basis is poorly understood. The role of p16(INK4A)/p14(ARF) and p53 mutations in sporadic glioma provides a strong rationale for investigating germline m...

  3. Interaction between the p21ras GTPase activating protein and the insulin receptor

    NARCIS (Netherlands)

    Pronk, G.J.; Medema, R.H.; Burgering, B.M.T.; Clark, R.; McCormick, F.; Bos, J.L.

    1992-01-01

    We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine

  4. ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling*

    Science.gov (United States)

    Kanamarlapudi, Venkateswarlu; Thompson, Aiysha; Kelly, Eamonn; López Bernal, Andrés

    2012-01-01

    The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a Gs-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization. PMID:22523074

  5. Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

    Science.gov (United States)

    Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

    2006-05-01

    To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

  6. GEP100/Arf6 is required for epidermal growth factor-induced ERK/Rac1 signaling and cell migration in human hepatoma HepG2 cells.

    Directory of Open Access Journals (Sweden)

    ZhenZhen Hu

    Full Text Available BACKGROUND: Epidermal growth factor (EGF signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined. METHODOLOGY/PRINCIPAL FINDINGS: We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N also largely abolished EGF-induced cell migration. CONCLUSIONS/SIGNIFICANCE: Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

  7. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

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    Martín-Sánchez, Paloma; Luengo, Alicia; Griera, Mercedes; Orea, María Jesús; López-Olañeta, Marina; Chiloeches, Antonio; Lara-Pezzi, Enrique; de Frutos, Sergio; Rodríguez-Puyol, Manuel; Calleros, Laura; Rodríguez-Puyol, Diego

    2018-02-01

    Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras -/- ) and control wild-type (H -ras +/+ ) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iβ protein expression in H -ras -/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras -/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3β-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iβ overexpression in H -ras -/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iβ overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

  8. The Role of ARF6 in Biliary Atresia.

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    Mylarappa Ningappa

    Full Text Available Altered extrahepatic bile ducts, gut, and cardiovascular anomalies constitute the variable phenotype of biliary atresia (BA.To identify potential susceptibility loci, Caucasian children, normal (controls and with BA (cases at two US centers were compared at >550000 SNP loci. Systems biology analysis was carried out on the data. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 2-5-day post-fertilization zebrafish embryos after morpholino-antisense oligonucleotide knockdown of the candidate gene ADP ribosylation factor-6 (ARF6, Mo-arf6.Among 39 and 24 cases at centers 1 and 2, respectively, and 1907 controls, which clustered together on principal component analysis, the SNPs rs3126184 and rs10140366 in a 3' flanking enhancer region for ARF6 demonstrated higher minor allele frequencies (MAF in each cohort, and 63 combined cases, compared with controls (0.286 vs. 0.131, P = 5.94x10-7, OR 2.66; 0.286 vs. 0.13, P = 5.57x10-7, OR 2.66. Significance was enhanced in 77 total cases, which included 14 additional BA genotyped at rs3126184 only (p = 1.58x10-2, OR = 2.66. Pathway analysis of the 1000 top-ranked SNPs in CHP cases revealed enrichment of genes for EGF regulators (p<1 x10-7, ERK/MAPK and CREB canonical pathways (p<1 x10-34, and functional networks for cellular development and proliferation (p<1 x10-45, further supporting the role of EGFR-ARF6 signaling in BA. In zebrafish embryos, Mo-arf6 injection resulted in a sparse intrahepatic biliary network, several biliary epithelial cell defects, and poor bile excretion to the gall bladder compared with uninjected embryos. Biliary defects were reproduced with the EGFR-blocker AG1478 alone or with Mo-arf6 at lower doses of each agent and rescued with arf6 mRNA.The BA-associated SNPs identify a chromosome 14q21.3 susceptibility locus encompassing the ARF6 gene. arf6 knockdown in zebrafish implicates early biliary dysgenesis as a basis for BA, and also

  9. Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

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    Parrell, Daniel; Zhang, Yang; Olenic, Sandra; Kroos, Lee

    2017-10-01

    RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli , we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli , including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His 6 -MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His 6 -MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are

  10. Aberrant DNA methylation of ESR1 and p14ARF genes could be useful as prognostic indicators in osteosarcoma

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    Sonaglio V

    2013-06-01

    Full Text Available Viviane Sonaglio,1 Ana C de Carvalho,2 Silvia R C Toledo,3,4 Carolina Salinas-Souza,3,4 André L Carvalho,5 Antonio S Petrilli,3 Beatriz de Camargo,6 André L Vettore21Pediatrics Department, A C Camargo Hospital, São Paulo, Brazil; 2Biological Science Department, Federal University of São Paulo, Diadema, Brazil; 3Department of Pediatrics, Pediatric Oncology Institute, GRAACC/Federal University of São Paulo, São Paulo, Brazil; 4Department of Morphology and Genetics, Federal University of São Paulo, São Paulo, Brazil; 5Department of Head and Neck Surgery, PIO XII Foundation, Barretos Cancer Hospital, Barretos, São Paulo, Brazil; 6Research Program Pediatric Oncology Program, CPNq, Instituto Nacional do Cancer, Rio de Janeiro, BrazilAbstract: Osteosarcoma (OS is the eighth most common form of childhood and adolescence cancer. Approximately 10%–20% of patients present metastatic disease at diagnosis and the 5-year overall survival remains around 70% for nonmetastatic patients and around 30% for metastatic patients. Metastatic disease at diagnosis and the necrosis grade induced by preoperative treatment are the only well-established prognostic factors for osteosarcoma. The DNA aberrant methylation is a frequent epigenetic alteration in humans and has been described as a molecular marker in different tumor types. This study evaluated the DNA aberrant methylation status of 18 genes in 34 OS samples without previous chemotherapy treatment and in four normal bone specimens and compared the methylation profile with clinicopathological characteristics of the patients. We were able to define a three-gene panel (AIM1, p14ARF, and ESR1 in which methylation was correlated with OS cases. The hypermethylation of p14ARF showed a significant association with the absence of metastases at diagnoses, while ESR1 hypermethylation was marginally associated with worse overall survival. This study demonstrated that aberrant promoter methylation is a common event

  11. Hypermethylation of the 5′ CpG island of the p14ARF flanking exon 1β in human colorectal cancer displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression

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    Nyiraneza Christine

    2012-06-01

    Full Text Available Abstract Background It has been suggested that inactivation of p14ARF, a tumor suppressor central to regulating p53 protein stability through interaction with the MDM2 oncoprotein, abrogates p53 activity in human tumors retaining the wild-type TP53 gene. Differences in expression of tumor suppressor genes are frequently associated with cancer. We previously reported on a pattern of restricted p53 immunohistochemical overexpression significantly associated with microsatellite instability (MSI, low TP53 mutation frequency, and MDM2 overexpression in colorectal cancers (CRCs. In this study, we investigated whether p14ARF alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs. Results Detailed maps of the alterations in the p14ARF gene were determined in a cohort of 98 CRCs to detect both nucleotide and copy-number changes. Methylation-specific PCR combined with bisulfite sequencing was used to evaluate the prevalence and distribution of p14ARF methylation. p14ARF alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14ARF mutations was extremely low (1/98; 1%, whereas coexistence of methylated and unmethylated alleles in both tumors and normal colon mucosa was common (91/98; 93%. Only seven of ninety-eight tumors (7% had a distinct pattern of methylation compared with normal colon mucosa. Evaluation of the prevalence and distribution of p14ARF promoter methylation in a region containing 27 CpG sites in 35 patients showed a range of methylated CpG sites in tumors (0 to 25 (95% CI 1 to 13 versus 0 to 17 (95% CI 0 to 2 in adjacent colon mucosa (P = 0.004. Hypermethylation of the p14ARF promoter was significantly correlated with the restricted p53 overexpression pattern (P = 0.03, and MDM2 overexpression (P = 0.02, independently of MSI phenotype. Although no significant correlation between p14ARF methylation and TP53 mutational

  12. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

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    Copp& #233; , Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith

    2008-10-24

    Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

  13. Use of human tissue to assess the oncogenic activity of melanoma-associated mutations.

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    Chudnovsky, Yakov; Adams, Amy E; Robbins, Paul B; Lin, Qun; Khavari, Paul A

    2005-07-01

    Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

  14. The inflammatory cytokine TNFα cooperates with Ras in elevating metastasis and turns WT-Ras to a tumor-promoting entity in MCF-7 cells

    International Nuclear Information System (INIS)

    Leibovich-Rivkin, Tal; Liubomirski, Yulia; Meshel, Tsipi; Abashidze, Anastasia; Brisker, Daphna; Solomon, Hilla; Rotter, Varda; Weil, Miguel; Ben-Baruch, Adit

    2014-01-01

    In the present study we determined the relative contribution of two processes to breast cancer progression: (1) Intrinsic events, such as activation of the Ras pathway and down-regulation of p53; (2) The inflammatory cytokines TNFα and IL-1β, shown in our published studies to be highly expressed in tumors of >80% of breast cancer patients with recurrent disease. Using MCF-7 human breast tumor cells originally expressing WT-Ras and WT-p53, we determined the impact of the above-mentioned elements and cooperativity between them on the expression of CXCL8 (ELISA, qRT-PCR), a member of a “cancer-related chemokine cluster” that we have previously identified. Then, we determined the mechanisms involved (Ras-binding-domain assays, Western blot, luciferase), and tested the impact of Ras + TNFα on angiogenicity (chorioallantoic membrane assays) and on tumor growth at the mammary fat pad of mice and on metastasis, in vivo. Using Ras G12V that recapitulates multiple stimulations induced by receptor tyrosine kinases, we found that Ras G12V alone induced CXCL8 expression at the mRNA and protein levels, whereas down-regulation of p53 did not. TNFα and IL-1β potently induced CXCL8 expression and synergized with Ras G12V , together leading to amplified CXCL8 expression. Testing the impact of WT-Ras, which is the common form in breast cancer patients, we found that WT-Ras was not active in promoting CXCL8; however, TNFα has induced the activation of WT-Ras: joining these two elements has led to cooperative induction of CXCL8 expression, via the activation of MEK, NF-κB and AP-1. Importantly, TNFα has led to increased expression of WT-Ras in an active GTP-bound form, with properties similar to those of Ras G12V . Jointly, TNFα + Ras activities have given rise to increased angiogenesis and to elevated tumor cell dissemination to lymph nodes. TNFα cooperates with Ras in promoting the metastatic phenotype of MCF-7 breast tumor cells, and turns WT-Ras into a tumor

  15. EMT-induced stemness and tumorigenicity are fueled by the EGFR/Ras pathway.

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    Dominic Chih-Cheng Voon

    Full Text Available Recent studies have revealed that differentiated epithelial cells would acquire stem cell-like and tumorigenic properties following an Epithelial-Mesenchymal Transition (EMT. However, the signaling pathways that participate in this novel mechanism of tumorigenesis have not been fully characterized. In Runx3 (-/- p53 (-/- murine gastric epithelial (GIF-14 cells, EMT-induced plasticity is reflected in the expression of the embryonal proto-oncogene Hmga2 and Lgr5, an exclusive gastrointestinal stem cell marker. Here, we report the concurrent activation of an EGFR/Ras gene expression signature during TGF-β1-induced EMT in GIF-14 cells. Amongst the altered genes was the induction of Egfr, which corresponded with a delayed sensitization to EGF treatment in GIF-14. Co-treatment with TGF-β1 and EGF or the expression of exogenous KRas led to increased Hmga2 or Lgr5 expression, sphere initiation and colony formation in soft agar assay. Interestingly, the gain in cellular plasticity/tumorigenicity was not accompanied by increased EMT. This uncoupling of EMT and the induction of plasticity reveals an involvement of distinct signaling cues, whereby the EGFR/Ras pathway specifically promotes stemness and tumorigenicity in EMT-altered GIF-14 cells. These data show that the EGFR/Ras pathway requisite for the sustenance of gastric stem cells in vivo and in vitro is involved in the genesis and promotion of EMT-induced tumor-initiating cells.

  16. Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

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    Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

    2015-12-15

    Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

  17. Increased p21ras activity in human fibroblasts transduced with survivin enhances cell proliferation

    International Nuclear Information System (INIS)

    Temme, Achim; Diestelkoetter-Bachert, Petra; Schmitz, Marc; Morgenroth, Agnieszka; Weigle, Bernd; Rieger, Michael A.; Kiessling, Andrea; Rieber, E. Peter

    2005-01-01

    Survivin is critically involved in mitosis and when overexpressed enhances the activity of the Aurora B kinase, a serine-threonine kinase belonging to the family of oncogenic Aurora/IpI1p-related kinases. Both proteins interact with Ras GTPase-activating protein suggesting an impact on the Ras pathway. This study aimed at defining the role of survivin in proliferation and potential transformation of cells. When survivin was overexpressed in normal human lung fibroblasts, the characteristic track lanes of fibroblasts were disturbed and the rate of cell proliferation was increased. An enhanced level of p21 ras mRNA and protein expression and concomitant rise in levels of activated p21 ras were observed. Despite increased proliferation cell survival remained dependent on serum and cells were not able to form colonies in soft agar assays. These data suggest that overexpression of survivin increases cell growth but, despite the increase in active p21 ras , is not sufficient to transform primary cells. Yet, in addition to its anti-apoptotic function it might contribute to the accelerated growth of tumour cells by increasing p21 ras activity

  18. Evaluation of K-ras and p53 expression in pancreatic adenocarcinoma using the cancer genome atlas.

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    Liming Lu

    Full Text Available Genetic alterations in K-ras and p53 are thought to be critical in pancreatic cancer development and progression. However, K-ras and p53 expression in pancreatic adenocarcinoma have not been systematically examined in The Cancer Genome Atlas (TCGA Data Portal. Information regarding K-ras and p53 alterations, mRNA expression data, and protein/protein phosphorylation abundance was retrieved from The Cancer Genome Atlas (TCGA databases, and analyses were performed by the cBioPortal for Cancer Genomics. The mutual exclusivity analysis showed that events in K-ras and p53 were likely to co-occur in pancreatic adenocarcinoma (Log odds ratio = 1.599, P = 0.006. The graphical summary of the mutations showed that there were hotspots for protein activation. In the network analysis, no solid association between K-ras and p53 was observed in pancreatic adenocarcinoma. In the survival analysis, neither K-ras nor p53 were associated with both survival events. As in the data mining study in the TCGA databases, our study provides a new perspective to understand the genetic features of K-ras and p53 in pancreatic adenocarcinoma.

  19. A Drosophila immune response against Ras-induced overgrowth

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    Thomas Hauling

    2014-03-01

    Full Text Available Our goal is to characterize the innate immune response against the early stage of tumor development. For this, animal models where genetic changes in specific cells and tissues can be performed in a controlled way have become increasingly important, including the fruitfly Drosophila melanogaster. Many tumor mutants in Drosophila affect the germline and, as a consequence, also the immune system itself, making it difficult to ascribe their phenotype to a specific tissue. Only during the past decade, mutations have been induced systematically in somatic cells to study the control of tumorous growth by neighboring cells and by immune cells. Here we show that upon ectopic expression of a dominant-active form of the Ras oncogene (RasV12, both imaginal discs and salivary glands are affected. Particularly, the glands increase in size, express metalloproteinases and display apoptotic markers. This leads to a strong cellular response, which has many hallmarks of the granuloma-like encapsulation reaction, usually mounted by the insect against larger foreign objects. RNA sequencing of the fat body reveals a characteristic humoral immune response. In addition we also identify genes that are specifically induced upon expression of RasV12. As a proof-of-principle, we show that one of the induced genes (santa-maria, which encodes a scavenger receptor, modulates damage to the salivary glands. The list of genes we have identified provides a rich source for further functional characterization. Our hope is that this will lead to a better understanding of the earliest stage of innate immune responses against tumors with implications for mammalian immunity.

  20. ARF1 recruits RAC1 to leading edge in neutrophil chemotaxis.

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    Mazaki, Yuichi; Onodera, Yasuhito; Higashi, Tsunehito; Horinouchi, Takahiro; Oikawa, Tsukasa; Sabe, Hisataka

    2017-10-02

    The small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex. We investigated the association between ARF1 and RAC1/CDC42 during the fMLP-stimulated chemotaxis of HL60 cells. We found that the silencing of GBF1 significantly impaired the recruitment of RAC1 to the leading edges, but not PAK1, αPIX, RAC2, or CDC42. A significant population of RAC1 colocalized with ARF1 at the leading edges in stimulated cells, whereas fMLP activated both ARF1 and ARF5. Consistently, the silencing of ARF1, but not ARF5, impaired the recruitment of RAC1, whereas the silencing of RAC1 did not affect the recruitment of ARF1 to the leading edges. Our results indicated that the activation of ARF1 triggers the plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling. Thus, membrane remodeling at the leading edges appears to precede actin remodeling in chemotaxis. Together with the fact that GIT2, which inactivates ARF1, is an integral component of the machinery activating RAC1, we proposed a model in which the ARF1-RAC1 linkage enables the regulation of ARF1 by repetitive on/off cycles during GPCR-mediated neutrophil chemotaxis.

  1. Diversification, phylogeny and evolution of auxin response factor (ARF) family: insights gained from analyzing maize ARF genes.

    Science.gov (United States)

    Wang, Yijun; Deng, Dexiang; Shi, Yating; Miao, Nan; Bian, Yunlong; Yin, Zhitong

    2012-03-01

    Auxin response factors (ARFs), member of the plant-specific B3 DNA binding superfamily, target specifically to auxin response elements (AuxREs) in promoters of primary auxin-responsive genes and heterodimerize with Aux/IAA proteins in auxin signaling transduction cascade. In previous research, we have isolated and characterized maize Aux/IAA genes in whole-genome scale. Here, we report the comprehensive analysis of ARF genes in maize. A total of 36 ARF genes were identified and validated from the B73 maize genome through an iterative strategy. Thirty-six maize ARF genes are distributed in all maize chromosomes except chromosome 7. Maize ARF genes expansion is mainly due to recent segmental duplications. Maize ARF proteins share one B3 DNA binding domain which consists of seven-stranded β sheets and two short α helixes. Twelve maize ARFs with glutamine-rich middle regions could be as activators in modulating expression of auxin-responsive genes. Eleven maize ARF proteins are lack of homo- and heterodimerization domains. Putative cis-elements involved in phytohormones and light signaling responses, biotic and abiotic stress adaption locate in promoters of maize ARF genes. Expression patterns vary greatly between clades and sister pairs of maize ARF genes. The B3 DNA binding and auxin response factor domains of maize ARF proteins are primarily subjected to negative selection during selective sweep. The mixed selective forces drive the diversification and evolution of genomic regions outside of B3 and ARF domains. Additionally, the dicot-specific proliferation of ARF genes was detected. Comparative genomics analysis indicated that maize, sorghum and rice duplicate chromosomal blocks containing ARF homologs are highly syntenic. This study provides insights into the distribution, phylogeny and evolution of ARF gene family.

  2. Oncogenic Ras-Induced Morphologic Change Is through MEK/ERK Signaling Pathway to Downregulate Stat3 at a Posttranslational Level in NIH3T3 Cells

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    Hsuan-Heng Yeh

    2008-01-01

    Full Text Available Ras is a key regulator of the MAP kinase-signaling cascade and may cause morphologic change of Ras-transformed cells. Signal transducer and activator of transcription 3 (Stat3 can be activated by cytokine stimulation. In this study, we unravel that Ha-rasV12 overexpression can downregulate the expression of Stat3 protein at a posttranslational level in NIH3T3 cells. Furthermore, we demonstrate that Stat3 expression downregulated by Ha-rasV12 overexpression is through proteosome degradation and not through a mTOR/p70S6K-related signaling pathway. The suppression of Stat3 accompanied by the morphologic change induced by Ha-rasV12 was through mitogen extracellular kinase (MEK/extracellular-regulated kinase (ERK signaling pathway. Microtubule disruption is involved in Ha-rasV12-induced morphologic change, which could be reversed by overexpression of Stat3. Taken together, we are the first to demonstrate that Stat3 protein plays a critical role in Ha-rasV12-induced morphologic change. Oncogenic Ras-triggered morphologic change is through the activation of MEK/ERK to posttranslationally downregulate Stat3 expression. Our finding may shed light on developing novel therapeutic strategies against Ras-related tumorigenesis.

  3. Manipulation of Auxin Response Factor 19 affects seed size in the woody perennial Jatropha curcas

    Science.gov (United States)

    Sun, Yanwei; Wang, Chunming; Wang, Ning; Jiang, Xiyuan; Mao, Huizhu; Zhu, Changxiang; Wen, Fujiang; Wang, Xianghua; Lu, Zhijun; Yue, Genhua; Xu, Zengfu; Ye, Jian

    2017-01-01

    Seed size is a major determinant of seed yield but few is known about the genetics controlling of seed size in plants. Phytohormones cytokinin and brassinosteroid were known to be involved in the regulation of herbaceous plant seed development. Here we identified a homolog of Auxin Response Factor 19 (JcARF19) from a woody plant Jatropha curcas and genetically demonstrated its functions in controlling seed size and seed yield. Through Virus Induced Gene Silencing (VIGS), we found that JcARF19 was a positive upstream modulator in auxin signaling and may control plant organ size in J. curcas. Importantly, transgenic overexpression of JcARF19 significantly increased seed size and seed yield in plants Arabidopsis thaliana and J. curcas, indicating the importance of auxin pathway in seed yield controlling in dicot plants. Transcripts analysis indicated that ectopic expression of JcARF19 in J. curcas upregulated auxin responsive genes encoding essential regulators in cell differentiation and cytoskeletal dynamics of seed development. Our data suggested the potential of improving seed traits by precisely engineering auxin signaling in woody perennial plants. PMID:28102350

  4. Mdm2 overexpression and p14(ARF) inactivation are two mutually exclusive events in primary human lung tumors.

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    Eymin, Béatrice; Gazzeri, Sylvie; Brambilla, Christian; Brambilla, Elisabeth

    2002-04-18

    Pathways involving p53 and pRb tumor suppressor genes are frequently deregulated during lung carcinogenesis. Through its location at the interface of these pathways, Mdm2 can modulate the function of both p53 and pRb genes. We have examined here the pattern of expression of Mdm2 in a series of 192 human lung carcinomas of all histological types using both immunohistochemical and Western blot analyses and four distinct antibodies mapping different epitopes onto the Mdm2 protein. Using Immunohistochemistry (IHC), Mdm2 was overexpressed as compared to normal lung in 31% (60 out of 192) of all tumors analysed, whatever their histological types. Western blotting was performed on 28 out of the 192 tumoral samples. Overexpression of p85/90, p74/76 and p57 Mdm2 isoforms was detected in 18% (5 out of 28), 25% (7 out of 28) and 39% (11 out of 28) of the cases respectively. Overall, overexpression of at least one isoform was observed in 14 out of 28 (50%) lung tumors and concomittant overexpression of at least two isoforms in 7 out of 28 (25%) cases. A good concordance (82%) was observed between immunohistochemical and Western blot data. Interestingly, a highly significant inverse relationship was detected between p14(ARF) loss and Mdm2 overexpression either in NSCLC (P=0.0089) or in NE lung tumors (P1 ratio was correlated with a high grade phenotype among NE tumors overexpressing Mdm2 (P=0.0021). Taken together, these data strongly suggest that p14(ARF)and Mdm2 act on common pathway(s) to regulate p53 and/or pRb-dependent or independent functions and that the Mdm2 : p14(ARF) ratio might act as a rheostat in modulating the activity of both proteins.

  5. Resistance and resilience of small-scale recirculating aquaculture systems (RAS) with or without algae to pH perturbation

    Science.gov (United States)

    Giatsis, Christos; Md Yusoff, Fatimah; Verreth, Johan; Verdegem, Marc

    2018-01-01

    The experimental set-up of this study mimicked recirculating aquaculture systems (RAS) where water quality parameters such as dissolved oxygen, pH, temperature, and turbidity were controlled and wastes produced by fish and feeding were converted to inorganic forms. A key process in the RAS was the conversion of ammonia to nitrite and nitrite to nitrate through nitrification. It was hypothesized that algae inclusion in RAS would improve the ammonia removal from the water; thereby improving RAS water quality and stability. To test this hypothesis, the stability of the microbiota community composition in a freshwater RAS with (RAS+A) or without algae (RAS-A) was challenged by introducing an acute pH drop (from pH 7 to 4 during three hours) to the system. Stigeoclonium nanum, a periphytic freshwater microalga was used in this study. No significant effect of the algae presence was found on the resistance to the acute pH drop on ammonia conversion to nitrite and nitrite conversion to nitrate. Also the resilience of the ammonia conversion to the pH drop disruption was not affected by the addition of algae. This could be due to the low biomass of algae achieved in the RAS. However, with regard to the conversion step of nitrite to nitrate, RAS+A was significantly more resilient than RAS-A. In terms of overall bacterial communities, the composition and predictive function of the bacterial communities was significantly different between RAS+A and RAS-A. PMID:29659617

  6. Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation

    Science.gov (United States)

    Bettoun, Audrey; Surdez, Didier; Vallerand, David; Gundogdu, Ramazan; Sharif, Ahmad A.D.; Gomez, Marta; Cascone, Ilaria; Meunier, Brigitte; White, Michael A.; Codogno, Patrice; Parrini, Maria Carla; Camonis, Jacques H.; Hergovich, Alexander

    2016-01-01

    Oncogenic Ras signalling occurs frequently in many human cancers. However, no effective targeted therapies are currently available to treat patients suffering from Ras-driven tumours. Therefore, it is imperative to identify downstream effectors of Ras signalling that potentially represent promising new therapeutic options. Particularly, considering that autophagy inhibition can impair the survival of Ras-transformed cells in tissue culture and mouse models, an understanding of factors regulating the balance between autophagy and apoptosis in Ras-transformed human cells is needed. Here, we report critical roles of the STK38 protein kinase in oncogenic Ras transformation. STK38 knockdown impaired anoikis resistance, anchorage-independent soft agar growth, and in vivo xenograft growth of Ras-transformed human cells. Mechanistically, STK38 supports Ras-driven transformation through promoting detachment-induced autophagy. Even more importantly, upon cell detachment STK38 is required to sustain the removal of damaged mitochondria by mitophagy, a selective autophagic process, to prevent excessive mitochondrial reactive oxygen species production that can negatively affect cancer cell survival. Significantly, knockdown of PINK1 or Parkin, two positive regulators of mitophagy, also impaired anoikis resistance and anchorage-independent growth of Ras-transformed human cells, while knockdown of USP30, a negative regulator of PINK1/Parkin-mediated mitophagy, restored anchorage-independent growth of STK38-depleted Ras-transformed human cells. Therefore, our findings collectively reveal novel molecular players that determine whether Ras-transformed human cells die or survive upon cell detachment, which potentially could be exploited for the development of novel strategies to target Ras-transformed cells. PMID:27283898

  7. A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK

    International Nuclear Information System (INIS)

    Abe, Yasuhito; Takeuchi, Takashi; Imai, Yoshinori; Murase, Ryuichi; Kamei, Yoshiaki; Fujibuchi, Taketsugu; Matsumoto, Suguru; Ueda, Norifumi; Ogasawara, Masahito; Shigemoto, Kazuhiro; Kito, Katsumi

    2006-01-01

    PRPK phosphorylates serine-15 residue of p53 and enhances transcriptional activity. PRPK possesses a bipartite nuclear localization signal and localizes in nucleus when over-expressed in cells. However, intrinsic PRPK localizes mainly in the cytosol in situ. While studying the mechanisms in the distribution of intrinsic PRPK, we identified a PRPK binding protein, an ubiquitously expressed Small Ras-like GTPase, Rab1c, also named Ray or Rab35. The over-expressed Ray was distributed in the nucleus, cytosol, and cell membrane. Both Ray wild type and GTP-restrictively binding mutant Ray-Q67L, but not guanine nucleotide unstable binding mutant Ray-N120I, partially distributed the over-expressed PRPK to the cytosol and also suppressed the PRPK-induced p53-transcriptional activity profoundly. A Small Ras-like GTPase protein Ray was thus indicated to modulate p53 transcriptional activity of PRPK

  8. TBC1D24, an ARF6-interacting protein, is mutated in familial infantile myoclonic epilepsy.

    Science.gov (United States)

    Falace, Antonio; Filipello, Fabia; La Padula, Veronica; Vanni, Nicola; Madia, Francesca; De Pietri Tonelli, Davide; de Falco, Fabrizio A; Striano, Pasquale; Dagna Bricarelli, Franca; Minetti, Carlo; Benfenati, Fabio; Fassio, Anna; Zara, Federico

    2010-09-10

    Idiopathic epilepsies (IEs) are a group of disorders characterized by recurrent seizures in the absence of detectable brain lesions or metabolic abnormalities. IEs include common disorders with a complex mode of inheritance and rare Mendelian traits suggesting the occurrence of several alleles with variable penetrance. We previously described a large family with a recessive form of idiopathic epilepsy, named familial infantile myoclonic epilepsy (FIME), and mapped the disease locus on chromosome 16p13.3 by linkage analysis. In the present study, we found that two compound heterozygous missense mutations (D147H and A509V) in TBC1D24, a gene of unknown function, are responsible for FIME. In situ hybridization analysis revealed that Tbc1d24 is mainly expressed at the level of the cerebral cortex and the hippocampus. By coimmunoprecipitation assay we found that TBC1D24 binds ARF6, a Ras-related family of small GTPases regulating exo-endocytosis dynamics. The main recognized function of ARF6 in the nervous system is the regulation of dendritic branching, spine formation, and axonal extension. TBC1D24 overexpression resulted in a significant increase in neurite length and arborization and the FIME mutations significantly reverted this phenotype. In this study we identified a gene mutation involved in autosomal-recessive idiopathic epilepsy, unveiled the involvement of ARF6-dependent molecular pathway in brain hyperexcitability and seizures, and confirmed the emerging role of subtle cytoarchitectural alterations in the etiology of this group of common epileptic disorders. 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  9. Hydrogen Peroxide Toxicity Induces Ras Signaling in Human Neuroblastoma SH-SY5Y Cultured Cells

    Directory of Open Access Journals (Sweden)

    Jirapa Chetsawang

    2010-01-01

    Full Text Available It has been reported that overproduction of reactive oxygen species occurs after brain injury and mediates neuronal cells degeneration. In the present study, we examined the role of Ras signaling on hydrogen peroxide-induced neuronal cells degeneration in dopaminergic neuroblastoma SH-SY5Y cells. Hydrogen peroxide significantly reduced cell viability in SH-SY5Y cultured cells. An inhibitor of the enzyme that catalyzes the farnesylation of Ras proteins, FTI-277, and a competitive inhibitor of GTP-binding proteins, GDP-beta-S significantly decreased hydrogen peroxide-induced reduction in cell viability in SH-SY5Y cultured cells. The results of this study might indicate that a Ras-dependent signaling pathway plays a role in hydrogen peroxide-induced toxicity in neuronal cells.

  10. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...

  11. PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Donghoon M Lee

    Full Text Available The recruitment of GDP/GTP exchange factors (GEFs to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.

  12. Detecção imunoistoquímica das oncoproteínas p21ras, c-myc E p53 no carcinoma hepatocelular e no tecido hepático não-neoplásico Immunohistochemical detection of p21ras, c-myc and p53 oncoproteins in hepatocellular carcinoma and in non-neoplastic liver tissue

    Directory of Open Access Journals (Sweden)

    Vera Lucia Nunes Pannain

    2004-12-01

    Full Text Available RACIONAL: A hepatocarcinogênese é um processo no qual as alterações genéticas e epigenéticas são bem conhecidas em modelos animais, mas carece de estudos no homem. OBJETIVOS: Analisar a freqüência das oncoproteínas p21ras, c-myc e p53 no carcinoma hepatocelular e no fígado não-neoplásico. Verificar ainda a associação destas oncoproteínas com os padrões e graus histológicos, assim como com as infecções pelos vírus das hepatites B e C. MÉTODOS: Foi analisada por método imunoistoquímico a detecção das oncoproteínas p21ras, c-myc e p53 em 47 casos de carcinoma hepatocelular e no tecido não-neoplásico circunjacente ao tumor (40 casos. RESULTADOS: As oncoproteínas p21ras, c-myc e p53 foram detectadas, respectivamente, em 44,7%, 53,2% e 36,2% dos casos de carcinoma hepatocelular. A imunorreatividade do p21ras e c-myc mostrou uma associação significativa. Contudo, não houve associação significativa entre a detecção do p21ras, c-myc e p53 com os diferentes graus e padrões histológicos, nem tampouco com as infecções pelos vírus das hepatites B e C. A mesma associação significativa entre o p21ras e c-myc foi encontrada no tecido não-neoplásico dos casos de cirrose em relação aos que não apresentaram cirrose, enquanto que o p53 foi negativo em todos os casos. CONCLUSÕES: A imunorreatividade das oncoproteínas p21ras, c-myc e p53 corrobora evidências prévias de sua detecção no carcinoma hepatocelular, o que sugere poder haver participação destas proteínas na hepatocarcinogênese humana. A significativa associação entre as proteínas p21ras, c-myc e p53 no carcinoma hepatocelular e na cirrose pode apontar uma interação entre as mesmas, sobretudo na hepatocarcinogênese pela via da cirrose.BACKGROUND: Genetic and epigenetic alterations have been described in animal hepatocarcinogenesis models but need to be studied in human being. AIMS: To assess the immunoreactivity of p21ras, c-myc and p53

  13. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

    Science.gov (United States)

    Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

    2000-05-01

    O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

  14. Possible involvement of MSX-2 homeoprotein in v-ras-induced transformation.

    Science.gov (United States)

    Takahashi, C; Akiyama, N; Kitayama, H; Takai, S; Noda, M

    1997-04-01

    A truncated MSX-2 homeoprotein was found to induce flat reversion when expressed in v-Ki-ras-transformed NIH3T3 cells. Although the expression of endogenous MSX-2 gene is low in most of the normal adult tissues examined, it is frequently activated in carcinoma-derived cell lines. Likewise, the gene is inactive in untransformed cells but is transcriptionally activated after transformation by v-Ki-ras oncogene, suggesting that the intact MSX-2 may play a positive, rather than suppressive, role in cell transformation. To test this possibility, we isolated a full-length human MSX-2 cDNA and tested its activities in two cell systems: fibroblast and myoblast. In NIH3T3 fibroblasts, although the gene by itself failed to confer a transformed phenotype, antisense MSX-2 cDNA as well as truncated MSX-2 cDNA interfered with the transforming activities of both v-Ki-ras and v-raf oncogene. In C2C12 myoblasts, MSX-2 was found to suppress MyoD gene expression, as do activated ras oncogenes, under certain culture conditions, and truncated MSX-2 cDNA was found to inhibit the activities of both MSX-2 and ras in this system as well. Our findings not only suggest that the truncated version MSX-2 may act as a dominant suppressor of intact MSX-2 but also raise the possibility that MSX-2 gene may be an important downstream target for the Ras signaling pathways.

  15. Neurotrophin responsiveness of sympathetic neurons is regulated by rapid mobilization of the p75 receptor to the cell surface through TrkA activation of Arf6.

    Science.gov (United States)

    Edward Hickman, F; Stanley, Emily M; Carter, Bruce D

    2018-05-22

    The p75 neurotrophin receptor (p75NTR) plays an integral role in patterning the sympathetic nervous system during development. Initially, p75NTR is expressed at low levels as sympathetic axons project toward their targets, which enables neurotrophin-3 (NT3) to activate TrkA receptors and promote growth. Upon reaching nerve growth factor (NGF) producing tissues, p75NTR is up regulated resulting in formation of TrkA-p75 complexes, which are high affinity binding sites selective for NGF, thereby blunting NT3 signaling. The level of p75NTR expressed on the neuron surface is instrumental in regulating trophic factor response; however, the mechanisms by which p75NTR expression is regulated are poorly understood. Here, we demonstrate a rapid, translation independent increase in surface expression of p75NTR in response to NGF in rat sympathetic neurons. p75NTR was mobilized to the neuron surface from GGA3-postitive vesicles through activation of the GTPase Arf6, which was stimulated by NGF, but not NT3 binding to TrkA. Arf6 activation required PI3 kinase activity and was prevented by an inhibitor of the cytohesin family of Arf6 GEFs. Overexpression of a constitutively active Arf6 mutant (Q67L) was sufficient to significantly increase surface expression of p75NTR even in the absence of NGF. Functionally, expression of active Arf6 markedly attenuated the ability of NT3 to promote neuronal survival and neurite outgrowth while the NGF response was unaltered. These data suggest that NGF activation of Arf6 through TrkA is critical for the increase in p75NTR surface expression that enables the switch in neurotrophin responsiveness during development in the sympathetic nervous system. SIGNIFICANCE STATEMENT p75NTR is instrumental in the regulation of neuronal survival and apoptosis during development and is also implicated as a contributor to aberrant neurodegeneration in numerous conditions. Therefore, a better understanding of the mechanisms that mediate p75NTR surface

  16. ERK mediated upregulation of death receptor 5 overcomes the lack of p53 functionality in the diaminothiazole DAT1 induced apoptosis in colon cancer models: efficiency of DAT1 in Ras-Raf mutated cells.

    Science.gov (United States)

    Thamkachy, Reshma; Kumar, Rohith; Rajasekharan, K N; Sengupta, Suparna

    2016-03-08

    p53 is a tumour suppressor protein that plays a key role in many steps of apoptosis, and malfunctioning of this transcription factor leads to tumorigenesis. Prognosis of many tumours also depends upon the p53 status. Most of the clinically used anticancer compounds activate p53 dependent pathway of apoptosis and hence require p53 for their mechanism of action. Further, Ras/Raf/MEK/ERK axis is an important signaling pathway activated in many cancers. Dependence of diaminothiazoles, compounds that have gained importance recently due to their anticancer and anti angiogenic activities, were tested in cancer models with varying p53 or Ras/Raf mutational status. In this study we have used p53 mutated and knock out colon cancer cells and xenograft tumours to study the role of p53 in apoptosis mediated by diaminothiazoles. Colon cancer cell lines with varying mutational status for Ras or Raf were also used. We have also examined the toxicity and in vivo efficacy of a lead diaminothiazole 4-Amino-5-benzoyl-2-(4-methoxy phenylamino)thiazole (DAT1) in colon cancer xenografts. We have found that DAT1 is active in both in vitro and in vivo models with nonfunctional p53. Earlier studies have shown that extrinsic pathway plays major role in DAT1 mediated apoptosis. In this study, we have found that DAT1 is causing p53 independent upregulation of the death receptor 5 by activating the Ras/Raf/MEK/ERK signaling pathway both in wild type and p53 suppressed colon cancer cells. These findings are also confirmed by the in vivo results. Further, DAT1 is more efficient to induce apoptosis in colon cancer cells with mutated Ras or Raf. Minimal toxicity in both acute and subacute studies along with the in vitro and in vivo efficacy of DAT1 in cancers with both wild type and nonfunctional p53 place it as a highly beneficial candidate for cancer chemotherapy. Besides, efficiency in cancer cells with mutations in the Ras oncoprotein or its downstream kinase Raf raise interest in

  17. Human Mut T Homolog 1 (MTH1): a roadblock for the tumor-suppressive effects of oncogenic RAS-induced ROS.

    Science.gov (United States)

    Rai, Priyamvada

    2012-01-01

    Oncogenic RAS-induced reactive oxygen species (ROS) trigger barriers to cell transformation and cancer progression through tumor-suppressive responses such as cellular senescence or cell death. We have recently shown that oncogenic RAS-induced DNA damage and attendant premature senescence can be prevented by overexpressing human MutT Homolog 1 (MTH1), the major mammalian detoxifier of the oxidized DNA precursor, 8-oxo-dGTP. Paradoxically, RAS-induced ROS are also able to participate in tumor progression via transformative processes such as mitogenic signaling, the epithelial-mesenchymal transition (EMT), anoikis inhibition, and PI3K/Akt-mediated survival signaling. Here we provide a preliminary insight into the influence of MTH1 levels on the EMT phenotype and Akt activation in RAS-transformed HMLE breast epithelial cells. Within this context, we will discuss the implications of MTH1 upregulation in oncogenic RAS-sustaining cells as a beneficial adaptive change that inhibits ROS-mediated cell senescence and participates in the maintenance of ROS-associated tumor-promoting mechanisms. Accordingly, targeting MTH1 in RAS-transformed tumor cells will not only induce proliferative defects but also potentially enhance therapeutic cytotoxicity by shifting cellular response away from pro-survival mechanisms.

  18. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    International Nuclear Information System (INIS)

    Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P.; Komori, Hideyuki; Ohtani, Kiyoshi

    2014-01-01

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  19. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    Energy Technology Data Exchange (ETDEWEB)

    Kurayoshi, Kenta [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Ozono, Eiko [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ (United Kingdom); Iwanaga, Ritsuko; Bradford, Andrew P. [Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, CO 80045 (United States); Komori, Hideyuki [Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 (United States); Ohtani, Kiyoshi, E-mail: btm88939@kwansei.ac.jp [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)

    2014-07-18

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  20. The influence of partial renal function impairment on thyroid hormones level in acute renal failure-ARF patients

    International Nuclear Information System (INIS)

    Ilic, S.; Vlajkovic, M.; Rajic, M.; Bogicevic, M.

    2002-01-01

    The aim of this study was to assess the relationship of thyroid hormone levels with glomerular and tubular renal function in ARF. In thirty one patients with different underlying diseases, glomerular filtration (GFR) was estimated by measuring 99mTc-DTPA clearance and tubular function by 131I-OIH clearance. The thyroid status was evaluated by radioimmunoassays of T4, FT4, T3, FT3, rT3 and TSH, while the cortisol level was used to indicate the stress role. All the measurements were performed within 7 days as well after 1,3,6 and 12 months from the ARF onset. The results obtained were compared with those of 30 healthy subjects. At the ARF beginning, the impairment of renal function was manifested by the fall of 99mTc-DTPA clearance to 21∫7ml/min and 131I-OIH clearance to 172±34ml/min, while end values were 77±19ml/min and 395±70ml/min, respectively. The hormone analysis within 7 days, showed T3 decrease to 1.21±0.16nmol/l and the increase rT3 to 38±7ng/1ooml, as well as cortisol to 350±71ng/ml, but at the end of the study these hormone levels were normalized. Other hormone levels didn't differ significantly from control values. Regression analysis established significant correlation of 99mTc-DTPA clearance with T3 (r=0.693, p 100, OIH>500), partial recovery (DTPA 51-100, OIH 250-500) and without any recovery (DTPA<50,OIH<250), showed hormone disturbances proportional to the degree of renal function damage. The most severe disorders were found in the third group, where both total and free T4 and T3 were lowered, while rT3 and cortisol elevated at 7th day from ARF onset. After 12 months all these values associated with that of TSH were observed as declined.This study suggests thyroid hormone levels to be dependent on the renal function in ARF. Greater impairment of glomerular related to tubular function appears to be more responsible for thyroid dysfunction, due to uremic toxins retention as well as stress induced by the extreme function failure

  1. Normal p21Ras/MAP kinase pathway expression and function in PBMC from patients with polycystic ovary disease.

    Science.gov (United States)

    Buchs, A; Chagag, P; Weiss, M; Kish, E; Levinson, R; Aharoni, D; Rapoport, M J

    2004-04-01

    Polycystic ovary disease (PCOD) is associated with insulin resistance and increased prevalence of type II diabetes mellitus (T2DM). The p21Ras/MAP kinase is a major intracellular signaling pathway mediating insulin signaling in insulin responsive tissues. The expression, regulation and function of the p21Ras/MAP kinase pathway in PCOD patients were examined. Peripheral blood mononuclear cells (PBMC) were isolated from ten patients with PCOD and ten controls. The expression of p21Ras and its regulatory proteins; hSOS1 and p120GAP were studied. The basal and phytohemaglutinin (PHA) or insulin stimulated phosphorylation of MAP kinase was determined. Expression of p21Ras, and its regulatory proteins hSOS1 and p120GAP were similar in PCOD patients and controls. Basal, PHA and insulin stimulated phosphorylation of MAP kinase, were also comparable in the two groups as well as their PBMC proliferative response. These data indicate that the expression and overall function of the p21Ras/MAP kinase pathway remain intact in non-diabetic patients with PCOD.

  2. ARF tumor suppression in the nucleolus.

    Science.gov (United States)

    Maggi, Leonard B; Winkeler, Crystal L; Miceli, Alexander P; Apicelli, Anthony J; Brady, Suzanne N; Kuchenreuther, Michael J; Weber, Jason D

    2014-06-01

    Since its discovery close to twenty years ago, the ARF tumor suppressor has played a pivotal role in the field of cancer biology. Elucidating ARF's basal physiological function in the cell has been the focal interest of numerous laboratories throughout the world for many years. Our current understanding of ARF is constantly evolving to include novel frameworks for conceptualizing the regulation of this critical tumor suppressor. As a result of this complexity, there is great need to broaden our understanding of the intricacies governing the biology of the ARF tumor suppressor. The ARF tumor suppressor is a key sensor of signals that instruct a cell to grow and proliferate and is appropriately localized in nucleoli to limit these processes. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Ubiquitin specific peptidase 5 mediates Histidine-rich protein Hpn induced cell apoptosis in hepatocellular carcinoma through P14-P53 signaling.

    Science.gov (United States)

    Liu, Yi; Wang, Wei-Mao; Zou, Li-Yi; Li, Li; Feng, Lu; Pan, Ming-Zhu; Lv, Min-Yi; Cao, Ying; Wang, Hua; Kung, Hsiang-Fu; Pang, Jian-Xin; Fu, Wei-Ming; Zhang, Jin-Fang

    2017-06-01

    Hpn is a small histidine-rich cytoplasmic protein from Helicobacter pylori and has been recognized as a high-risk factor for several cancers including gastric cancer, colorectal cancer, and MALT lymphoma. However, the relationship between Hpn and cancers remains elusive. In this study, we discovered that Hpn protein effectively suppressed cell growth and induced apoptosis in hepatocellular carcinoma (HCC). A two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics was performed to find the molecular targets of Hpn in HCC cells. It was identified that twelve proteins were differentially expressed, with USP5 being one of the most significantly downregulated protein. The P14 ARF -P53 signaling was activated by USP5 knockdown in HCC cells. Furthermore, USP5 overexpression significantly rescued the suppressive effect of Hpn on the viability of HCC cells. In conclusion, our study suggests that Hpn plays apoptosis-inducing roles through suppressing USP5 expression and activating the P14 ARF -P53 signaling. Therefore, Hpn may be a potential candidate for developing novel anti-HCC drugs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Vesicular transport protein Arf6 modulates cytoskeleton dynamics for polar body extrusion in mouse oocyte meiosis.

    Science.gov (United States)

    Duan, Xing; Zhang, Hao-Lin; Pan, Meng-Hao; Zhang, Yu; Sun, Shao-Chen

    2018-02-01

    Arf6 (ADP-ribosylation factor 6) is known to play important roles in membrane dynamics through the regulation of actin filament reorganization for multiple cellular processes such as cytokinesis, phagocytosis, cell migration and tumor cell invasion. However, the functions of Arf6 in mammalian oocyte meiosis have not been clarified. In present study we showed that Arf6 expressed in mouse oocytes and was mainly distributed around the spindle during meiosis. Depletion of Arf6 by morpholino microinjection caused oocytes failing to extrude first polar body. Further analysis indicated that Arf6 knock down caused the aberrant actin distribution, which further induced the failure of meiotic spindle movement. And the loss of oocyte polarity also confirmed this. The regulation of Arf6 on actin filaments in mouse oocytes might be due to its effects on the phosphorylation level of cofilin and the expression of Arp2/3 complex. Moreover, we found that the decrease of Arf6 caused the disruption of spindle formation, indicating the multiple roles of Arf6 on cytoskeleton dynamics in meiosis. In summary, our results indicated that Arf6 was involved in mouse oocyte meiosis through its functional roles in actin-mediated spindle movement and spindle organization. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. The p21 ras C-terminus is required for transformation and membrane association

    DEFF Research Database (Denmark)

    Willumsen, B M; Christensen, A; Hubbert, N L

    1984-01-01

    The Harvey murine sarcoma virus (Ha-MuSV) transforming gene, v-rasH, encodes a 21,000 molecular weight protein (p21) that is closely related to the p21 proteins encoded by the cellular transforming genes of the ras gene family. The primary translation product (prop21), which is found in the cytosol...... of these biochemical features of the protein, we have now studied a series of deletion mutants located at or near the C-terminus of the viral p21 protein. Our tissue culture studies indicate that amino acids located at or near the C-terminus are required for cellular transformation, membrane association and lipid...

  6. The Arf GTPase-activating protein family is exploited by Salmonella enterica serovar Typhimurium to invade nonphagocytic host cells.

    Science.gov (United States)

    Davidson, Anthony C; Humphreys, Daniel; Brooks, Andrew B E; Hume, Peter J; Koronakis, Vassilis

    2015-02-10

    To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by subverting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasma membrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Surprisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be considered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Furthermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intracellular infections. To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP

  7. Bilobalide induces neuronal differentiation of P19 embryonic carcinoma cells via activating Wnt/β-catenin pathway.

    Science.gov (United States)

    Liu, Mei; Guo, Jingjing; Wang, Juan; Zhang, Luyong; Pang, Tao; Liao, Hong

    2014-08-01

    Bilobalide, a natural product extracted from Ginkgo biloba leaf, is known to exhibit a number of pharmacological activities. So far, whether it could affect embryonic stem cell differentiation is still unknown. The main aim of this study was to investigate the effect of bilobalide on P19 embryonic carcinoma cells differentiation and the underlying mechanisms. Our results showed that bilobalide induced P19 cells differentiation into neurons in a concentration- and time-dependent manner. We also found that bilobalide promoted neuronal differentiation through activation of Wnt/β-catenin signaling pathway. Exposure to bilobalide increased inactive GSK-3β phosphorylation, further induced the nuclear accumulation of β-catenin, and also up-regulated the expression of Wnt ligands Wnt1 and Wnt7a. Neuronal differentiation induced by bilobalide was totally abolished by XAV939, an inhibitor of Wnt/β-catenin pathway. These results revealed a novel role of bilobalide in neuronal differentiation from P19 embryonic cells acting through Wnt/β-catenin signaling pathway, which would provide a better insight into the beneficial effects of bilobalide in brain diseases.

  8. Oxidative Stress Posttranslationally Regulates the Expression of Ha-Ras and Ki-Ras in Cultured Astrocytes

    Directory of Open Access Journals (Sweden)

    Samantha Messina

    2012-01-01

    Full Text Available Addition of hydrogen peroxide to cultured astrocytes induced a rapid and transient increase in the expression of Ha-Ras and Ki-Ras. Pull-down experiments with the GTP-Ras-binding domain of Raf-1 showed that oxidative stress substantially increased the activation of Ha-Ras, whereas a putative farnesylated activated form of Ki-Ras was only slightly increased. The increase in both Ha-Ras and Ki-Ras was insensitive to the protein synthesis inhibitor, cycloheximide, and was occluded by the proteasomal inhibitor, MG-132. In addition, exposure to hydrogen peroxide reduced the levels of ubiquitinated Ras protein, indicating that oxidative stress leads to a reduced degradation of both isoforms through the ubiquitin/proteasome pathway. Indeed, the late reduction in Ha-Ras and Ki-Ras was due to a recovery of proteasomal degradation because it was sensitive to MG-132. The late reduction of Ha-Ras levels was abrogated by compound PD98059, which inhibits the MAP kinase pathway, whereas the late reduction of Ki-Ras was unaffected by PD98059. We conclude that oxidative stress differentially regulates the expression of Ha-Ras and Ki-Ras in cultured astrocytes, and that activation of the MAP kinase pathway by oxidative stress itself or by additional factors may act as a fail-safe mechanism limiting a sustained expression of the potentially detrimental Ha-Ras.

  9. Simvastatin attenuates acrolein-induced mucin production in rats: involvement of the Ras/extracellular signal-regulated kinase pathway.

    Science.gov (United States)

    Chen, Ya-Juan; Chen, Peng; Wang, Hai-Xia; Wang, Tao; Chen, Lei; Wang, Xun; Sun, Bei-Bei; Liu, Dai-Shun; Xu, Dan; An, Jing; Wen, Fu-Qiang

    2010-06-01

    Airway mucus overproduction is a cardinal feature of airway inflammatory diseases, such as chronic obstructive pulmonary disease and cystic fibrosis. Since the small G-protein Ras is known to modulate cellular functions in the lung, we sought to investigate whether the Ras inhibitor simvastatin could attenuate acrolein-induced mucin production in rat airways. Rats were exposed to acrolein for 12 days, after first being pretreated intragastrically for 24 h with either simvastatin alone or simvastatin in combination with mevalonate, which prevents the isoprenylation needed for Ras activation. Lung tissue was analyzed for extracellular signal-regulated kinase (ERK) activity, goblet cell metaplasia and mucin production. To analyze the effect of simvastatin on mucin production in more detail, acrolein-exposed human airway epithelial NCI-H292 cells were pretreated with simvastatin alone or together with mevalonate. Culture medium was collected to detect mucin secretion, and cell lysates were examined for Ras-GTPase activity and epidermal growth factor receptor (EGFR)/ERK phosphorylation. In vivo, simvastatin treatment dose-dependently suppressed acrolein-induced goblet cell hyperplasia and metaplasia in bronchial epithelium and inhibited ERK phosphorylation in rat lung homogenates. Moreover, simvastatin inhibited Muc5AC mucin synthesis at both the mRNA and protein levels in the lung. In vitro, simvastatin pretreatment attenuated the acrolein-induced significant increase in MUC5AC mucin expression, Ras-GTPase activity and EGFR/ERK phosphorylation. These inhibitory effects of simvastatin were neutralized by mevalonate administration both in vitro and in vivo. Our results suggest that simvastatin may attenuate acrolein-induced mucin protein synthesis in the airway and airway inflammation, possibly by blocking ERK activation mediated by Ras protein isoprenylation. Thus, the evidence from the experiment suggests that human trials are warranted to determine the potential

  10. MEKK1 is a Novel Regulator of the Dmp1-Arf-p53 Pathway and Prognostic Indicator in Breast Cancer

    Science.gov (United States)

    2012-12-01

    hDMP1, INK4a/ARF, p53 or Hdm2 amplification. Kaplan -Meier analyses have been conducted to study the impact for the impact of loss or gain of each locus on...Palma P, Pellegrini S, Fina P et al. Mdm2 gene alterations and mdm2 protein expression in breast carcinomas. J Pathol 1995; 175: 31–38. 21 Turbin DA

  11. Characterization of a novel oncogenic K-ras mutation in colon cancer

    International Nuclear Information System (INIS)

    Akagi, Kiwamu; Uchibori, Ryosuke; Yamaguchi, Kensei; Kurosawa, Keiko; Tanaka, Yoichiro; Kozu, Tomoko

    2007-01-01

    Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity

  12. Interleukin (IL)‐39 [IL‐23p19/Epstein–Barr virus‐induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus‐prone mice

    Science.gov (United States)

    Liu, X.; Zhang, Y.; Wang, Z.; Zhu, G.; Han, G.; Chen, G.; Hou, C.; Wang, T.; Ma, N.; Shen, B.; Li, Y.; Wang, R.

    2016-01-01

    Summary Interleukin (IL)‐12 family cytokines play critical roles in autoimmune diseases. Our previous study has shown that IL‐23p19 and Epstein–Barr virus‐induced 3 (Ebi3) form a new IL‐12 family heterodimer, IL‐23p19/Ebi3, termed IL‐39, and knock‐down of p19 or Ebi3 reduced diseases by transferred GL7+ B cells in lupus‐prone mice. In the present study, we explore further the possible effect of IL‐39 on murine lupus. We found that IL‐39 in vitro and in vivo induces differentiation and/or expansion of neutrophils. GL7+ B cells up‐regulated neutrophils by secreting IL‐39, whereas IL‐39‐deficient GL7+ B cells lost the capacity to up‐regulate neutrophils in lupus‐prone mice and homozygous CD19cre (CD19‐deficient) mice. Finally, we found that IL‐39‐induced neutrophils had a positive feedback on IL‐39 expression in activated B cells by secreting B cell activation factor (BAFF). Taken together, our results suggest that IL‐39 induces differentiation and/or expansion of neutrophils in lupus‐prone mice. PMID:27400195

  13. CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling.

    Science.gov (United States)

    Manara, Maria Cristina; Terracciano, Mario; Mancarella, Caterina; Sciandra, Marika; Guerzoni, Clara; Pasello, Michela; Grilli, Andrea; Zini, Nicoletta; Picci, Piero; Colombo, Mario P; Morrione, Andrea; Scotlandi, Katia

    2016-11-29

    CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.

  14. Involvement of Bmi-1 gene in the development of gastrointestinal stromal tumor by regulating p16Ink4A/p14ARF gene expressions: An in vivo and in vitro study.

    Science.gov (United States)

    Wang, Jiang-Li; Wu, Jiang-Hong; Hong, Cai; Wang, Ya-Nong; Zhou, Ye; Long, Zi-Wen; Zhou, Ying; Qin, Hai-Shu

    2017-12-01

    This study was conducted in order to explore the role that Bmi-1 plays during the development of a gastrointestinal stromal tumor (GIST) by regulation of the p16 Ink4A and p14 ARF expressions. Eighty-six patients diagnosed with GIST were selected to take part in this experiment. The Bmi-1 protein expressions in GIST and adjacent normal tissues were detected using immunohistochemistry and further analyzed by using photodensitometry. To monitor and track the progression of the GIST, a 3-year follow-up was conducted for all affected patients. After cell transfection, the GIST cells were assigned into the control group (without transfection), the negative control (NC) group (transfected with Bmi-1-Scramble plasmid), and the Bmi-1 shRNA group (transfected with the pcDNA3.1-Bmi-1 shRNA plasmid). Protein and mRNA expressions collected from Bmi-1, p16 lnk4A , P14 ARF , cyclin D1, and CDK4 were measured using both the RT-qPCR and western blotting methods Cell senescence was assessed and obtained by using the β-Galactosidase (β-Gal) activity assay. The use of a Soft agar colony formation assay and CCK-8 assay were performed in order to detect the cell growth and subsequent proliferation. Cell invasion and migration were analyzed using the Transwell assay and scratch test. Bmi-1 in the GIST tissues was found to be significantly higher and the p16 lnk4A and P14 ARF expressions were lower than those in the adjacent normal tissues. Bmi-1 was negatively correlated with p16 lnk4A and P14 ARF expressions according to the correlation analysis. Bmi-1 expression was associated with the TNM stage, postoperative recurrence, metastasis, tumor size, and the 5-year survival rate. Area under ROC curve was calculated at 0.884, and sensitivity, specificity, and accuracy of Bmi-1 predicting the GIST were 67.44%, 97.67%, and 65.12%, respectively. Patients exhibiting a high Bmi-1 expression in the GIST tissues had lower survival rates than those with low Bmi-1 expression. In comparison with

  15. Immunoscintigraphy of human tumors transplanted in nude mice with radiolabeled anti-ras p21 monoclonal antibodies

    International Nuclear Information System (INIS)

    Katoh, Y.; Nakata, K.; Kohno, K.; Shima, M.; Satoh, A.; Kusumoto, Y.; Ishii, N.; Kohji, T.; Shiku, H.; Nagataki, S.

    1990-01-01

    Anti-ras p21 monoclonal antibody (RASK-3) was used for immunoscintigraphy of human cancer cell lines in nude mice. Iodine-125-labeled RASK-3 was injected into nude mice with either human colon cancers (FCC-1 or BM-314) or lung cancer (KNS-62). Clear images were obtained in all three cancers 7 days after the injection of antibody. No localization of 125 I-labeled control monoclonal antibody was observed. The ratio of tissue/blood radioactivity and % ID/g in the tumor were significantly higher than other organs by Day 8. The specific localization index examined by 131 I-RASK-3 and 125 I-control monoclonal antibody was also higher in the tumor than in other tissues. In the in vitro study, binding of RASK-3 to tumor cells increased significantly by treatment of cells with either lysolecithin or periodate-lysine-paraformaldehyde, which confirmed the intracellular localization of ras p21. The mechanism by which anti-ras p21 antibodies accumulate in tumor sites could be the necrotic changes in tumor cells or changes in membrane permeability of non-necrotic cells. These results provide a strong rationale for the utilization of ras p21 as a target antigen in the imaging of a variety of human cancers

  16. GRIM-19 disrupts E6/E6AP complex to rescue p53 and induce apoptosis in cervical cancers.

    Directory of Open Access Journals (Sweden)

    Ying Zhou

    Full Text Available BACKGROUND: Our previous studies showed a down-regulation of GRIM-19 in primary human cervical cancers, and restoration of GRIM-19 induced tumor regression. The induction of tumor suppressor protein p53 ubiquitination and degradation by E6 oncoportein of high risk-HPV through forming a stable complex with E6AP is considered as a critical mechanism for cervical tumor development. The aims of this study were to determine the potential role of GRIM-19 in rescuing p53 protein and inducing cervical cancer cell apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: The protein levels of GRIM-19 and p53 were detected in normal cervical tissues from 45 patients who underwent hysterectomy for reasons other than neoplasias of either the cervix or endometrium, and cervical cancer tissues from 60 patients with non-metastatic squamous epithelial carcinomas. Coimmunoprecipitation and GST pull-down assay were performed to examine the interaction of GRIM-19 with 18E6 and E6AP in vivo and in vitro respectively. The competition of 18E6 with E6AP in binding GRIM-19 by performing competition pull-down assays was designed to examine the disruption of E6/E6AP complex by GRIM-19. The augment of E6AP ubiquitination by GRIM-19 was detected in vivo and in vitro ubiquitination assay. The effects of GRIM-19-dependent p53 accumulation on cell proliferation, cell cycle, apoptosis were explored by MTT, flow cytometry and transmission electron microscopy respectively. The tumor suppression was detected by xenograft mouse model. CONCLUSION/SIGNIFICANCE: The levels of GRIM-19 and p53 were concurrently down regulated in cervical cancers. The restoration of GRIM-19 can induce ubiquitination and degradation of E6AP, and disrupt the E6/E6AP complex through the interaction of N-terminus of GRIM-19 with both E6 and E6AP, which protected p53 from degradation and promoted cell apoptosis. Tumor xenograft studies also revealed the suppression of p53 degradation in presence of GRIM-19. These data

  17. HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons.

    Science.gov (United States)

    Shen, Qing-Tao; Ren, Xuefeng; Zhang, Rui; Lee, Il-Hyung; Hurley, James H

    2015-10-23

    The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat. Copyright © 2015, American Association for the Advancement of Science.

  18. [Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

    Science.gov (United States)

    Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

    2002-01-01

    Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P ras4B cell growth (P ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

  19. Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells

    Directory of Open Access Journals (Sweden)

    Jesse E. Jun

    2013-09-01

    Full Text Available The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and SOS-family GEFs.Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood.One large group of biomolecules critically involved in the control of Ras-GEFs´functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells.

  20. Immunodetection of rasP21 and c-myc oncogenes in oral mucosal swab preparation from clove cigarette smokers

    Directory of Open Access Journals (Sweden)

    Silvi Kintawati

    2008-12-01

    Full Text Available Background: Smoking is the biggest factor for oral cavity malignancy. Some carcinogens found in cigar will stimulate epithel cell in oral cavity and cause mechanism disturbance on tissue resistance and produce abnormal genes (oncogenes. Oncogenes ras and myc are found on malignant tumor in oral cavity which are associated with smoking. Purpose: This research is to find the expression of oncogenes rasP21 and c-myc in oral mucosa epithelial of smoker with immunocytochemistry reaction. Methods: An oral mucosal swab was performed to 30 smokers categorized as light, moderate, and chain, and 10 non smokers which was followed by immunocytochemistry reaction using antibody towards oncogene rasP21 and c-myc is reacted to identify the influence of smoking towards malignant tumor in oral cavity. The result is statistically analyzed using Kruskal-Wallis test. Result: Based on the observation result of oncogene rasP21reaction, it shows that there is significant difference between non smoker group and light smoker, compared to moderate and chain smoker group (p < 0.01. On the other side, the observation result of oncogene c-myc indicates that there is no significant difference between the group of non smokers and the group of light, moderate, and chain smokers (p > 0.05. Conclusion: The higher the possibility of oral cavity malignancy and that the antibody for rasP21 oncogene can be used as a marker for early detection of oral cavity malignancy caused by smoking.

  1. Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells

    International Nuclear Information System (INIS)

    Wolff, Natascha A.; Lee, Wing-Kee; Abouhamed, Marouan; Thevenod, Frank

    2008-01-01

    Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membrane (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 μM for 24 h) toxicity was significantly attenuated from 27.3 ± 3.9% in ARF6-WT to 11.1 ± 4.0% in ARF6-DN cells (n = 6, P < 0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0 ± 4.6% versus 3.9 ± 3.9% of ARF6-WT cells, n = 3, P < 0.01) and/or remained near the PM (89.3 ± 5. 6% versus 45.2 ± 14.3% of ARF6-WT cells, n = 3, P < 0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells

  2. Role of pp60(c-src) and p(44/42) MAPK in ANG II-induced contraction of rat tonic gastrointestinal smooth muscles.

    Science.gov (United States)

    Puri, Rajinder N; Fan, Ya-Ping; Rattan, Satish

    2002-08-01

    We examined the role of mitogen-activated protein kinase (p(44/42) MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a pp60(c-src) inhibitor), and antibodies to pp60(c-src) and p(120) ras GTPase-activating protein (p(120) rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of p(120) rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of p(44/42) MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that pp60(c-src) and p(44/42) MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles.

  3. Involvement of H- and N-Ras isoforms in transforming growth factor-β1-induced proliferation and in collagen and fibronectin synthesis

    International Nuclear Information System (INIS)

    Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Garcia-Cenador, Begona; Santos, Eugenio; Lopez-Novoa, Jose M.

    2006-01-01

    Transforming growth factor β1 (TGF-β1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-β and Ras signaling pathways are closely related: TGF-β1 overcomes Ras mitogenic effects and Ras counteracts TGF-β signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-β1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras -/- /N-ras -/- ) isoforms and from heterozygote mice (H-ras +/- /N-ras +/- ). ECM synthesis is increased in basal conditions in H-ras -/- /N-ras -/- fibroblasts, this increase being higher after stimulation with TGF-β1. TGF-β1-induced fibroblast proliferation is smaller in H-ras -/- /N-ras -/- than in H-ras +/- /N-ras +/- fibroblasts. Erk activation is decreased in H-ras -/- /N-ras -/- fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras

  4. Activated H-Ras regulates hematopoietic cell survival by modulating Survivin

    International Nuclear Information System (INIS)

    Fukuda, Seiji; Pelus, Louis M.

    2004-01-01

    Survivin expression and Ras activation are regulated by hematopoietic growth factors. We investigated whether activated Ras could circumvent growth factor-regulated Survivin expression and if a Ras/Survivin axis mediates growth factor independent survival and proliferation in hematopoietic cells. Survivin expression is up-regulated by IL-3 in Ba/F3 and CD34 + cells and inhibited by the Ras inhibitor, farnesylthiosalicylic acid. Over-expression of constitutively activated H-Ras (CA-Ras) in Ba/F3 cells blocked down-modulation of Survivin expression, G 0 /G 1 arrest, and apoptosis induced by IL-3 withdrawal, while dominant-negative (DN) H-Ras down-regulated Survivin. Survivin disruption by DN T34A Survivin blocked CA-Ras-induced IL-3-independent cell survival and proliferation; however, it did not affect CA-Ras-mediated enhancement of S-phase, indicating that the anti-apoptotic activity of CA-Ras is Survivin dependent while its S-phase enhancing effect is not. These results indicate that CA-Ras modulates Survivin expression independent of hematopoietic growth factors and that a CA-Ras/Survivin axis regulates survival and proliferation of transformed hematopoietic cells

  5. A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors.

    Science.gov (United States)

    Loboda, Andrey; Nebozhyn, Michael; Klinghoffer, Rich; Frazier, Jason; Chastain, Michael; Arthur, William; Roberts, Brian; Zhang, Theresa; Chenard, Melissa; Haines, Brian; Andersen, Jannik; Nagashima, Kumiko; Paweletz, Cloud; Lynch, Bethany; Feldman, Igor; Dai, Hongyue; Huang, Pearl; Watters, James

    2010-06-30

    Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.

  6. A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors

    Directory of Open Access Journals (Sweden)

    Paweletz Cloud

    2010-06-01

    Full Text Available Abstract Background Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. Methods We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. Results The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90% sensitivity but relatively low (50% specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. Conclusions These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical

  7. Hydrophilicity and morphological investigation of polycarbonate irradiated by ArF excimer laser

    Energy Technology Data Exchange (ETDEWEB)

    Jaleh, B. [Bu-Ali-Sina University, Physics Department, Postal Code 65174, Hamedan (Iran, Islamic Republic of)], E-mail: jaleh@basu.ac.ir; Parvin, P. [Amir Kabir University of Technology, Physics Department, P.O. Box: 15875-4413, Tehran (Iran, Islamic Republic of); Laser Research Center, AEOI, P.O. Box: 11365-8486, Tehran (Iran, Islamic Republic of); Sheikh, N. [Nuclear Science and Technology Research Institute, Radiation Applications Research School, Tehran (Iran, Islamic Republic of); Zamanipour, Z. [Laser Research Center, AEOI, P.O. Box: 11365-8486, Tehran (Iran, Islamic Republic of); Sajad, B. [Azzahra University, Physics Department, Tehran (Iran, Islamic Republic of)

    2007-12-15

    Lasers are used to modify polymeric materials. In this work, a number of polycarbonate (PC) pieces were exposed by ArF excimer laser, 193 nm, at various UV doses from 10 to 100 J/cm{sup 2} with 50-500 mJ/pulse at 10 Hz pulse repetition rate. Morphology of PC has been investigated by scanning electron microscope (SEM) at three regimes pre-ablation, slow and fast ablation. SEM identifies that the conical defects are created on the polymer surface to grow opposite to the direction of laser irradiation. It increases the superficial absorptivity of the material dependent on the ArF laser induced conical microstructure geometry. The contact angle measurement was performed here, in order to determine the hydrophilicity of the irradiated polymer at various coherent doses. It is shown that the contact angle of PC samples which are exposed to the ArF laser significantly alters with UV dose below 7 J/cm{sup 2}.

  8. The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH

    DEFF Research Database (Denmark)

    Willumsen, B M; Vass, W C; Velu, T J

    1991-01-01

    We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass...... in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype......., and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming...

  9. ArF laser-based quantum well intermixing in InGaAs/InGaAsP heterostructures

    International Nuclear Information System (INIS)

    Genest, Jonathan; Beal, Romain; Aimez, Vincent; Dubowski, Jan J.

    2008-01-01

    Radiation from a 193 nm ArF laser was investigated to modify surface properties of InGaAs/InGaAsP quantum well (QW) heterostructures and introduce defects required to enhance intermixing during the annealing process. A top 200 nm thick sacrificial layer of InP served as a reservoir for laser generated defects. The irradiation with up to 90 pulses at 65-150 mJ/cm 2 allowed to generate an array of 1.2x1 mm 2 sites of QW intermixed material, with bandgap energy blueshifted up to 107 nm. We discuss the mechanism and advantages of this approach for postgrowth wafer level fabrication of multibandgap QW material

  10. Ras Activity Oscillates in the Mouse Suprachiasmatic Nucleus and Modulates Circadian Clock Dynamics.

    Science.gov (United States)

    Serchov, Tsvetan; Jilg, Antje; Wolf, Christian T; Radtke, Ina; Stehle, Jörg H; Heumann, Rolf

    2016-04-01

    Circadian rhythms, generated in the mouse suprachiasmatic nucleus (SCN), are synchronized to the environmental day-night changes by photic input. The activation of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) and cAMP response element-binding protein (CREB)-mediated transcription play a critical role in this photoentrainment. The small GTPase Ras is one of the major upstream regulators of the ERK1,2/CREB pathway. In contrast to the well-described role of Ras in structural and functional synaptic plasticity in the adult mouse brain, the physiological regulation of Ras by photic sensory input is yet unknown. Here, we describe for the first time a circadian rhythm of Ras activity in the mouse SCN. Using synRas transgenic mice, expressing constitutively activated V12-Ha-Ras selectively in neurons, we demonstrate that enhanced Ras activation causes shortening of the circadian period length. We found upregulated expression and decreased inhibitory phosphorylation of the circadian period length modulator, glycogen synthase kinase-3 beta (GSK3β), in the SCN of synRas mice. Conversely, downregulation of Ras activity by blocking its function with an antibody in oscillating cell cultures reduced protein levels and increased phosphorylation of GSK3β and lengthened the period of BMAL1 promoter-driven luciferase activity. Furthermore, enhanced Ras activity in synRas mice resulted in a potentiation of light-induced phase delays at early subjective night, and increased photic induction of pERK1,2/pCREB and c-Fos. In contrast, at late subjective night, photic activation of Ras/ERK1,2/CREB in synRas mice was not sufficient to stimulate c-Fos protein expression and phase advance the clock. Taken together, our results demonstrate that Ras activity fine tunes the period length and modulates photoentrainment of the circadian clock.

  11. Upstream CREs participate in the basal activity of minute virus of mice promoter P4 and in its stimulation in ras-transformed cells.

    Science.gov (United States)

    Perros, M; Deleu, L; Vanacker, J M; Kherrouche, Z; Spruyt, N; Faisst, S; Rommelaere, J

    1995-01-01

    The activity of the P4 promoter of the parvovirus minute virus of mice (prototype strain MVMp) is stimulated in ras-transformed FREJ4 cells compared with the parental FR3T3 line. This activation may participate in the oncolytic effect of parvoviruses, given that P4 drives a transcriptional unit encoding cytotoxic nonstructural proteins. Our results suggest that the higher transcriptional activity of promoter P4 in FREJ4 cells is mediated at least in part by upstream CRE elements. Accordingly, mutations in the CRE motifs impair P4 function more strongly in the FREJ4 derivative than in its FR3T3 parent. Further evidence that these elements contribute to hyperactivity of the P4 promoter in the ras transformant is the fact that they form distinct complexes with proteins from FREJ4 and FR3T3 cell extracts. This difference can be abolished by treating the FREJ4 cell extracts with cyclic AMP-dependent protein kinase (PKA) or treating original cultures with a PKA activator. These findings can be linked with two previously reported features of ras-transformed cells: the activation of a PKA-inhibited protein kinase cascade and the reduction of PKA-induced protein phosphorylation. In keeping with these facts, P4-directed gene expression can be up- or downmodulated in vivo by exposing cells to known inhibitors or activators of PKA, respectively. PMID:7636996

  12. Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

    Science.gov (United States)

    Wu, Yue; Zhuang, Yuan; Han, Min; Xu, Tian; Deng, Kejing

    2009-10-20

    Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

  13. K-ras2 Activation and Genome Instability Increase Proliferation and Size of FAP Adenomas

    Directory of Open Access Journals (Sweden)

    Anna Rapallo

    1999-01-01

    Full Text Available The possible role of K‐ras2 mutations and aneuploidy toward increase of proliferation and adenoma size in Familial Adenomatous Polyposis (FAP adenomas is not known. The present study addresses these issues by investigating 147 colorectal adenomas obtained from four FAP patients. The majority of adenomas had size lower than or equal to 10 mm (86%, low grade dysplasia (63%, and were preferentially located in the right colon (60%. Normal mucosa samples were obtained from 19 healthy donors. Three synchronous adenocarcinomas were also investigated. K‐ras2 mutation spectrum was analysed by PCR and Sequence Specific Oligonucleotide (SSO hybridization, while flow cytometry (FCM was used for evaluating degree of DNA ploidy and S‐phase fraction. Overall, incidences of K‐ras2 mutations, DNA aneuploidy and high S‐phase values (>7.2% were 6.6%, 5.4% and 10.5%, respectively. In particular, among the adenomas with size lower than 5 mm, K‐ras2 mutation and DNA aneuploidy frequencies were only slightly above 1%. Statistically significant correlations were found between K‐ras2 and size, DNA ploidy and size and K‐ras2 and S‐phase (p. In particular, among the wild type K‐ras2 adenomas, high S‐phase values were detected in 8% of the cases versus 57% among the K‐ras2 mutated adenomas (p=0.0005. The present series of FAP adenomas indicates that K‐ras2 activation and gross genomic changes play a role toward a proliferative gain and tumour growth in size.

  14. Limited role of murine ATM in oncogene-induced senescence and p53-dependent tumor suppression.

    Directory of Open Access Journals (Sweden)

    Alejo Efeyan

    Full Text Available Recent studies in human fibroblasts have provided a new general paradigm of tumor suppression according to which oncogenic signaling produces DNA damage and this, in turn, results in ATM/p53-dependent cellular senescence. Here, we have tested this model in a variety of murine experimental systems. Overexpression of oncogenic Ras in murine fibroblasts efficiently induced senescence but this occurred in the absence of detectable DNA damage signaling, thus suggesting a fundamental difference between human and murine cells. Moreover, lung adenomas initiated by endogenous levels of oncogenic K-Ras presented abundant senescent cells, but undetectable DNA damage signaling. Accordingly, K-Ras-driven adenomas were also senescent in Atm-null mice, and the tumorigenic progression of these lesions was only modestly accelerated by Atm-deficiency. Finally, we have examined chemically-induced fibrosarcomas, which possess a persistently activated DNA damage response and are highly sensitive to the activity of p53. We found that the absence of Atm favored genomic instability in the resulting tumors, but did not affect the persistent DNA damage response and did not impair p53-dependent tumor suppression. All together, we conclude that oncogene-induced senescence in mice may occur in the absence of a detectable DNA damage response. Regarding murine Atm, our data suggest that it plays a minor role in oncogene-induced senescence or in p53-dependent tumor suppression, being its tumor suppressive activity probably limited to the maintenance of genomic stability.

  15. Lack of HXK2 Induces Localization of Active Ras in Mitochondria and Triggers Apoptosis in the Yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Loredana Amigoni

    2013-01-01

    Full Text Available We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in the hxk2Δ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis in S. cerevisiae. In particular, cells lacking HXK2 and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion of HXK2 causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast.

  16. Lack of HXK2 induces localization of active Ras in mitochondria and triggers apoptosis in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Amigoni, Loredana; Martegani, Enzo; Colombo, Sonia

    2013-01-01

    We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in the hxk2Δ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis in S. cerevisiae. In particular, cells lacking HXK2 and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion of HXK2 causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast.

  17. Role of Arf GTPases in fungal morphogenesis and virulence.

    Directory of Open Access Journals (Sweden)

    Hayet Labbaoui

    2017-02-01

    Full Text Available Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth, which requires sustained membrane traffic and polarized growth. In many organisms, small GTPases of the Arf (ADP-ribosylation factor family regulate membrane/protein trafficking, yet little is known about their role in fungal filamentous growth. To investigate these GTPases in C. albicans, we generated loss of function mutants in all 3 Arf proteins, Arf1-Arf3, and 2 Arf-like proteins, Arl1 and Arl3. Our results indicate that of these proteins, Arf2 is required for viability and sensitivity to antifungal drugs. Repressible ARF2 expression results in defects in filamentous growth, cell wall integrity and virulence, likely due to alteration of the Golgi. Arl1 is also required for invasive filamentous growth and, although arl1/arl1 cells can initiate hyphal growth, hyphae are substantially shorter than that of the wild-type, due to the inability of this mutant to maintain hyphal growth at a single site. We show that this defect does not result from an alteration of phospholipid distribution and is unlikely to result from the sole Golgin Imh1 mislocalization, as Imh1 is not required for invasive filamentous growth. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion in this mutant. Strikingly, the arl1/arl1 mutant is drastically reduced in virulence during oropharyngeal candidiasis. Together, our results highlight the importance of Arl1 and Arf2 as key regulators of hyphal growth and virulence in C. albicans and identify a unique function of Arl1 in secretion.

  18. Point mutation in activated c-Ha-ras gene of a chemically induced transplantable human pancreas carcinoma

    International Nuclear Information System (INIS)

    Maheshwari, K.K.; Parsa, I.

    1986-01-01

    The authors have reported a model of human pancreas carcinogenesis where repeated treatment with MNU of explants results in the development of transplantable carcinoma. This report compares the endonuclease digests of DNAs from normal human pancreas (HP) and MNU-induced transplantable tumor (HP-T1) analyzed with 32 P-labelled Ha-ras probe prepared from clone BS-9. The hybridization patterns of BamHI, BglII, EcoRI and HindIII digests of HP were significantly different from those of HP-T1. In EcoRI digests a 3.0 kb fragments of HP-T1 DNA hybridized with Ha-ras probe instead of a 4.3 kb fragments seen in HP DNA. The pattern for HindIII digests was similar to those of EcoRI. The BgIII digests of HP DNA revealed two hybridizing fragments of 8.0 and 4.3 kb whereas those of HP-T1 DNA fragments measured 8.5 and 4.0 kb. BamHI treated HP DNA showed only hybridizing fragments of 6.6 kb while the HP-T1 DNA showed to hybridizing fragments of 6.8 and 7.2 kb. The digested DNAs by HhaI, HinfI, KpnI, pstI, PvuII, SaII, SstI, TaqI and XbaI showed similar hybridization profiles. The point mutation in c-Ha-ras was examined in the HpaII and MspI double digests of both DNAs by 0.6 Kb SmaI fragments of pEJ. The hybridized fragments measured 412 and 355 bp in DNA digests from tumor and normal pancreas respectively

  19. Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion

    Science.gov (United States)

    Rennoll-Bankert, Kristen E.; Rahman, M. Sayeedur; Gillespie, Joseph J.; Guillotte, Mark L.; Kaur, Simran J.; Lehman, Stephanie S.; Beier-Sexton, Magda; Azad, Abdu F.

    2015-01-01

    Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legionella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole. In contrast, previous in vitro studies showed R. prowazekii (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma membrane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across Rickettsia species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of R. typhi (Typhus Group) interacts with the Rickettsia T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion. For R. typhi and R. felis (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remarkably, R. bellii (Ancestral Group) RalF showed perinuclear localization reminiscent of ectopically expressed Legionella RalF, for which it shares several structural features. For R. typhi, RalF co-localization with Arf6 and PI(4,5)P2 at entry foci on the host plasma membrane was determined to be critical for invasion. Thus, we propose recruitment of PI(4,5)P2 at entry foci, mediated by RalF activation of Arf6, initiates actin remodeling and ultimately facilitates bacterial invasion. Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for Ral

  20. RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

    Science.gov (United States)

    Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

    2007-01-01

    The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

  1. p55-hGRF, a short natural form of the Ras-GDP exchange factor high yield production and characterization.

    Science.gov (United States)

    Meyer, P; Janin, J; Baudet-Nessler, S

    1999-08-01

    p55-hGRF, a natural short form of the guanine-nucleotide-releasing factor for p21-Ras from human brain, was expressed at high level in Escherichia coli as well as an engineered truncated form, p39-hGRF. A T7 polymerase expression system was used, resulting in the formation of insoluble cytoplasmic protein aggregates. The recombinant products were resolubilized, renatured and purified to homogeneity. The exchange activity of the refolded hGRF samples on H-Ras was comparable with that published for the soluble catalytic domain of the mouse counterpart, CDC25 Mm. Both p55-hGRF and p39-hGRF form dimers. We established a procedure to prepare and purify the complex with Ras. The results of the characterization study are consistent with a stoichiometry of 1:1 and an equilibrium between dimeric and monomeric forms of the complex.

  2. The role of autophagy in cytotoxicity induced by new oncogenic B-Raf inhibitor UI-152 in v-Ha-ras transformed fibroblasts

    International Nuclear Information System (INIS)

    Ahn, Jun-Ho; Ahn, Soon Kil; Lee, Michael

    2012-01-01

    Highlights: ► We recently discovered a potent and selective B-Raf inhibitor, UI-152. ► UI-152 displayed a selective cytotoxicity toward v-Ha-ras transformed cells. ► UI-152-induced growth inhibition was largely meditated by autophagy. ► UI-152 induced paradoxical activation of Raf-1. -- Abstract: In human cancers, B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade, making it an important therapeutic target. We recently discovered a potent and selective B-Raf inhibitor, UI-152, by using a structure-based drug design strategy. In this study, we examined whether B-Raf inhibition by UI-152 may be an effective therapeutic strategy for eliminating cancer cells transformed with v-Ha-ras (Ras-NIH 3T3). UI-152 displayed selective cytotoxicity toward Ras-NIH 3T3 cells while having little to no effect on non-transformed NIH 3T3 cells. We found that treatment with UI-152 markedly increased autophagy and, to a lesser extent, apoptosis. However, inhibition of autophagy by addition of 3-MA failed to reverse the cytotoxic effects of UI-152 on Ras-NIH 3T3 cells, demonstrating that apoptosis and autophagy can act as cooperative partners to induce growth inhibition in Ras-NIH 3T3 cells treated with UI-152. Most interestingly, cell responses to UI-152 appear to be paradoxical. Here, we showed that although UI-152 inhibited ERK, it induced B-Raf binding to Raf-1 as well as Raf-1 activation. This paradoxical activation of Raf-1 by UI-152 is likely to be coupled with the inhibition of the mTOR pathway, an intracellular signaling pathway involved in autophagy. We also showed for the first time that, in multi-drug resistant cells, the combination of UI-152 with verapamil significantly decreased cell proliferation and increased autophagy. Thus, our findings suggest that the inhibition of autophagy, in combination with UI-152, offers a more effective therapeutic strategy for v-Ha-ras-transformed cells harboring wild-type B-Raf.

  3. The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells

    DEFF Research Database (Denmark)

    Bracken, Adrian P; Kleine-Kohlbrecher, Daniela; Dietrich, Nikolaj

    2007-01-01

    The p16INK4A and p14ARF proteins, encoded by the INK4A-ARF locus, are key regulators of cellular senescence, yet the mechanisms triggering their up-regulation are not well understood. Here, we show that the ability of the oncogene BMI1 to repress the INK4A-ARF locus requires its direct association...... and is dependent on the continued presence of the EZH2-containing Polycomb-Repressive Complex 2 (PRC2) complex. Significantly, EZH2 is down-regulated in stressed and senescing populations of cells, coinciding with decreased levels of associated H3K27me3, displacement of BMI1, and activation of transcription...

  4. Identification, isolation and expression analysis of auxin response factor (ARF) genes in Solanum lycopersicum.

    Science.gov (United States)

    Wu, Jian; Wang, Feiyan; Cheng, Lin; Kong, Fuling; Peng, Zhen; Liu, Songyu; Yu, Xiaolin; Lu, Gang

    2011-11-01

    Auxin response factors (ARFs) encode transcriptional factors that bind specifically to the TGTCTC-containing auxin response elements found in the promoters of primary/early auxin response genes that regulate plant development. In this study, investigation of the tomato genome revealed 21 putative functional ARF genes (SlARFs), a number comparable to that found in Arabidopsis (23) and rice (25). The full cDNA sequences of 15 novel SlARFs were isolated and delineated by sequencing of PCR products. A comprehensive genome-wide analysis of this gene family is presented, including the gene structures, chromosome locations, phylogeny, and conserved motifs. In addition, a comparative analysis between ARF family genes in tomato and maize was performed. A phylogenetic tree generated from alignments of the full-length protein sequences of 21 OsARFs, 23 AtARFs, 31 ZmARFs, and 21 SlARFs revealed that these ARFs were clustered into four major groups. However, we could not find homologous genes in rice, maize, or tomato with AtARF12-15 and AtARF20-23. The expression patterns of tomato ARF genes were analyzed by quantitative real-time PCR. Our comparative analysis will help to define possible functions for many of these newly isolated ARF-family genes in plant development.

  5. A population-based study on the association between acute renal failure (ARF and the duration of polypharmacy

    Directory of Open Access Journals (Sweden)

    Chang Yi-Ping

    2012-08-01

    Full Text Available Abstract Background Because of the rapid growth in elderly population, polypharmacy has become a serious public health issue worldwide. Although acute renal failure (ARF is one negative consequence of polypharmacy, the association between the duration of polypharmacy and ARF remains unclear. We therefore assessed this association using a population-based database. Methods Data were collected from the Taiwan National Health Insurance Research Database (NHIRD from 2003 through 2006. The case group included patients hospitalized for ARF during 2006, but not admitted due to trauma, surgery, burn trauma, car accident, transplantation, or infectious diseases; the control group included patients hospitalized without ARF. The cumulative number of days of polypharmacy (defined as more than 5 prescriptions per day for 1 year prior to admission was determined, with patients further subdivided into 4 categories: less than 30 days, 31–90 days, 91–180 days, and over 181 days. The dependent variable was ARF, and the control variables were age, gender, comorbidities in patients hospitalized for ARF, stay in ICUs during ARF hospitalization and site of operation for prior admissions within one month of ARF hospitalization. Results Of 20,790 patients who were admitted to hospitals for ARF in 2006, 12,314 (59.23 % were male and more than 60 % were older than 65 years. Of patients with and without ARF, 16.14 % and 10.61 %, respectively, received polypharmacy for 91–180 days and 50.22 % and 24.12 %, respectively, for over 181 days. A statistical model indicated that, relative to patients who received polypharmacy for less than 30 days, those who received polypharmacy for 31–90, 91–180 and over 181 days had odds ratios of developing ARF of 1.33 (p Conclusions We observed statistically significant associations between the duration of polypharmacy and the occurrence of ARF.

  6. The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription

    DEFF Research Database (Denmark)

    Nielsen, O; Davey, William John; Egel, R

    1992-01-01

    Loss of ras1+ function renders fission yeast cells unable to undergo morphological changes in response to mating pheromones, whereas cells carrying activated mutations in ras1 are hyper-responsive. This has led to the suggestion that the ras1 gene product plays a role in mating pheromone signal...

  7. Oncogenic RAS enables DNA damage- and p53-dependent differentiation of acute myeloid leukemia cells in response to chemotherapy.

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    Mona Meyer

    Full Text Available Acute myeloid leukemia (AML is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells.

  8. Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice.

    Science.gov (United States)

    Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

    2014-05-22

    Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.

  9. Decisive factors affecting plasma resistance and roughness formation in ArF photoresist

    Energy Technology Data Exchange (ETDEWEB)

    Jinnai, Butsurin; Uesugi, Takuji; Koyama, Koji; Samukawa, Seiji [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Kato, Keisuke; Yasuda, Atsushi; Maeda, Shinichi [Yokohama Research Laboratories, Mitsubishi Rayon Co., Ltd, 10-1 Daikoku-cho, Tsurumi-ku, Yokohama 230-0053 (Japan); Momose, Hikaru, E-mail: samukawa@ifs.tohoku.ac.j [Corporate Research Laboratories, Mitsubishi Rayon Co., Ltd, 2-1 Miyuki-cho, Otake, Hiroshima 739-0693 (Japan)

    2010-10-06

    Low plasma resistance and roughness formation in an ArF photoresist are serious issues in plasma processes. To resolve these issues, we investigated several factors that affect the roughness formation and plasma resistance in an ArF photoresist. We used our neutral beam process to categorize the effects of species from the plasma on the ArF photoresist into physical bombardment, chemical reactions and ultraviolet/vacuum ultraviolet (UV/VUV) radiation. The UV/VUV radiation drastically increased the etching rates of the ArF photoresist films, and, in contrast, chemical reactions increased the formation of surface roughness. FTIR analysis indicated that the UV/VUV radiation preferentially dissociates C-H bonds in the ArF photoresist, rather than C=O bonds, because of the dissociation energies of the bonds. This indicated that the etching rates of the ArF photoresist are determined by the UV/VUV radiation because this radiation can break C-H bonds, which account for the majority of structures in the ArF photoresist. In contrast, FTIR analysis showed that chemical species such as radicals and ions were likely to react with C=O bonds, in particular C=O bonds in the lactone groups of the ArF photoresist, due to the structural and electronic effects of the lactone groups. As a result, the etching rates of the ArF photoresist can vary in different bond structures, leading to increased surface roughness in the ArF photoresist.

  10. The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

    Science.gov (United States)

    Vanli, Güliz; Peltzer, Nieves; Dubuis, Gilles; Widmann, Christian

    2014-12-01

    The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.

    Directory of Open Access Journals (Sweden)

    Clara L Oeste

    2011-01-01

    Full Text Available Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184 in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14-PGJ(2 (15d-PGJ(2 and Δ(12-PGJ(2 selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO, a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2. Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

  12. Genetic alterations in Ki-ras and Ha-ras genes in Juvenile Nasopharyngeal Angiofibromas and head and neck cancer

    Directory of Open Access Journals (Sweden)

    Cláudia Malheiros Coutinho

    1999-05-01

    Full Text Available CONETXT: Ras gene mutations have been associated to a wide range of human solid tumors. Members of the ras gene family (Ki-ras, Ha-ras and N-ras are structurally related and code for a protein (p21 known to play an important role in the regulation of normal signal transduction and cell growth. The frequency of ras mutations is different from one type of tumor to another, suggesting that point mutations might be carcinogen-specific. OBJECTIVES: To study the occurrence of Ki-ras and Ha-ras mutations. We also studied the relative level of Ha-ras mRNA in 32 of the head and neck tumors. DESIGN: Case series. SETTING: University referral unit. PARTICIPANTS: 60 head and neck tumors and in 28 Juvenile Nasopharyngeal Angiofibromas (JNA. DIAGNOSTIC TEST: Using PCR-SSCP we examined the occurrence of Ki-ras and Ha-ras mutations. The relative level of Ha-ras mRNA was examined by Northern blot analysis. RESULTS: None of the head and neck tumors or JNA samples showed evidence of mutations within codons 12, 13, 59 and 61 of Ki-ras or Ha-ras genes. However, 17 (53% of the tumors where gene expression could be examined exhibited increased levels of Ha-ras mRNA compared with the normal tissue derived from the same patient. CONCLUSIONS: Our results demonstrate for the first time that mutations of Ki-ras and Ha-ras genes are not associated with the development of JNA and confirm previous reports indicating that activating ras mutations are absent or rarely involved in head and neck tumors from western world patients. Furthermore, our findings suggest that overexpression of Ha-ras, rather than mutations, might be an important factor in the development and progression of head and neck tumors.

  13. Transcriptional Profile of Ki-Ras-Induced Transformation of Thyroid Cells

    DEFF Research Database (Denmark)

    Visconti, Roberta; Federico, Antonella; Coppola, Valeria

    2007-01-01

    Abstract In the last years, an increasing number of experiments has provided compelling evidence for a casual role of Ras protein mutations, resulting in their constitutive activation, in thyroid carcinogenesis. However, despite the clear involvement of Ras proteins in thyroid carcinogenesis, the...

  14. Molecular Analysis of a Multistep Lung Cancer Model Induced by Chronic Inflammation Reveals Epigenetic Regulation of p16, Activation of the DNA Damage Response Pathway

    Directory of Open Access Journals (Sweden)

    David Blanco

    2007-10-01

    Full Text Available The molecular hallmarks of inflammation-mediated lung carcinogenesis have not been fully clarified, mainly due to the scarcity of appropriate animal models. We have used a silica-induced multistep lung carcinogenesis model driven by chronic inflammation to study the evolution of molecular markers, genetic alterations. We analyzed markers of DNA damage response (DDR, proliferative stress, telomeric stress: δ-H2AX, p16, p53, TERT. Lung cancer-related epigenetic, genetic alterations, including promoter hypermethylation status of p16(CDKN2A, APC, CDH13, Rassf1, Nore1A, as well as mutations of Tp53, epidermal growth factor receptor, K-ras, N-ras, c-H-ras, have been also studied. Our results showed DDR pathway activation in preneoplastic lesions, in association with inducible nitric oxide synthase, p53 induction. p16 was also induced in early tumorigenic progression, was inactivated in bronchiolar dysplasias, tumors. Remarkably, lack of mutations of Ras, epidermal growth factor receptor, a very low frequency of Tp53 mutations suggest that they are not required for tumorigenesis in this model. In contrast, epigenetic alterations in p16(CDKN2A, CDH13, APC, but not in Rassf1, Nore1A, were clearly observed. These data suggest the existence of a specific molecular signature of inflammation-driven lung carcinogenesis that shares some, but not all, of the molecular landmarks of chemically induced lung cancer.

  15. ArF Laser -Induced Chemical Vapour Deposition of Polythiene Films from Carbon Disulfide

    Czech Academy of Sciences Publication Activity Database

    Tomovska, R.; Bastl, Zdeněk; Vorlíček, Vladimír; Vacek, Karel; Šubrt, Jan; Plzák, Zbyněk; Pola, Josef

    2003-01-01

    Roč. 107, č. 36 (2003), s. 9793-9801 ISSN 1089-5647 R&D Projects: GA MŠk ME 612 Institutional research plan: CEZ:AV0Z4032918; CEZ:AV0Z4040901 Keywords : laser photolysis * ArF * chemical vapour deposition Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.679, year: 2003

  16. Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12 -induced cell transformation.

    Science.gov (United States)

    Lin, Hsiu-Kuan; Lin, Hsi-Hui; Chiou, Yu-Wei; Wu, Ching-Lung; Chiu, Wen-Tai; Tang, Ming-Jer

    2018-05-01

    Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-Ras V12 gene) transformation by Ha-Ras V12 . Cav1 overexpression abrogates the Ha-Ras V12 -driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-Ras V12 -inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-Ras V12 , was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-Ras V12 - and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-Ras V12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-Ras V12 -Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-Ras V12 -driven cell transformation. © 2018 The Authors

  17. SCD1 Expression is dispensable for hepatocarcinogenesis induced by AKT and Ras oncogenes in mice.

    Directory of Open Access Journals (Sweden)

    Lei Li

    Full Text Available Increased de novo lipogenesis is one of the major metabolic events in cancer. In human hepatocellular carcinoma (HCC, de novo lipogenesis has been found to be increased and associated with the activation of AKT/mTOR signaling. In mice, overexpression of an activated form of AKT results in increased lipogenesis and hepatic steatosis, ultimately leading to liver tumor development. Hepatocarcinogenesis is dramatically accelerated when AKT is co-expressed with an oncogenic form of N-Ras. SCD1, the major isoform of stearoyl-CoA desaturases, catalyzing the conversion of saturated fatty acids (SFA into monounsaturated fatty acids (MUFA, is a key enzyme involved in de novo lipogenesis. While many studies demonstrated the requirement of SCD1 for tumor cell growth in vitro, whether SCD1 is necessary for tumor development in vivo has not been previously investigated. Here, we show that genetic ablation of SCD1 neither inhibits lipogenesis and hepatic steatosis in AKT-overexpressing mice nor affects liver tumor development in mice co-expressing AKT and Ras oncogenes. Molecular analysis showed that SCD2 was strongly upregulated in liver tumors from AKT/Ras injected SCD1(-/- mice. Noticeably, concomitant silencing of SCD1 and SCD2 genes was highly detrimental for the growth of AKT/Ras cells in vitro. Altogether, our study provides the evidence, for the first time, that SCD1 expression is dispensable for AKT/mTOR-dependent hepatic steatosis and AKT/Ras-induced hepatocarcinogenesis in mice. Complete inhibition of stearoyl-CoA desaturase activity may be required to efficiently suppress liver tumor development.

  18. Arf4 is required for Mammalian development but dispensable for ciliary assembly.

    Directory of Open Access Journals (Sweden)

    John A Follit

    2014-02-01

    Full Text Available The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.

  19. DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells.

    Science.gov (United States)

    Watanabe-Takano, Haruko; Takano, Kazunori; Hatano, Masahiko; Tokuhisa, Takeshi; Endo, Takeshi

    2015-01-01

    Myofibroblasts play critical roles in the development of idiopathic pulmonary fibrosis by depositing components of extracellular matrix. One source of lung myofibroblasts is thought to be alveolar epithelial type 2 cells that undergo epithelial-mesenchymal transition (EMT). Rat RLE-6TN alveolar epithelial type 2 cells treated with transforming growth factor-β1 (TGF-β1) are converted into myofibroblasts through EMT. TGF-β induces both canonical Smad signaling and non-canonical signaling, including the Ras-induced ERK pathway (Raf-MEK-ERK). However, the signaling mechanisms regulating TGF-β1-induced EMT are not fully understood. Here, we show that the Ras-ERK pathway negatively regulates TGF-β1-induced EMT in RLE-6TN cells and that DA-Raf1 (DA-Raf), a splicing isoform of A-Raf and a dominant-negative antagonist of the Ras-ERK pathway, plays an essential role in EMT. Stimulation of the cells with fibroblast growth factor 2 (FGF2), which activated the ERK pathway, prominently suppressed TGF-β1-induced EMT. An inhibitor of MEK, but not an inhibitor of phosphatidylinositol 3-kinase, rescued the TGF-β1-treated cells from the suppression of EMT by FGF2. Overexpression of a constitutively active mutant of a component of the Ras-ERK pathway, i.e., H-Ras, B-Raf, or MEK1, interfered with EMT. Knockdown of DA-Raf expression with siRNAs facilitated the activity of MEK and ERK, which were only weakly and transiently activated by TGF-β1. Although DA-Raf knockdown abrogated TGF-β1-induced EMT, the abrogation of EMT was reversed by the addition of the MEK inhibitor. Furthermore, DA-Raf knockdown impaired the TGF-β1-induced nuclear translocation of Smad2, which mediates the transcription required for EMT. These results imply that intrinsic DA-Raf exerts essential functions for EMT by antagonizing the TGF-β1-induced Ras-ERK pathway in RLE-6TN cells.

  20. Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus

    DEFF Research Database (Denmark)

    Willumsen, B M; Norris, K; Papageorge, A G

    1984-01-01

    localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein...... not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue...

  1. Aberrations of the p53 pathway components p53, MDM2 and CDKN2A appear independent in diffuse large B cell lymphoma

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Ino, Y; Gerdes, A M

    1999-01-01

    The two gene products of the CDKN2A gene, p16 and p19ARF, have recently been linked to each of two major tumour suppressor pathways in human carcinogenesis, the RB1 pathway and the p53 pathway. p16 inhibits the phosphorylation of the retinoblastoma gene product by cyclin D-dependent kinases...

  2. Using a preclinical mouse model of high-grade astrocytoma to optimize p53 restoration therapy.

    Science.gov (United States)

    Shchors, Ksenya; Persson, Anders I; Rostker, Fanya; Tihan, Tarik; Lyubynska, Natalya; Li, Nan; Swigart, Lamorna Brown; Berger, Mitchel S; Hanahan, Douglas; Weiss, William A; Evan, Gerard I

    2013-04-16

    Based on clinical presentation, glioblastoma (GBM) is stratified into primary and secondary types. The protein 53 (p53) pathway is functionally incapacitated in most GBMs by distinctive type-specific mechanisms. To model human gliomagenesis, we used a GFAP-HRas(V12) mouse model crossed into the p53ER(TAM) background, such that either one or both copies of endogenous p53 is replaced by a conditional p53ER(TAM) allele. The p53ER(TAM) protein can be toggled reversibly in vivo between wild-type and inactive conformations by administration or withdrawal of 4-hydroxytamoxifen (4-OHT), respectively. Surprisingly, gliomas that develop in GFAP-HRas(V12);p53(+/KI) mice abrogate the p53 pathway by mutating p19(ARF)/MDM2 while retaining wild-type p53 allele. Consequently, such tumors are unaffected by restoration of their p53ER(TAM) allele. By contrast, gliomas arising in GFAP-HRas(V12);p53(KI/KI) mice develop in the absence of functional p53. Such tumors retain a functional p19(ARF)/MDM2-signaling pathway, and restoration of p53ER(TAM) allele triggers p53-tumor-suppressor activity. Congruently, growth inhibition upon normalization of mutant p53 by a small molecule, Prima-1, in human GBM cultures also requires p14(ARF)/MDM2 functionality. Notably, the antitumoral efficacy of p53 restoration in tumor-bearing GFAP-HRas(V12);p53(KI/KI) animals depends on the duration and frequency of p53 restoration. Thus, intermittent exposure to p53ER(TAM) activity mitigated the selective pressure to inactivate the p19(ARF)/MDM2/p53 pathway as a means of resistance, extending progression-free survival. Our results suggest that intermittent dosing regimes of drugs that restore wild-type tumor-suppressor function onto mutant, inactive p53 proteins will prove to be more efficacious than traditional chronic dosing by similarly reducing adaptive resistance.

  3. Multiple fractions of gamma rays do not induce overexpression of c-myc or c-Ki-ras oncogenes in human cervical carcinoma cells

    International Nuclear Information System (INIS)

    Osmak, M.; Soric, J.; Matulic, M.

    1993-01-01

    Multiple fractions of gamma rays (0.5 Gy daily, 30 fractions) had previously been found to change the sensitivity of human cervical carcinoma HeLa cells to anticancer drugs. Preirradiated cells became resistant to cisplatin, methotrexate and vincristine but retained the same sensitivity to gamma rays and ultraviolet light. Some mechanisms involved in the resistance of preirradiated cells to cisplatin and vincristine were determined, i.e. the increased levels of metallothioneins and increased expression of plasma membrane P glycoprotein. As recent reports indicated that the resistance to cisplatin and ionizing radiation may involve the expression of oncogenes, the problem was studied whether multiple fractions of gamma rays can change the expression of c-myc and c-Ki-ras oncogenes in HeLa cells and whether there is a correlation between the expression of these oncogenes and the sensitivity of preirradiated cells to cisplatin and gamma rays. The expression of c-myc and c-Ki-ras oncogenes was examined using the DNA dot blot, the RNA dot blot and Northern blot analysis. The results show that preirradiation induced neither amplification nor elevated expression of c-myc and c-Ki-ras oncogenes. Furthermore, there is no correlation between the expression of c-myc and c-Ki-ras oncogenes and the acquired resistance to cisplatin. (author) 3 figs., 32 refs

  4. Characteristics and Efficacy of a Sterile Hyphomycete (ARF18), a New Biocontrol Agent for Heterodera glycines and Other Nematodes

    Science.gov (United States)

    Kim, D. G.; Riggs, R. D.

    1991-01-01

    A filamentous, nonsporulating fungus, designated Arkansas Fungus 18 (ARF18), was isolated from 9 of 95 populations of Heterodera glycines, the soybean cyst nematode, in Arkansas. In petri dishes, ARF18 parasitized 89% of H. glycines eggs in cysts. The fungus also infected eggs of Meloidogyne incognita and eggs in cysts of Cactodera betulae, H. graminophila, H. lespedezae, H. leuceilyma, H. schachtii, and H. trifolii. In pot tests, reproduction of SCN was 70% less in untreated field soil that was naturally infested by ARF18 than in autoclaved field soil. Although ARF18 grew well at 25 C on cornmeal agar over a wide pH range, it did not sporulate on 28 media and thus could not be identified to genus or species. PMID:19283127

  5. Characteristics and Efficacy of a Sterile Hyphomycete (ARF18), a New Biocontrol Agent for Heterodera glycines and Other Nematodes.

    Science.gov (United States)

    Kim, D G; Riggs, R D

    1991-07-01

    A filamentous, nonsporulating fungus, designated Arkansas Fungus 18 (ARF18), was isolated from 9 of 95 populations of Heterodera glycines, the soybean cyst nematode, in Arkansas. In petri dishes, ARF18 parasitized 89% of H. glycines eggs in cysts. The fungus also infected eggs of Meloidogyne incognita and eggs in cysts of Cactodera betulae, H. graminophila, H. lespedezae, H. leuceilyma, H. schachtii, and H. trifolii. In pot tests, reproduction of SCN was 70% less in untreated field soil that was naturally infested by ARF18 than in autoclaved field soil. Although ARF18 grew well at 25 C on cornmeal agar over a wide pH range, it did not sporulate on 28 media and thus could not be identified to genus or species.

  6. Coupling between p210bcr-abl and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to ras activation pathway.

    Science.gov (United States)

    Tauchi, T; Boswell, H S; Leibowitz, D; Broxmeyer, H E

    1994-01-01

    Enforced expression of p210bcr-abl transforms interleukin 3 (IL-3)-dependent hematopoietic cell lines to growth factor-independent proliferation. It has been demonstrated that nonreceptor tyrosine kinase oncogenes may couple to the p21ras pathway to exert their transforming effect. In particular, p210bcr-abl was recently found to effect p21ras activation in hematopoietic cells. In this context, experiments were performed to evaluate a protein signaling pathway by which p210bcr-abl might regulate p21ras. It was asked whether Shc p46/p52, a protein containing a src-homology region 2 (SH2) domain, and known to function upstream from p21ras, might form specific complexes with p210bcr-abl and thus, possibly alter p21ras activity by coupling to the guanine nucleotide exchange factor (Sos/CDC25) through the Grb2 protein-Sos complex. This latter complex has been previously demonstrated to occur ubiquitously. We found that p210bcr-abl formed a specific complex with Shc and with Grb2 in three different murine cell lines transfected with a p210bcr-abl expression vector. There appeared to be a higher order complex containing Shc, Grb2, and bcr-abl proteins. In contrast to p210bcr-abl transformed cells, in which there was constitutive tight association between Grb2 and Shc, binding between Grb2 and Shc was Steel factor (SLF)-dependent in a SLF-responsive, nontransformed parental cell line. The SLF-dependent association between Grb2 and Shc in nontransformed cells involved formation of a complex of Grb2 with c-kit receptor after SLF treatment. Thus, p210bcr-abl appears to function in a hematopoietic p21ras activation pathway to allow growth factor-independent coupling between Grb2, which exists in a complex with the guanine nucleotide exchange factor (Sos), and p21ras. Shc may not be required for Grb2-c-kit interaction, because it fails to bind strongly to c-kit.

  7. Genomewide identification and expression analysis of the ARF gene ...

    Indian Academy of Sciences (India)

    Figure 1. Phylogenetic relation of apple ARF genes. The phylogenetic tree was constructed based on a complete protein sequence align- ment of MdARFs by the neighbour-joining method with bootstrapping analysis (1000 replicates). The scale bar represents 0.05 amino acid substitutions per site. Paralogous gene pairs ...

  8. Epac activation sensitizes rat sensory neurons via activation of Ras

    Science.gov (United States)

    Shariati, Behzad; Thompson, Eric L.; Nicol, Grant D.; Vasko, Michael R.

    2015-01-01

    Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2′-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. PMID:26596174

  9. Epac activation sensitizes rat sensory neurons through activation of Ras.

    Science.gov (United States)

    Shariati, Behzad; Thompson, Eric L; Nicol, Grant D; Vasko, Michael R

    2016-01-01

    Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2'-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. TBC1D24 regulates neuronal migration and maturation through modulation of the ARF6-dependent pathway

    Science.gov (United States)

    Falace, Antonio; Buhler, Emmanuelle; Fadda, Manuela; Watrin, Françoise; Lippiello, Pellegrino; Pallesi-Pocachard, Emilie; Baldelli, Pietro; Benfenati, Fabio; Zara, Federico; Represa, Alfonso; Fassio, Anna; Cardoso, Carlos

    2014-01-01

    Alterations in the formation of brain networks are associated with several neurodevelopmental disorders. Mutations in TBC1 domain family member 24 (TBC1D24) are responsible for syndromes that combine cortical malformations, intellectual disability, and epilepsy, but the function of TBC1D24 in the brain remains unknown. We report here that in utero TBC1D24 knockdown in the rat developing neocortex affects the multipolar-bipolar transition of neurons leading to delayed radial migration. Furthermore, we find that TBC1D24-knockdown neurons display an abnormal maturation and retain immature morphofunctional properties. TBC1D24 interacts with ADP ribosylation factor (ARF)6, a small GTPase crucial for membrane trafficking. We show that in vivo, overexpression of the dominant-negative form of ARF6 rescues the neuronal migration and dendritic outgrowth defects induced by TBC1D24 knockdown, suggesting that TBC1D24 prevents ARF6 activation. Overall, our findings demonstrate an essential role of TBC1D24 in neuronal migration and maturation and highlight the physiological relevance of the ARF6-dependent membrane-trafficking pathway in brain development. PMID:24469796

  11. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain

    DEFF Research Database (Denmark)

    Adari, H; Lowy, D R; Willumsen, B M

    1988-01-01

    A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as w...

  12. DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, is a putative tumor suppressor.

    Science.gov (United States)

    Kanno, Emiri; Kawasaki, Osamu; Takahashi, Kazuya; Takano, Kazunori; Endo, Takeshi

    2018-01-01

    Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf-MEK-ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Oncogenic K-Ras Activates p38 to Maintain Colorectal Cancer Cell Proliferation during MEK Inhibition

    Directory of Open Access Journals (Sweden)

    Winan J. van Houdt

    2010-01-01

    Full Text Available Background: Colon carcinomas frequently contain activating mutations in the K-ras proto-oncogene. K-ras itself is a poor drug target and drug development efforts have mostly focused on components of the classical Ras-activated MEK/ERK pathway. Here we have studied whether endogenous oncogenic K-ras affects the dependency of colorectal tumor cells on MEK/ERK signaling.

  14. Somatic INK4a-ARF locus mutations: a significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck.

    Science.gov (United States)

    Poi, M J; Yen, T; Li, J; Song, H; Lang, J C; Schuller, D E; Pearl, D K; Casto, B C; Tsai, M D; Weghorst, C M

    2001-01-01

    The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16(INK4a) (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14(ARF) tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G(1) arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14(ARF) genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1alpha, 1beta, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1alpha. No mutations were found in exon 1beta. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14(ARF) proteins. Specific somatic alterations included microdeletions or

  15. A-RAF kinase functions in ARF6 regulated endocytic membrane traffic.

    Directory of Open Access Journals (Sweden)

    Elena Nekhoroshkova

    Full Text Available BACKGROUND: RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149 that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. CONCLUSIONS/SIGNIFICANCE: A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking.

  16. Auxin Response Factors (ARFs are potential mediators of auxin action in tomato response to biotic and abiotic stress (Solanum lycopersicum.

    Directory of Open Access Journals (Sweden)

    Sarah Bouzroud

    Full Text Available Survival biomass production and crop yield are heavily constrained by a wide range of environmental stresses. Several phytohormones among which abscisic acid (ABA, ethylene and salicylic acid (SA are known to mediate plant responses to these stresses. By contrast, the role of the plant hormone auxin in stress responses remains so far poorly studied. Auxin controls many aspects of plant growth and development, and Auxin Response Factors play a key role in the transcriptional activation or repression of auxin-responsive genes through direct binding to their promoters. As a mean to gain more insight on auxin involvement in a set of biotic and abiotic stress responses in tomato, the present study uncovers the expression pattern of SlARF genes in tomato plants subjected to biotic and abiotic stresses. In silico mining of the RNAseq data available through the public TomExpress web platform, identified several SlARFs as responsive to various pathogen infections induced by bacteria and viruses. Accordingly, sequence analysis revealed that 5' regulatory regions of these SlARFs are enriched in biotic and abiotic stress-responsive cis-elements. Moreover, quantitative qPCR expression analysis revealed that many SlARFs were differentially expressed in tomato leaves and roots under salt, drought and flooding stress conditions. Further pointing to the putative role of SlARFs in stress responses, quantitative qPCR expression studies identified some miRNA precursors as potentially involved in the regulation of their SlARF target genes in roots exposed to salt and drought stresses. These data suggest an active regulation of SlARFs at the post-transcriptional level under stress conditions. Based on the substantial change in the transcript accumulation of several SlARF genes, the data presented in this work strongly support the involvement of auxin in stress responses thus enabling to identify a set of candidate SlARFs as potential mediators of biotic and abiotic

  17. RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

    Science.gov (United States)

    Chow, Jimmy Y C; Quach, Khai T; Cabrera, Betty L; Cabral, Jennifer A; Beck, Stayce E; Carethers, John M

    2007-11-01

    Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

  18. RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

    Science.gov (United States)

    Lim, C J; Spiegelman, G B; Weeks, G

    2001-08-15

    Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

  19. Identification of H-Ras-Specific Motif for the Activation of Invasive Signaling Program in Human Breast Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Hae-Young Yong

    2011-02-01

    Full Text Available Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR, consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

  20. Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Edwards Jeremy

    2003-07-01

    Full Text Available Abstract Background The identification of known mutations in a cell population is important for clinical applications and basic cancer research. In this work an immobilized form of the polymerase chain reaction, referred to as polony technology, was used to detect mutations as well as gene deletions, resulting in loss of heterozygosity (LOH, in cancer cell lines. Specifically, the mutational hotspots in p53, namely codons 175, 245, 248, 249, 273, and 282, and K-ras2, codons 12, 13 and 61, were genotyped in the pancreatic cell line, Panc-1. In addition LOH analysis was also performed for these same two genes in Panc-1 by quantifying the relative gene copy number of p53 and K-ras2. Results Using polony technology, Panc-1 was determined to possess only one copy of p53, which possessed a mutation in codon 273, and two copies of K-ras2, one wildtype and one with a mutation in codon 12. To further demonstrate the general approach of this method, polonies were also used to detect K-ras2 mutations in the pancreatic cell lines, AsPc-1 and CAPAN-1. Conclusions In conclusion, we have developed an assay that can detect mutations in hotspots of p53 and K-ras2 as well as diagnose LOH in these same genes.

  1. [Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

    Science.gov (United States)

    Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

    2004-01-01

    To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P ras4B can enhance the expression of ERalpha and

  2. Use of Human Tissue to Assess the Oncogenic Activity of Melanoma-Associated Mutations

    OpenAIRE

    Chudnovsky, Yakov; Adams, Amy E.; Robbins, Paul B.; Lin, Qun; Khavari, Paul A.

    2005-01-01

    Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence1,2. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Ras and Raf induction can occur via active N-Ras and B-Raf mutants as well as by gene amplification3–5. Activation of PI3K pathway components occurs by PTEN loss and by AKT amplification6–8. Melanomas also commonly display impairment of p16INK4A-CDK4-Rb and ARF-HDM2-p53 tumor s...

  3. Decay kinetics of Krsup(*) levels populated by ArF laser radiation

    International Nuclear Information System (INIS)

    Wren, D.J.; Setser, D.W.

    1981-01-01

    The Kr 6p [3/2] 2 (or 3p 6 ) level was pumped via a two-photon absorption using a focused ArF laser (193 nm). The time dependent fluorescence from excited Kr levels was observed using a monochromator and photomultiplier tube and transient digitizer. Fluorescence from the pumped level, observed via the 3p 6 → 1s 5 transition, has a lifetime of 8 +- 2 ns independent of the Kr pressure (2-30 Torr). Fluorescence from other levels of the Kr (4p 5 6p) manifold was either unobserved or weak. Strong fluorescence was observed from the Kr (4p 5 5p) levels via transitions to the Kr (4p 5 5s) levels, the highest populated levels being 2p 6 , 2p 8 and 2p 9 . All Kr (4p 5 5p) levels display effective lifetimes much longer than the radiative lifetimes; effective zero pressure lifetimes for Kr 2p 3 , 2p 6 and 2p 7 are 72 +- 15, 66 +- 15 and 90 +- 15 ns, respectively. The short lifetime of the Kr (3p 6 ) level is explained by very fast collisional depopulation by photoelectrons produced in three photon ionization of Kr by the ArF laser. The Kr (4p 5 5p) levels are populated indirectly from the Kr (3p 6 ) level and the long lifetimes of the Kr (4p 5 5p) levels represent the lifetime of the intermediate state (or states) which lead to the Kr (4p 5 5p) levels. Strong possibilities for the intermediate states are the Kr (4p 5 4d) levels. (orig.)

  4. In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis.

    Science.gov (United States)

    Qian, Jiaying; Niu, Jiangong; Li, Ming; Chiao, Paul J; Tsao, Ming-Sound

    2005-06-15

    Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.

  5. PI3K: A Crucial Piece in the RAS Signaling Puzzle.

    Science.gov (United States)

    Krygowska, Agata Adelajda; Castellano, Esther

    2018-06-01

    RAS proteins are key signaling switches essential for control of proliferation, differentiation, and survival of eukaryotic cells. RAS proteins are mutated in 30% of human cancers. In addition, mutations in upstream or downstream signaling components also contribute to oncogenic activation of the pathway. RAS proteins exert their functions through activation of several signaling pathways and dissecting the contributions of these effectors in normal cells and in cancer is an ongoing challenge. In this review, we summarize our current knowledge about how RAS regulates type I phosphatidylinositol 3-kinase (PI3K), one of the main RAS effectors. RAS signaling through PI3K is necessary for normal lymphatic vasculature development and for RAS-induced transformation in vitro and in vivo, especially in lung cancer, where it is essential for tumor initiation and necessary for tumor maintenance. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

  6. Molecular analysis of ARF1 expression profiles during development of physic nut (Jatropha curcas L.).

    Science.gov (United States)

    Qin, Xiaobo; Lin, Fanrong; Lii, Yifan; Gou, Chunbao; Chen, Fang

    2011-03-01

    A cDNA clone designated arf1 was isolated from a physic nut (Jatropha curcas L.) endosperm cDNA library which encodes a small GTP-binding protein and has significant homology to ADP-ribosylation factors (ARF) in plants, animals and microbes. The cDNA contains an open reading frame that encodes a polypeptide of 181 amino acids with a calculated molecular mass of 20.7 kDa. The deduced amino acid sequence showed high homology to known ARFs from other organisms. The products of the arf1 obtained by overexpression in E. coli revealed the specific binding activity toward GTP. The expression of arf1 was observed in flowers, roots, stems and leaves as analyzed by RT-PCR, and its transcriptional level was highest in flowers. In particular, the accumulation of arf1 transcripts was different under various environmental stresses in seedlings. The results suggest that arf1 plays distinct physiological roles in Jatropha curcas cells.

  7. The RAS Initiative

    Science.gov (United States)

    NCI established the RAS Initiative to explore innovative approaches for attacking the proteins encoded by mutant forms of RAS genes and to ultimately create effective, new therapies for RAS-related cancers.

  8. The Ras effector RASSF2 is a novel tumor-suppressor gene in human colorectal cancer.

    Science.gov (United States)

    Akino, Kimishige; Toyota, Minoru; Suzuki, Hiromu; Mita, Hiroaki; Sasaki, Yasushi; Ohe-Toyota, Mutsumi; Issa, Jean-Pierre J; Hinoda, Yuji; Imai, Kohzoh; Tokino, Takashi

    2005-07-01

    Activation of Ras signaling is a hallmark of colorectal cancer (CRC), but the roles of negative regulators of Ras are not fully understood. Our aim was to address that question by surveying genetic and epigenetic alterations of Ras-Ras effector genes in CRC cells. The expression and methylation status of 6 RASSF family genes were examined using RT-PCR and bisulfite PCR in CRC cell lines and in primary CRCs and colorectal adenomas. Colony formation assays and flow cytometry were used to assess the tumor suppressor activities of RASSF1 and RASSF2. Immunofluorescence microscopy was used to determine the effect of altered RASSF2 expression on cell morphology. Mutations of K- ras , BRAF, and p53 were identified using single-strand conformation analysis and direct sequencing. Aberrant methylation and histone deacetylation of RASSF2 was associated with the gene's silencing in CRC. The activities of RASSF2, which were distinct from those of RASSF1, included induction of morphologic changes and apoptosis; moreover, its ability to prevent cell transformation suggests that RASSF2 acts as a tumor suppressor in CRC. Primary CRCs that showed K- ras /BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced K- ras -induced oncogenic transformation. RASSF2 methylation was also frequently identified in colorectal adenomas. RASSF2 is a novel tumor suppressor gene that regulates Ras signaling and plays a pivotal role in the early stages of colorectal tumorigenesis.

  9. The calcium-sensing receptor changes cell shape via a beta-arrestin-1 ARNO ARF6 ELMO protein network.

    Science.gov (United States)

    Bouschet, Tristan; Martin, Stéphane; Kanamarlapudi, Venkateswarlu; Mundell, Stuart; Henley, Jeremy M

    2007-08-01

    G-protein-coupled receptors (GPCRs) transduce the binding of extracellular stimuli into intracellular signalling cascades that can lead to morphological changes. Here, we demonstrate that stimulation of the calcium-sensing receptor (CaSR), a GPCR that promotes chemotaxis by detecting increases in extracellular calcium, triggers plasma membrane (PM) ruffling via a pathway that involves beta-arrestin 1, Arf nucleotide binding site opener (ARNO), ADP-ribosylating factor 6 (ARF6) and engulfment and cell motility protein (ELMO). Expression of dominant negative beta-arrestin 1 or its knockdown with siRNA impaired the CaSR-induced PM ruffling response. Expression of a catalytically inactive ARNO also reduced CaSR-induced PM ruffling. Furthermore, beta-arrestin 1 co-immunoprecipitated with the CaSR and ARNO under resting conditions. Agonist treatment did not markedly alter beta-arrestin 1 binding to the CaSR or to ARNO but it did elicit the translocation and colocalisation of the CaSR, beta-arrestin 1 and ARNO to membrane protrusions. Furthermore, ARF6 and ELMO, two proteins known to couple ARNO to the cytoskeleton, were required for CaSR-dependent morphological changes and translocated to the PM ruffles. These data suggest that cells ruffle upon CaSR stimulation via a mechanism that involves translocation of beta-arrestin 1 pre-assembled with the CaSR or ARNO, and that ELMO plays an essential role in this CaSR-signalling-induced cytoskeletal reorganisation.

  10. Inhibitor of DNA binding 1 (Id1) induces differentiation and proliferation of mouse embryonic carcinoma P19CL6 cells

    International Nuclear Information System (INIS)

    Meng, Qingzhen; Jia, Zhuqing; Wang, Weiping; Li, Binhong; Ma, Kangtao; Zhou, Chunyan

    2011-01-01

    Highlights: → Id1 was upregulated during the cardiac differentiation process of P19CL6 cells. → Id1 upregulated expression of cardiac specific genes Gata4, α-MHC and ISL1. → Id1 promoted proliferation of P19CL6 cells. → Overexpression of Id1 increased activity of TOP flash. → Wnt3a or LiCl treatment promoted Id1 expression in P19CL6 cells. -- Abstract: The inhibitor of DNA binding (Id) family of genes encodes negative regulators of basic helix-loop-helix transcription factors and has been implicated in such diverse cellular processes as differentiation, proliferation, apoptosis and migration. Id knockout mouse embryos display multiple cardiac defects but the specific role of Id1 in cardiac differentiation is unclear. In the present study, we investigated the function of Id1 in DMSO-induced P19CL6 cells, a widely-accepted cell model of cardiac differentiation. We found that Id1 was upregulated during the cardiac differentiation of P19CL6 cells. The expression of cardiac specific marker genes, Gata4, α-MHC and ISL1, was upregulated in P19CL6 cells stably transfected with Id1 (P19CL6-Id1) during cardiac differentiation. The overexpression of Id1 reduced the number of cells in G1 phase and increased the cell population in G2, M and S phases, while knockdown of Id1 increased the number of cells in G1 phase from 48.6 ± 2.51% to 62.2 ± 1.52% at day 0 of cardiac induction, and from 52.5 ± 3.41% to 63.7 ± 1.02% at day 3 after cardiac induction, indicating that Id1 promoted proliferation of P19CL6 cells. Luciferase assays showed that the activity of TOP flash was higher in P19CL6-Id1 cells than wildtype P19CL6 cells, while Id1 expression was also upregulated in P19CL6 cells treated with Wnt3a or LiCl. This indicates that there may be positive feedback between Id1 and Wnt signaling which plays an important role in cardiac differentiation.

  11. BMP suppresses PTEN expression via RAS/ERK signaling.

    Science.gov (United States)

    Beck, Stayce E; Carethers, John M

    2007-08-01

    Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

  12. Activation of RAS/ERK alone is insufficient to inhibit RXRα function and deplete retinoic acid in hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ai-Guo, E-mail: wangaiguotl@hotmail.com; Song, Ya-Nan; Chen, Jun; Li, Hui-Ling; Dong, Jian-Yi; Cui, Hai-Peng; Yao, Liang; Li, Xue-Feng; Gao, Wen-Ting; Qiu, Ze-Wen; Wang, Fu-Jin; Wang, Jing-Yu, E-mail: wangjingyus@163.com

    2014-09-26

    Highlights: • The activation of RAS/ERK is insufficient to inhibit RXRα function and deplete RA. • The retinoid metabolism-related genes are down-regulated by ras oncogene. • The atRA has no effect on preventing hepatic tumorigenesis or curing the developed hepatic nodules. - Abstract: Activation of RAS/ERK signaling pathway, depletion of retinoid, and phosphorylation of retinoid X receptor alpha (RXRα) are frequent events found in liver tumors and thought to play important roles in hepatic tumorigenesis. However, the relationships among them still remained to be elucidated. By exploring the transgenic mouse model of hepatic tumorigenesis induced by liver-specific expression of H-ras12V oncogene, the activation of RAS/ERK, the mRNA expression levels of retinoid metabolism-related genes, the contents of retinoid metabolites, and phosphorylation of RXRα were determined. RAS/ERK signaling pathway was gradually and significantly activated in hepatic tumor adjacent normal liver tissues (P) and hepatic tumor tissues (T) of H-ras12V transgenic mice compared with normal liver tissues (Wt) of wild type mice. On the contrary, the mRNA expression levels of retinoid metabolism-related genes were significantly reduced in P and T compared with Wt. Interestingly, the retinoid metabolites 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (atRA), the well known ligands for nuclear transcription factor RXR and retinoic acid receptor (RAR), were significantly decreased only in T compared with Wt and P, although the oxidized polar metabolite of atRA, 4-keto-all-trans-retinoic-acid (4-keto-RA) was significantly decreased in both P and T compared with Wt. To our surprise, the functions of RXRα were significantly blocked only in T compared with Wt and P. Namely, the total protein levels of RXRα were significantly reduced and the phosphorylation levels of RXRα were significantly increased only in T compared with Wt and P. Treatment of H-ras12V transgenic mice at 5-week

  13. Ras and Rheb Signaling in Survival and Cell Death

    International Nuclear Information System (INIS)

    Ehrkamp, Anja; Herrmann, Christian; Stoll, Raphael; Heumann, Rolf

    2013-01-01

    One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively

  14. Ras and Rheb Signaling in Survival and Cell Death

    Energy Technology Data Exchange (ETDEWEB)

    Ehrkamp, Anja [Molecular Neurobiochemistry, Ruhr University of Bochum, 44780 Bochum (Germany); Herrmann, Christian [Department of Physical Chemistry1, Protein Interaction, Ruhr University of Bochum, 44780 Bochum (Germany); Stoll, Raphael [Biomolecular NMR, Ruhr University of Bochum, 44780 Bochum (Germany); Heumann, Rolf, E-mail: rolf.heumann@rub.de [Molecular Neurobiochemistry, Ruhr University of Bochum, 44780 Bochum (Germany)

    2013-05-28

    One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively.

  15. ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate

    International Nuclear Information System (INIS)

    Masse, Julie; Piquet-Pellorce, Claire; Viet, Justine; Guerrier, Daniel; Pellerin, Isabelle; Deschamps, Stephane

    2011-01-01

    The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible. We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells. Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.

  16. Ras and relatives--job sharing and networking keep an old family together.

    Science.gov (United States)

    Ehrhardt, Annette; Ehrhardt, Götz R A; Guo, Xuecui; Schrader, John W

    2002-10-01

    Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

  17. Assessment of the chemosensitizing activity of TAT-RasGAP317-326 in childhood cancers.

    Directory of Open Access Journals (Sweden)

    Nadja Chevalier

    Full Text Available Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology. TAT-RasGAP317-326 is a RasGAP-derived cell-permeable peptide that acts as a sensitizer to various anti-cancer treatments in adult tumor cells. In the present study, we assessed the effect of TAT-RasGAP317-326 in several childhood cancer cell lines. The RasGAP-derived peptide-induced cell death was analyzed in several neuroblastoma, Ewing sarcoma and leukemia cell lines (as well as in normal lymphocytes. Cell death was evaluated using flow cytometry methods in the absence or in the presence of the peptide in combination with various genotoxins used in the clinics (4-hydroperoxycyclophosphamide, etoposide, vincristine and doxorubicin. All tested pediatric tumors, in response to at least one genotoxin, were sensitized by TAT-RasGAP317-326. The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies. Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

  18. Andrographolide Sensitizes Ras-Transformed Cells to Radiation in vitro and in vivo

    International Nuclear Information System (INIS)

    Hung, Shih-Kai; Hung, Ling-Chien; Kuo, Cheng-Deng

    2010-01-01

    Purpose: Increasing the sensitivity of tumor cells to radiation is a major goal of radiotherapy. The present study investigated the radiosensitizing effects of andrographolide and examined the molecular mechanisms of andrographolide-mediated radiosensitization. Methods and Materials: An H-ras-transformed rat kidney epithelial (RK3E) cell line was used to measure the radiosensitizing effects of andrographolide in clonogenic assays, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assays, and a xenograft tumor growth model. The mechanism of andrographolide-sensitized cell death was analyzed using annexin V staining, caspase 3 activity assays, and terminal transferase uridyl nick end labeling assays. The roles of nuclear factor kappa B (NF-κB) and Akt in andrographolide-mediated sensitization were examined using reporter assays, electrophoretic mobility shift assays, and Western blotting. Results: Concurrent andrographolide treatment (10 μM, 3 h) sensitized Ras-transformed cells to radiation in vitro (sensitizer enhancement ratio, 1.73). Andrographolide plus radiation (one dose of 300 mg/kg peritumor andrographolide and one dose of 6 Gy radiation) resulted in significant tumor growth delay (27 ± 2.5 days) compared with radiation alone (22 ± 1.5 days; p <.05). Radiation induced apoptotic markers (e.g., caspase-3, membrane reversion, DNA fragmentation), and andrographolide treatment did not promote radiation-induced apoptosis. However, the protein level of activated Akt was significantly reduced by andrographolide. NF-κB activity was elevated in irradiated Ras-transformed cells, and andrographolide treatment significantly reduced radiation-induced NF-κB activity. Conclusion: Andrographolide sensitized Ras-transformed cells to radiation both in vitro and in vivo. Andrographolide-mediated radiosensitization was associated with downregulation of Akt and NF-κB activity. These observations indicate that andrographolide is a novel radiosensitizing agent

  19. High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

    Science.gov (United States)

    Burns, Michael C; Howes, Jennifer E; Sun, Qi; Little, Andrew J; Camper, DeMarco V; Abbott, Jason R; Phan, Jason; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

    2018-05-01

    K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Syndecan-2 promotes perineural invasion and cooperates with K-ras to induce an invasive pancreatic cancer cell phenotype

    Directory of Open Access Journals (Sweden)

    De Oliveira Tiago

    2012-04-01

    Full Text Available Abstract Background We have identified syndecan-2 as a protein potentially involved in perineural invasion of pancreatic adenocarcinoma (PDAC cells. Methods Syndecan-2 (SDC-2 expression was analyzed in human normal pancreas, chronic pancreatitis and PDAC tissues. Functional in vitro assays were carried out to determine its role in invasion, migration and signaling. Results SDC-2 was expressed in the majority of the tested pancreatic cancer cell lines while it was upregulated in nerve-invasive PDAC cell clones. There were 2 distinct expression patterns of SDC-2 in PDAC tissue samples: SDC-2 positivity in the cancer cell cytoplasm and a peritumoral expression. Though SDC-2 silencing (using specific siRNA oligonucleotides did not affect anchorage-dependent growth, it significantly reduced cell motility and invasiveness in the pancreatic cancer cell lines T3M4 and Su8686. On the transcriptional level, migration-and invasion-associated genes were down-regulated following SDC-2 RNAi. Furthermore, SDC-2 silencing reduced K-ras activity, phosphorylation of Src and - further downstream - phosphorylation of ERK2 while levels of the putative SDC-2 signal transducer p120GAP remained unaltered. Conclusion SDC-2 is a novel (perineural invasion-associated gene in PDAC which cooperates with K-ras to induce a more invasive phenotype.

  1. Induction of non-apoptotic programmed cell death by oncogenic RAS in human epithelial cells and its suppression by MYC overexpression.

    Science.gov (United States)

    Dendo, Kasumi; Yugawa, Takashi; Nakahara, Tomomi; Ohno, Shin-Ichi; Goshima, Naoki; Arakawa, Hirofumi; Kiyono, Tohru

    2018-02-09

    Oncogenic mutations of RAS genes, found in about 30% of human cancers, are considered to play important roles in cancer development. However, oncogenic RAS can also induce senescence in mouse and human normal fibroblasts. In some cell lines, oncogenic RAS has been reported to induce non-apoptotic programed cell death (PCD). Here, we investigated effects of oncogenic RAS expression in several types of normal human epithelial cells. Oncogenic RAS but not wild-type RAS stimulated macropinocytosis with accumulation of large-phase lucent vacuoles in the cytoplasm, subsequently leading to cell death which was indistinguishable from a recently proposed new type of PCD, methuosis. A RAC1 inhibitor suppressed accumulation of macropinosomes and overexpression of MYC attenuated oncogenic RAS-induced such accumulation, cell cycle arrest and cell death. MYC suppression or rapamycin treatment in some cancer cell lines harbouring oncogenic mutations in RAS genes induced cell death with accumulation of macropinosomes. These results suggest that this type of non-apoptotic PCD is a tumour-suppressing mechanism acting against oncogenic RAS mutations in normal human epithelial cells, which can be overcome by MYC overexpression, raising the possibility that its induction might be a novel approach to treatment of RAS-mutated human cancers. © The Author(s) 2017. Published by Oxford University Press.

  2. Exploring environmental causes of altered ras effects: fragmentation plus integration?

    Science.gov (United States)

    Porta, Miquel; Ayude, Daniel; Alguacil, Juan; Jariod, Manuel

    2003-02-01

    Mutations in ras genes are the most common abnormality of oncogenes in human cancer and a major example of activation by point mutation. Experimental and epidemiological studies support the notion that Ki-ras activation and expression may be chemically related. We discuss the potential role of several environmental compounds in the induction or promotion of ras mutations in humans, with a focus on exocrine pancreatic cancer, the human tumor with the highest prevalence at diagnosis of Ki-ras mutations. Organochlorine compounds, organic solvents, and coffee compounds may play an indirect role in causing Ki-ras mutations, rather than as direct inducers of the mutations. Although for some organochlorine compounds the induction of point mutations in ras oncogenes cannot be excluded, it seems more likely that the effects of these compounds are mediated through nongenomic or indirectly genotoxic mechanisms of action. Organic solvents also may act via enzymatic induction of ras mutagens or by providing a proliferation advantage to ras-mutated cell clones. In exocrine pancreatic cancer, caffeine, other coffee compounds, or other factors with which coffee drinking is associated could modulate Ki-ras activation by interfering with DNA repair, cell-cycle checkpoints, and apoptosis. Asbestos, cigarette smoking, and some dietary factors also may be involved in the initiation or the promotion of Ki-ras mutations in lung and colon cancers. Further development of the mechanistic scenarios proposed here could contribute to a meaningful integration of biological, clinical, and environmental knowledge on the causes of altered ras effects. Copyright 2003 Wiley-Liss, Inc.

  3. Epigenetic changes in the CDKN2A locus are associated with differential expression of P16INK4A and P14ARF in HPV-positive oropharyngeal squamous cell carcinoma

    International Nuclear Information System (INIS)

    Schlecht, Nicolas F; Ben-Dayan, Miriam; Anayannis, Nicole; Lleras, Roberto A; Thomas, Carlos; Wang, Yanhua; Smith, Richard V; Burk, Robert D; Harris, Thomas M; Childs, Geoffrey; Ow, Thomas J; Prystowsky, Michael B; Belbin, Thomas J

    2015-01-01

    Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease entity associated with improved survival. DNA hypermethylation profiles differ significantly by HPV status suggesting that a specific subset of methylated CpG loci could give mechanistic insight into HPV-driven OPSCC. We analyzed genome-wide DNA methylation of primary tumor samples and adjacent normal mucosa from 46 OPSCC patients undergoing treatment at Montefiore Medical Center, Bronx, NY using the Illumina HumanMethylation27 beadchip. For each matched tissue set, we measured differentially methylated CpG loci using a change in methylation level (M value). From these analyses, we identified a 22 CpG loci panel for HPV+ OPSCC that included four CDKN2A loci downstream of the p16(INK4A) and p14(ARF) transcription start sites. This panel was significantly associated with overall HPV detection (P < 0.05; ROC area under the curve = 0.96, 95% CI: 0.91–1.0) similar to the subset of four CDKN2A-specific CpG loci (0.90, 95% CI: 0.82–0.99) with equivalence to the full 22 CpG panel. DNA hypermethylation correlated with a significant increase in alternative open reading frame (ARF) expression in HPV+ OPSCC primary tumors, but not to the other transcript variant encoded by the CDKN2A locus. Overall, this study provides evidence of epigenetic changes to the downstream region of the CDKN2A locus in HPV+ oropharyngeal cancer that are associated with changes in expression of the coded protein products

  4. Dietary turmeric modulates DMBA-induced p21ras, MAP kinases and AP-1/NF-κB pathway to alter cellular responses during hamster buccal pouch carcinogenesis

    International Nuclear Information System (INIS)

    Garg, Rachana; Ingle, Arvind; Maru, Girish

    2008-01-01

    The chemopreventive efficacy of turmeric has been established in experimental systems. However, its mechanism(s) of action are not fully elucidated in vivo. The present study investigates the mechanism of turmeric-mediated chemoprevention in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis at 2, 4, 6, 10 and 12 weeks. Dietary turmeric (1%) led to decrease in DMBA-induced tumor burden and multiplicity, and enhanced the latency period in parallel, to its modulatory effects on oncogene products and various cellular responses during HBP tumorigenesis. DMBA-induced expression of ras oncogene product, p21 and downstream target, the mitogen-activated protein kinases were significantly decreased by turmeric during HBP carcinogenesis. Turmeric also diminished the DMBA-induced mRNA expression of proto-oncogenes (c-jun, c-fos) and NF-κB, leading to decreased protein levels and in further attenuation of DMBA-induced AP-1/NF-κB DNA-binding in the buccal pouch nuclear extracts. Besides, buccal pouch of hamsters receiving turmeric diet showed significant alterations in DMBA-induced effects: (a) decrease in cell proliferation (diminished PCNA and Bcl2 expression), (b) enhanced apoptosis (increased expression of Bax, caspase-3 and apoptotic index), (c) decrease in inflammation (levels of Cox-2, the downstream target of AP-1/NF-κB, and PGE2) and (d) aberrant expression of differentiation markers, the cytokeratins (1, 5, 8, and 18). Together, the protective effects of dietary turmeric converge on augmenting apoptosis of the initiated cells and decreasing cell proliferation in DMBA-treated animals, which in turn, is reflected in decreased tumor burden, multiplicity and enhanced latency period. Some of these biomarkers are likely to be helpful in monitoring clinical trials and evaluating drug effect measurements

  5. ArF photo resist pattern sample preparation method using FIB without protective coating

    Science.gov (United States)

    Okushima, Hirohisa; Onozuka, Toshihiko; Kuroda, Yasushi; Yaguchi, Toshie; Umemura, Kaoru; Tamochi, Ryuichiro; Watanabe, Kenji; Hasegawa, Norio; Kawata, Isao; Rijpers, Bart

    2006-03-01

    This paper presents a novel method of FIB (FIB: focused ion beam) sample preparation to accurately evaluate critical dimensions and profiles of ArF photo resist patterns without the use of a protective coating on the photo resist. In order to accomplish this, the FIB micro-sampling method that is one of effective FIB milling and fabrication method was employed. First a Si cap is picked up from a silicon wafer and fixed to ArF photo resist patterns to protect against ion beam irradiation. Then, a micro-sample, a piece of Si-capped ArF photo resist, was extracted from the bulk ArF photo resist. In this procedure, this silicon cap always protects ArF photo resist patterns against ion beam irradiation. For the next step, the micro-sample is fixed to a needle stub of the FIB-STEM (STEM: scanning transmission electron microscopy) compatible rotation holder. This sample on the needle stub was rotated 180 degrees and milled from the side of Si substrate. Lastly, the sample is milled to the thickness of 2μm. In this process, the ion beam is irradiating from the silicon substrate side to minimize the ion beam irradiation damages on the ArF photo resist patterns. EDX (EDX: Energy dispersive X-ray spectroscopy) analysis proved that no gallium ions were detected on the surface of the ArF photo resist patterns. The feasibility of high accelerating voltage observation of STEM to observe line edge roughness of a thick sample like 2μm without shrinkage has been demonstrated.

  6. Loss of Arf4 causes severe degeneration of the exocrine pancreas but not cystic kidney disease or retinal degeneration.

    Directory of Open Access Journals (Sweden)

    Jillian N Pearring

    2017-04-01

    Full Text Available Arf4 is proposed to be a critical regulator of membrane protein trafficking in early secretory pathway. More recently, Arf4 was also implicated in regulating ciliary trafficking, however, this has not been comprehensively tested in vivo. To directly address Arf4's role in ciliary transport, we deleted Arf4 specifically in either rod photoreceptor cells, kidney, or globally during the early postnatal period. Arf4 deletion in photoreceptors did not cause protein mislocalization or retinal degeneration, as expected if Arf4 played a role in protein transport to the ciliary outer segment. Likewise, Arf4 deletion in kidney did not cause cystic disease, as expected if Arf4 were involved in general ciliary trafficking. In contrast, global Arf4 deletion in the early postnatal period resulted in growth restriction, severe pancreatic degeneration and early death. These findings are consistent with Arf4 playing a critical role in endomembrane trafficking, particularly in the pancreas, but not in ciliary function.

  7. Genome-Wide Identification, Phylogeny, and Expression Analysis of ARF Genes Involved in Vegetative Organs Development in Switchgrass

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    Jianli Wang

    2018-01-01

    Full Text Available Auxin response factors (ARFs have been reported to play vital roles during plant growth and development. In order to reveal specific functions related to vegetative organs in grasses, an in-depth study of the ARF gene family was carried out in switchgrass (Panicum virgatum L., a warm-season C4 perennial grass that is mostly used as bioenergy and animal feedstock. A total of 47 putative ARF genes (PvARFs were identified in the switchgrass genome (2n = 4x = 36, 42 of which were anchored to the seven pairs of chromosomes and found to be unevenly distributed. Sixteen PvARFs were predicted to be potential targets of small RNAs (microRNA160 and 167. Phylogenetically speaking, PvARFs were divided into seven distinct subgroups based on the phylogeny, exon/intron arrangement, and conserved motif distribution. Moreover, 15 pairs of PvARFs have different temporal-spatial expression profiles in vegetative organs (2nd, 3rd, and 4th internode and leaves, which implies that different PvARFs have specific functions in switchgrass growth and development. In addition, at least 14 pairs of PvARFs respond to naphthylacetic acid (NAA treatment, which might be helpful for us to study on auxin response in switchgrass. The comprehensive analysis, described here, will facilitate the future functional analysis of ARF genes in grasses.

  8. Activation and amplification of c-Ki-ras in a chemically induced transplantable human pancreas carcinoma

    International Nuclear Information System (INIS)

    Parsa, I.; Maheshwari, K.K.

    1986-01-01

    Increasing evidence suggests that carcinogenesis is associated with the stepwise activation of oncogenes. The c-Ki-ras oncogene has been demonstrated in several human solid tumors and is shown to be amplified in tumor cell lines. The authors have probed endonuclease cleaved human pancreas (HP) DNAs and DNAs from an in vitro induced transplantable human pancreas carcinoma (HP-T1) for the presence and/or amplification of c-Ki-ras oncogene. The DNAs were cleaved with BamHI, BgIII, EcoRI, HhaI, HinfI, KpnI, PSTI, PvuII, SaII, SstI, TaqI or XbaI and were subjected to Southern blot analysis using 32 P-labelled EcoRI fragments from HiHi3 clone. The hybridization profiles were similar in both DNAs when digested with BamHI, BgIII, HinfI, KpnI, SaII, SstI, or TaqI. The EcoRI cleaved DNAs from HP and HP-T1 revealed two hybridizing fragments of 6.8 and 3.0 kbp. The 3.0 kbp fragments in DNA from HP-T1 showed more than a 100 folds enhancement as compared to that of HP. The 6.8 hybridizing fragments also appeared 10 fold greater in HP-T1 DNA. Similar enhancements were also present in HP-T1 DNA cleaved with PstI and PvuII. Preliminary results from comparison of poly(A) + RNAs, prepared from total HP and HP-T1 RNAs, by Northern blot analysis using the same probe reflect similar enhancement in RNA from transplantable pancreas carcinoma

  9. Mutant PIK3CA Induces EMT in a Cell Type Specific Manner.

    Directory of Open Access Journals (Sweden)

    Divya Bhagirath

    Full Text Available Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various in-vitro and in-vivo analyses. Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44+/CD24low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent

  10. RAS Insight

    Science.gov (United States)

    David Heimbrook, now CEO of the Frederick National Laboratory for Cancer Research, played a major role in a large pharma as it tried to develop an anti-RAS drug. Lessons from that failure inform the RAS Initiative today.

  11. Evidence implicating the Ras pathway in multiple CD28 costimulatory functions in CD4+ T cells.

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    Sujit V Janardhan

    Full Text Available CD28 costimulation is a critical event in the full activation of CD4(+ T cells that augments cytokine gene transcription, promotes cytokine mRNA stability, prevents induction of anergy, increases cellular metabolism, and increases cell survival. However, despite extensive biochemical analysis of the signaling events downstream of CD28, molecular pathways sufficient to functionally replace the diverse aspects of CD28-mediated costimulation in normal T cells have not been identified. Ras/MAPK signaling is a critical pathway downstream of T cell receptor stimulation, but its role in CD28-mediated costimulation has been controversial. We observed that physiologic CD28 costimulation caused a relocalization of the RasGEF RasGRP to the T cell-APC interface by confocal microscopy. In whole cell biochemical analysis, CD28 cross-linking with either anti-CD28 antibody or B7.1-Ig augmented TCR-induced Ras activation. To determine whether Ras signaling was sufficient to functionally mimic CD28 costimulation, we utilized an adenoviral vector encoding constitutively active H-Ras (61L to transduce normal, Coxsackie-Adenovirus Receptor (CAR transgenic CD4(+ T cells. Like costimulation via CD28, active Ras induced AKT, JNK and ERK phosphorylation. In addition, constitutive Ras signaling mimicked the ability of CD28 to costimulate IL-2 protein secretion, prevent anergy induction, increase glucose uptake, and promote cell survival. Importantly, we also found that active Ras mimicked the mechanism by which CD28 costimulates IL-2 production: by increasing IL-2 gene transcription, and promoting IL-2 mRNA stability. Finally, active Ras was able to induce IL-2 production when combined with ionomycin stimulation in a MEK-1-dependent fashion. Our results are consistent with a central role for Ras signaling in CD28-mediated costimulation.

  12. A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells

    International Nuclear Information System (INIS)

    Mouron, Silvana Andrea; Grillo, Claudia Alejandra; Dulout, Fernando Noel; Golijow, Carlos Daniel

    2004-01-01

    Cadmium (Cd) is a toxic heavy metal of continuing occupational and environmental concern with a wide variety of adverse effects. Several studies have shown that cadmium produces DNA strand breaks, DNA-protein cross-links, oxidative DNA damage, chromosomal aberrations, dysregulation of gene expression resulting in enhanced proliferation, depressed apoptosis and/or altered DNA repair. This study was undertaken to investigate the ability of cadmium chloride (CdCl 2 ) and cadmium sulphate (CdSO 4 ) to induce point mutations in codon 12 of the K-ras protooncogene assessed by polymerase chain reaction-single strand conformation polymorphisms (PCR-SSCP) and RFLP-enriched PCR methods. Also their genotoxic effects were analyzed by the comet assay and sister chromatid exchanges test. The human lung fibroblast cell line MRC-5 was used for the experiments. Sister chromatid exchanges assay (SCEs) frequencies were significantly increased in cells exposed to cadmium salts in relation to controls (p < 0.001). Despite the slow increment observed in the three comet parameters considered when cells were treated with cadmium chloride, significant differences between groups were only found in the variable comet moment (CM) (p < 0.005). On the other hand, when cells were exposed to cadmium sulphate, the Kruskal-Wallis test showed highly significant differences between groups for migration, tail moment and comet moment parameters (p < 0.001). Nevertheless, a null or weak point mutation induction in K-ras protooncogene was detected using polymerase chain reaction-low ionic strength-single strand conformation polymorphisms (PCR-LIS-SSCP) and RFLP-enriched PCR methods when cells were treated with cadmium salts. Thus, inorganic cadmium produces genotoxicity in human lung fibroblast MRC-5 cells, in the absence of significant point mutation of the K-ras gene

  13. Improving plasma resistance and lowering roughness in an ArF photoresist by adding a chemical reaction inhibitor

    International Nuclear Information System (INIS)

    Jinnai, Butsurin; Uesugi, Takuji; Koyama, Koji; Samukawa, Seiji; Kato, Keisuke; Yasuda, Atsushi; Maeda, Shinichi; Momose, Hikaru

    2010-01-01

    Major challenges associated with 193 nm lithography using an ArF photoresist are low plasma resistance and roughness formation in the ArF photoresist during plasma processes. We have previously found decisive factors affecting the plasma resistance and roughness formation in an ArF photoresist: plasma resistance is determined by UV/VUV radiation, and roughness formation is dominated by chemical reactions. In this study, based on our findings on the interaction between plasma radiation species and ArF photoresist polymers, we proposed an ArF photoresist with a chemical reaction inhibitor, which can trap reactive species from the plasma, and characterized the performances of the resultant ArF photoresist through neutral beam experiments. Hindered amine light stabilizers, i.e. 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (HO-TEMPO), were used as the chemical reaction inhibitor. Etching rates of the ArF photoresist films were not dependent on the HO-TEMPO content in the irradiations without chemical reactions or under UV/VUV radiation. However, in the irradiation with chemical reactions, the etching rates of the ArF photoresist films decreased as the HO-TEMPO content increased. In addition, the surface roughness decreased with the increase in the additive amount of chemical reaction inhibitor. According to FTIR analysis, a chemical reaction inhibitor can inhibit the chemical reactions in ArF photoresist films through plasma radicals. These results indicate that a chemical reaction inhibitor is effective against chemical reactions, resulting in improved plasma resistance and less roughness in an ArF photoresist. These results also support our suggested mechanism of plasma resistance and roughness formation in an ArF photoresist.

  14. Non-Serotonergic Neurotoxicity by MDMA (Ecstasy in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells.

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    Dina Popova

    Full Text Available 3,4-methylenedioxymethamphetamine (MDMA; ecstasy is a commonly abused recreational drug that causes neurotoxic effects in both humans and animals. The mechanism behind MDMA-induced neurotoxicity is suggested to be species-dependent and needs to be further investigated on the cellular level. In this study, the effects of MDMA in neuronally differentiated P19 mouse embryonal carcinoma cells have been examined. MDMA produces a concentration-, time- and temperature-dependent toxicity in differentiated P19 neurons, as measured by intracellular MTT reduction and extracellular LDH activity assays. The P19-derived neurons express both the serotonin reuptake transporter (SERT, that is functionally active, and the serotonin metabolizing enzyme monoamine oxidase A (MAO-A. The involvement of these proteins in the MDMA-induced toxicity was investigated by a pharmacological approach. The MAO inhibitors clorgyline and deprenyl, and the SERT inhibitor fluoxetine, per se or in combination, were not able to mimic the toxic effects of MDMA in the P19-derived neurons or block the MDMA-induced cell toxicity. Oxidative stress has been implicated in MDMA-induced neurotoxicity, but pre-treatment with the antioxidants α-tocopherol or N-acetylcysteine did not reveal any protective effects in the P19 neurons. Involvement of mitochondria in the MDMA-induced cytotoxicity was also examined, but MDMA did not alter the mitochondrial membrane potential (ΔΨm in the P19 neurons. We conclude that MDMA produce a concentration-, time- and temperature-dependent neurotoxicity and our results suggest that the mechanism behind MDMA-induced toxicity in mouse-derived neurons do not involve the serotonergic system, oxidative stress or mitochondrial dysfunction.

  15. Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells.

    Science.gov (United States)

    Popova, Dina; Forsblad, Andréas; Hashemian, Sanaz; Jacobsson, Stig O P

    2016-01-01

    3,4-methylenedioxymethamphetamine (MDMA; ecstasy) is a commonly abused recreational drug that causes neurotoxic effects in both humans and animals. The mechanism behind MDMA-induced neurotoxicity is suggested to be species-dependent and needs to be further investigated on the cellular level. In this study, the effects of MDMA in neuronally differentiated P19 mouse embryonal carcinoma cells have been examined. MDMA produces a concentration-, time- and temperature-dependent toxicity in differentiated P19 neurons, as measured by intracellular MTT reduction and extracellular LDH activity assays. The P19-derived neurons express both the serotonin reuptake transporter (SERT), that is functionally active, and the serotonin metabolizing enzyme monoamine oxidase A (MAO-A). The involvement of these proteins in the MDMA-induced toxicity was investigated by a pharmacological approach. The MAO inhibitors clorgyline and deprenyl, and the SERT inhibitor fluoxetine, per se or in combination, were not able to mimic the toxic effects of MDMA in the P19-derived neurons or block the MDMA-induced cell toxicity. Oxidative stress has been implicated in MDMA-induced neurotoxicity, but pre-treatment with the antioxidants α-tocopherol or N-acetylcysteine did not reveal any protective effects in the P19 neurons. Involvement of mitochondria in the MDMA-induced cytotoxicity was also examined, but MDMA did not alter the mitochondrial membrane potential (ΔΨm) in the P19 neurons. We conclude that MDMA produce a concentration-, time- and temperature-dependent neurotoxicity and our results suggest that the mechanism behind MDMA-induced toxicity in mouse-derived neurons do not involve the serotonergic system, oxidative stress or mitochondrial dysfunction.

  16. Inhibition of carcinogen induced c-Ha-ras and c-fos proto-oncogenes expression by dietary curcumin

    Directory of Open Access Journals (Sweden)

    Lipigorngoson Suwiwek

    2001-01-01

    Full Text Available Abstract Background We investigated the chemopreventive action of dietary curcumin on 7,12-dimethylbenz(aanthracene (DMBA-initiated and 12,0-tetradecanoylphorbol-13-acetate (TPA-promoted skin tumor formation in Swiss albino mice. Curcumin, a yellow coloring matter isolated from roots of Curcuma longa Linn, is a phenolic compound possessing antioxidant, free radical scavenger, and antiinflammatory properties. It has been shown by previously reported work that TPA-induced skin tumors were inhibited by topical application of curcumin, and curcumin has been shown to inhibit a variety of biological activities of TPA. Topical application of curcumin was reported to inhibit TPA-induced c-fos, c-jun and c-myc gene expression in mouse skin. This paper reports the effects of orally administered curcumin, which was consumed as a dietary component at concentrations of 0.2 % or 1 %, in ad libitum feeding. Results Animals in which tumors had been initiated with DMBA and promoted with TPA experienced significantly fewer tumors and less tumor volume if they ingested either 0.2% or 1% curcumin diets. Also, the dietary consumption of curcumin resulted in a significantly decreased expression of ras and fos proto-oncogenes in the tumorous skin, as measured by enhanced chemiluminesence Western blotting detection system (Amersham. Conclusions Whereas earlier work demonstrated that topical application of curcumin to mouse skin inhibited TPA-induced expression of c-fos, c-jun and c-myc oncogenes, our results are the first to show that orally consumed curcumin significantly inhibited DMBA- and TPA-induced ras and fos gene expression in mouse skin.

  17. Inhibition of carcinogen induced c-Ha-ras and c-fos proto-oncogenes expression by dietary curcumin

    International Nuclear Information System (INIS)

    Limtrakul, Porn-ngarm; Anuchapreeda, Songyot; Lipigorngoson, Suwiwek; Dunn, Floyd W

    2001-01-01

    We investigated the chemopreventive action of dietary curcumin on 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12,0-tetradecanoylphorbol-13-acetate (TPA)-promoted skin tumor formation in Swiss albino mice. Curcumin, a yellow coloring matter isolated from roots of Curcuma longa Linn, is a phenolic compound possessing antioxidant, free radical scavenger, and antiinflammatory properties. It has been shown by previously reported work that TPA-induced skin tumors were inhibited by topical application of curcumin, and curcumin has been shown to inhibit a variety of biological activities of TPA. Topical application of curcumin was reported to inhibit TPA-induced c-fos, c-jun and c-myc gene expression in mouse skin. This paper reports the effects of orally administered curcumin, which was consumed as a dietary component at concentrations of 0.2 % or 1 %, in ad libitum feeding. Animals in which tumors had been initiated with DMBA and promoted with TPA experienced significantly fewer tumors and less tumor volume if they ingested either 0.2% or 1% curcumin diets. Also, the dietary consumption of curcumin resulted in a significantly decreased expression of ras and fos proto-oncogenes in the tumorous skin, as measured by enhanced chemiluminesence Western blotting detection system (Amersham). Whereas earlier work demonstrated that topical application of curcumin to mouse skin inhibited TPA-induced expression of c-fos, c-jun and c-myc oncogenes, our results are the first to show that orally consumed curcumin significantly inhibited DMBA- and TPA-induced ras and fos gene expression in mouse skin

  18. RasGRP3 regulates the migration of glioma cells via interaction with Arp3

    Science.gov (United States)

    Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Poisson, Laila M.; Blumberg, Peter M.; Brodie, Chaya

    2015-01-01

    Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM. PMID:25682201

  19. Sustained oxidative stress causes late acute renal failure via duplex regulation on p38 MAPK and Akt phosphorylation in severely burned rats.

    Directory of Open Access Journals (Sweden)

    Yafei Feng

    Full Text Available BACKGROUND: Clinical evidence indicates that late acute renal failure (ARF predicts high mortality in severely burned patients but the pathophysiology of late ARF remains undefined. This study was designed to test the hypothesis that sustained reactive oxygen species (ROS induced late ARF in a severely burned rat model and to investigate the signaling mechanisms involved. MATERIALS AND METHODS: Rats were exposed to 100°C bath for 15 s to induce severe burn injury (40% of total body surface area. Renal function, ROS generation, tubular necrosis and apoptosis, and phosphorylation of MAPK and Akt were measured during 72 hours after burn. RESULTS: Renal function as assessed by serum creatinine and blood urea nitrogen deteriorated significantly at 3 h after burn, alleviated at 6 h but worsened at 48 h and 72 h, indicating a late ARF was induced. Apoptotic cells and cleavage caspase-3 in the kidney went up slowly and turned into significant at 48 h and 72 h. Tubular cell ROS production shot up at 6 h and continuously rose during the 72-h experiment. Scavenging ROS with tempol markedly attenuated tubular apoptosis and renal dysfunction at 72 h after burn. Interestingly, renal p38 MAPK phosphorylation elevated in a time dependent manner whereas Akt phosphorylation increased during the first 24 h but decreased at 48 h after burn. The p38 MAPK specific inhibitor SB203580 alleviated whereas Akt inhibitor exacerbated burn-induced tubular apoptosis and renal dysfunction. Furthermore, tempol treatment exerted a duplex regulation through inhibiting p38 MAPK phosphorylation but further increasing Akt phosphorylation at 72 h postburn. CONCLUSIONS: These results demonstrate that sustained renal ROS overproduction induces continuous tubular cell apoptosis and thus a late ARF at 72 h after burn in severely burned rats, which may result from ROS-mediated activation of p38 MAPK but a late inhibition of Akt phosphorylation.

  20. Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors

    Energy Technology Data Exchange (ETDEWEB)

    Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas; Irving, Thomas C.; Bilsel, Osman; Lambright, David G.

    2018-01-01

    Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.

  1. High miR156 Expression Is Required for Auxin-Induced Adventitious Root Formation via MxSPL26 Independent of PINs and ARFs in Malus xiaojinensis

    Directory of Open Access Journals (Sweden)

    Xiaozhao Xu

    2017-06-01

    Full Text Available Adventitious root formation is essential for the vegetative propagation of perennial woody plants. During the juvenile-to-adult phase change mediated by the microRNA156 (miR156, the adventitious rooting ability decreases dramatically in many species, including apple rootstocks. However, the mechanism underlying how miR156 affects adventitious root formation is unclear. In the present study, we showed that in the presence of the synthetic auxin indole-3-butyric acid (IBA, semi-lignified leafy cuttings from juvenile phase (Mx-J and rejuvenated (Mx-R Malus xiaojinensis trees exhibited significantly higher expression of miR156, PIN-FORMED1 (PIN1, PIN10, and rootless concerning crown and seminal roots-like (RTCS-like genes, thus resulting in higher adventitious rooting ability than those from adult phase (Mx-A trees. However, the expression of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE26 (SPL26 and some auxin response factor (ARF gene family members were substantially higher in Mx-A than in Mx-R cuttings. The expression of NbRTCS-like but not NbPINs and NbARFs varied with miR156 expression in tobacco (Nicotiana benthamiana plants transformed with 35S:MdMIR156a6 or 35S:MIM156 constructs. Overexpressing the miR156-resistant MxrSPL genes in tobacco confirmed the involvement of MxSPL20, MxSPL21&22, and MxSPL26 in adventitious root formation. Together, high expression of miR156 was necessary for auxin-induced adventitious root formation via MxSPL26, but independent of MxPINs and MxARFs expression in M. xiaojinensis leafy cuttings.

  2. Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

    Science.gov (United States)

    Han, Chang Woo; Jeong, Mi Suk; Jang, Se Bok

    2017-09-16

    Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. The radiosensitivity of human keratinocytes: influence of activated c-H-ras oncogene expression and tumorigenicity

    International Nuclear Information System (INIS)

    Mendonca, M.S.; Redpath, J.L.; Stanbridge, E.J.

    1991-01-01

    The authors investigated γ-ray sensitivity of several activated c-H-ras (EJ) containing clones established after transfection of the spontaneously immortalized non-tumorigenic human keratinocyte cell line HaCaT. The clones were grouped according to tumorigenic potential after subcutaneous injection into nude mice, and fell into three classes: Class I clones A-4 and I-6 are non-tumorigenic and express very low levels of c-H-ras mRNA and no mutated ras protein (p 21 ); Class II clones I-5 and I-7 grow to large (benign) epidermal cysts, express intermediate to high c-H-ras mRNA and variable levels of mutated ras p 21 protein with clone I-5 expressing little and clone I-7 expressing high levels of p 21 ; Class III clones II-3 and II-4 grow to solid squamous cell carcinomas, express high c-H-ras mRNA and high level of mutated p 21 ras protein similar to clone I-7. Comparison of single-hit multitarget or linear-quadratic survival curve parameters, and survival at 2Gy (S 2 ) indicate no general correlation with either activated c-H-ras expression level or tumorigenic potential, and increased radioresistance. (author)

  4. Analysis of beta-catenin, Ki-ras, and microsatellite stability in azoxymethane-induced colon tumors of BDIX/Orl Ico rats

    DEFF Research Database (Denmark)

    Sørensen, Nanna Møller; Kobaek-Larsen, Morten; Bonne, Anita

    2003-01-01

    The aim of the study reported here was to investigate whether the azoxymethane (AOM)-induced colon cancer rat model mimics the human situation with regard to microsatellite stability, changes in expression of beta-catenin, and/or changes in the sequence of the proto-oncogene Ki-ras. Colon cancer ...

  5. Oncogenic N-Ras Stimulates SRF-Mediated Transactivation via H3 Acetylation at Lysine 9

    Directory of Open Access Journals (Sweden)

    Sun-Ju Yi

    2018-01-01

    Full Text Available Signal transduction pathways regulate the gene expression by altering chromatin dynamics in response to mitogens. Ras proteins are key regulators linking extracellular stimuli to a diverse range of biological responses associated with gene regulation. In mammals, the three ras genes encode four Ras protein isoforms: H-Ras, K-Ras4A, K-Ras4B, and N-Ras. Although emerging evidence suggests that Ras isoforms differentially regulate gene expressions and are functionally nonredundant, the mechanisms underlying Ras specificity and Ras signaling effects on gene expression remain unclear. Here, we show that oncogenic N-Ras acts as the most potent regulator of SRF-, NF-κB-, and AP-1-dependent transcription. N-Ras-RGL2 axis is a distinct signaling pathway for SRF target gene expression such as Egr1 and JunB, as RGL2 Ras binding domain (RBD significantly impaired oncogenic N-Ras-induced SRE activation. By monitoring the effect of Ras isoforms upon the change of global histone modifications in oncogenic Ras-overexpressed cells, we discovered that oncogenic N-Ras elevates H3K9ac/H3K23ac levels globally in the chromatin context. Importantly, chromatin immunoprecipitation (ChIP assays revealed that H3K9ac is significantly enriched at the promoter and coding regions of Egr1 and JunB. Collectively, our findings define an undocumented role of N-Ras in modulating of H3 acetylation and in gene regulation.

  6. CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling

    OpenAIRE

    Manara, Maria Cristina; Terracciano, Mario; Mancarella, Caterina; Sciandra, Marika; Guerzoni, Clara; Pasello, Michela; Grilli, Andrea; Zini, Nicoletta; Picci, Piero; Colombo, Mario P.; Morrione, Andrea; Scotlandi, Katia

    2016-01-01

    CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD9...

  7. Comparison of KrF and ArF excimer laser treatment of biopolymer surface

    Energy Technology Data Exchange (ETDEWEB)

    Michaljaničová, I. [Department of Solid State Engineering, University of Chemistry and Technology, 166 28 Prague (Czech Republic); Slepička, P., E-mail: petr.slepicka@vscht.cz [Department of Solid State Engineering, University of Chemistry and Technology, 166 28 Prague (Czech Republic); Heitz, J.; Barb, R.A. [Institute of Applied Physics, Johannes Kepler University Linz, A-4040 Linz (Austria); Sajdl, P. [Department of Power Engineering, University of Chemistry and Technology, 166 28 Prague (Czech Republic); Švorčík, V. [Department of Solid State Engineering, University of Chemistry and Technology, 166 28 Prague (Czech Republic)

    2015-06-01

    Highlights: • The influence of ArF and KrF laser on biopolymer surface was determined. • ArF laser acts predominantly on biopolymer surface. • PHB roughness is increased similarly for both applied wavelengths. • Roughness of nanostructures can be precisely controlled. • ArF laser introduces nitrogen on PHB surface. - Abstract: The goal of this work was the investigation of the impact of two different excimer lasers on two biocompatible and biodegradable polymers (poly-L-lactide and poly hydroxybutyrate). Both polymers find usage in medical and pharmaceutical fields. The polymers were modified by KrF and ArF excimer lasers. Subsequently the impact on surface morphology, surface chemistry changes, and thermal properties was studied by means of confocal and AFM microscopy, FTIR and XPS spectroscopy and DSC calorimetry. Under the same conditions of laser treatment it was observed that ArF laser causes more significant changes on surface chemistry, surface morphology and pattern formation on the polymers under investigation. The data obtained in this work can be used for a wide range of possible applications, in tissue engineering or in combination with metallization in electronics, e.g. for biosensors.

  8. Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains

    DEFF Research Database (Denmark)

    Willumsen, B M; Papageorge, A G; Hubbert, N

    1985-01-01

    or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences...... that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids...... that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor...

  9. New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling.

    Science.gov (United States)

    Erickson, Keesha E; Rukhlenko, Oleksii S; Posner, Richard G; Hlavacek, William S; Kholodenko, Boris N

    2018-03-05

    RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisition of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Lead acetate induces EGFR activation upstream of SFK and PKCα linkage to the Ras/Raf-1/ERK signaling

    International Nuclear Information System (INIS)

    Wang, C.-Y.; Wang, Y.-T.; Tzeng, D.-W.; Yang, J.-L.

    2009-01-01

    Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC → ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1 S338 and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Goe6976 or depleting PKCα using specific small interfering RNA blocked Pb induction of Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKCα, Ras-GTP, phospho-Raf-1 S338 and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKCα activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKCα activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKCα and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade

  11. The role of hypoxia inducible factor-1 alpha in bypassing oncogene-induced senescence.

    Directory of Open Access Journals (Sweden)

    Mehtap Kilic Eren

    Full Text Available Oncogene induced senescence (OIS is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR, senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on RasV12-induced senescence in human diploid fibroblasts (HDFs. We showed here that hypoxia prevents execution of oncogene induced senescence (OIS, through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21CIP1 and p16INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α. In addition, hypoxia also decreased marks of H-RasV12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21CIP1 but not p16INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.

  12. The Role of Conserved Waters in Conformational Transitions of Q61H K-ras

    Science.gov (United States)

    Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Gorfe, Alemayehu A.

    2012-01-01

    To investigate the stability and functional role of long-residence water molecules in the Q61H variant of the signaling protein K-ras, we analyzed all available Ras crystal structures and conformers derived from a series of independent explicit solvent molecular dynamics (MD) simulations totaling 1.76 µs. We show that the protein samples a different region of phase space in the presence and absence of several crystallographically conserved and buried water molecules. The dynamics of these waters is coupled with the local as well as the global motions of the protein, in contrast to less buried waters whose exchange with bulk is only loosely coupled with the motion of loops in their vicinity. Aided by two novel reaction coordinates involving the distance (d) between the Cα atoms of G60 at switch 2 and G10 at the P-loop and the N-Cα-C-O dihedral (ξ) of G60, we further show that three water molecules located in lobe1, at the interface between the lobes and at lobe2, are involved in the relative motion of residues at the two lobes of Q61H K-ras. Moreover, a d/ξ plot classifies the available Ras x-ray structures and MD-derived K-ras conformers into active GTP-, intermediate GTP-, inactive GDP-bound, and nucleotide-free conformational states. The population of these states and the transition between them is modulated by water-mediated correlated motions involving the functionally critical switch 2, P-loop and helix 3. These results suggest that water molecules act as allosteric ligands to induce a population shift among distinct switch 2 conformations that differ in effector recognition. PMID:22359497

  13. Effect of saline loading on uranium-induced acute renal failure in rats

    International Nuclear Information System (INIS)

    Hishida, A.; Yonemura, K.; Ohishi, K.; Yamada, M.; Honda, N.

    1988-01-01

    Studies were performed to examine the effect of saline loading on uranium-induced acute renal failure (ARF) in rats. Forty-eight hours after the i.v. injection of uranyl acetate (UA, 5 mg/kg), inulin clearance rate (Cin) decreased to approximately 43% of the control value in water drinking rats (P less than 0.005). Animals receiving continuous isotonic saline infusion following UA showed higher urine flow and Cin (60% of control, P less than 0.01), and lessened intratubular cast formation when compared with water-drinking ARF rats. A short-term saline infusion following UA did not attenuate the decline in Cin (43% of control). An inverse relationship was found between Cin and the number of casts (r = -0.75, P less than 0.01). Multiple regression analysis showed that standardized partial regression coefficient is statistically significant between Cin and cast formation (-0.69, P less than 0.05), but not between Cin and tubular necrosis (-0.07, P greater than 0.05). Renin depletion caused by DOCA plus saline drinking did not attenuate the decline in Cin in ARF (47% of control). No significant difference was found in urinary uranium excretion between water-drinking and saline-infused ARF rats. The findings suggest that continuous saline infusion following UA attenuates the decline in Cin in ARF rats; and that this beneficial effect of saline loading is associated with lessened cast formation rather than with suppressed renin-angiotensin activity or enhanced urinary-uranium excretion

  14. The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis

    Czech Academy of Sciences Publication Activity Database

    Evangelou, K.; Bartkova, J.; Kotsinas, A.; Pateras, I.S.; Liontos, M.; Velimezi, G.; Košař, Martin; Liloglou, T.; Trougakos, I.P.; Dyrskjot, L.; Andersen, C.J.; Papaioannou, G.; Drosos, A.; Papafotiou, M.; Hodný, Zdeněk; Sosa-Pineda, B.; Wu, X.R.; Klinakis, A.; Orntoft, T.; Lukas, J.; Bartek, Jiří; Gorgoulis, V.G.

    2013-01-01

    Roč. 20, č. 11 (2013), s. 1485-1497 ISSN 1350-9047 Institutional support: RVO:68378050 Keywords : ARF * carcinogenesis * DDR * E2F1 * p16(INK4A) Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.385, year: 2013

  15. RAS - Target Identification - Informatics

    Science.gov (United States)

    The RAS Informatics lab group develops tools to track and analyze “big data” from the RAS Initiative, as well as analyzes data from external projects. By integrating internal and external data, this group helps improve understanding of RAS-driven cancers.

  16. Residues in the alternative reading frame tumor suppressor that influence its stability and p53-independent activities

    International Nuclear Information System (INIS)

    Tommaso, Anne di; Hagen, Jussara; Tompkins, Van; Muniz, Viviane; Dudakovic, Amel; Kitzis, Alain; Ladeveze, Veronique; Quelle, Dawn E.

    2009-01-01

    The Alternative Reading Frame (ARF) protein suppresses tumorigenesis through p53-dependent and p53-independent pathways. Most of ARF's anti-proliferative activity is conferred by sequences in its first exon. Previous work showed specific amino acid changes occurred in that region during primate evolution, so we programmed those changes into human p14ARF to assay their functional impact. Two human p14ARF residues (Ala 14 and Thr 31 ) were found to destabilize the protein while two others (Val 24 and Ala 41 ) promoted more efficient p53 stabilization and activation. Despite those effects, all modified p14ARF forms displayed robust p53-dependent anti-proliferative activity demonstrating there are no significant biological differences in p53-mediated growth suppression associated with simian versus human p14ARF residues. In contrast, p53-independent p14ARF function was considerably altered by several residue changes. Val 24 was required for p53-independent growth suppression whereas multiple residues (Val 24 , Thr 31 , Ala 41 and His 60 ) enabled p14ARF to block or reverse the inherent chromosomal instability of p53-null MEFs. Together, these data pinpoint specific residues outside of established p14ARF functional domains that influence its expression and signaling activities. Most intriguingly, this work reveals a novel and direct role for p14ARF in the p53-independent maintenance of genomic stability.

  17. Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis.

    Science.gov (United States)

    Overmeyer, Jean H; Kaul, Aparna; Johnson, Erin E; Maltese, William A

    2008-06-01

    Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.

  18. Ras-Induced and Extracellular Signal-Regulated Kinase 1 and 2 Phosphorylation-Dependent Isomerization of Protein Tyrosine Phosphatase (PTP)-PEST by PIN1 Promotes FAK Dephosphorylation by PTP-PEST ▿

    Science.gov (United States)

    Zheng, Yanhua; Yang, Weiwei; Xia, Yan; Hawke, David; Liu, David X.; Lu, Zhimin

    2011-01-01

    Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression. PMID:21876001

  19. Inhibitors of Ras-SOS Interactions.

    Science.gov (United States)

    Lu, Shaoyong; Jang, Hyunbum; Zhang, Jian; Nussinov, Ruth

    2016-04-19

    Activating Ras mutations are found in about 30 % of human cancers. Ras activation is regulated by guanine nucleotide exchange factors, such as the son of sevenless (SOS), which form protein-protein interactions (PPIs) with Ras and catalyze the exchange of GDP by GTP. This is the rate-limiting step in Ras activation. However, Ras surfaces lack any evident suitable pockets where a molecule might bind tightly, rendering Ras proteins still 'undruggable' for over 30 years. Among the alternative approaches is the design of inhibitors that target the Ras-SOS PPI interface, a strategy that is gaining increasing recognition for treating Ras mutant cancers. Herein we focus on data that has accumulated over the past few years pertaining to the design of small-molecule modulators or peptide mimetics aimed at the interface of the Ras-SOS PPI. We emphasize, however, that even if such Ras-SOS therapeutics are potent, drug resistance may emerge. To counteract this development, we propose "pathway drug cocktails", that is, drug combinations aimed at parallel (or compensatory) pathways. A repertoire of classified cancer, cell/tissue, and pathway/protein combinations would be beneficial toward this goal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. The Prognostic Impact of K-RAS Mutations in Adult Acute Myeloid Leukemia Patients Treated with High Dose Cytarabine

    International Nuclear Information System (INIS)

    Ahmad, E.I.; Gawish, H.H.; Al-Azizi, N.M.A.; El-Hefni, A.M.

    2009-01-01

    Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML). However there is still controversy about its clinical relevance on the treatment outcome of this leukemia. Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C) used in post induction consolidation chemotherapy in adult AML patients. Patients and Methods: The study comprised 71de novo AML patients with a male: Female ratio of 1.4: 1; their ages ranged from 21-59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G banding and K-RAS mutation detection using realtime PCR. The patients were randomized into 2 groups (gps) according to the ara-C dose used in consolidation treatment, HDAC gp receiving 400 mg ara-C and LDAC gp receiving 100 mg ara-C. They were followed over a period of 5 years. Results: Mutations in the K-RAS gene (mutRAS) were detected in 23 patients (32%) with the remaining 48 patients (68%) having wild type RAS (wtRAS). Blast cell percentage was significantly lower in mutRAS compared to wtRAS patients (p=<0.001). The M4 subtype of AML and cases with Inv 16 showed significantly higher frequencies in mutRAS compared to wtRAS patients, (p=0.015, 0.003, respectively). The patients were followed up for a median of 43 months (range 11-57 months). There was no significant difference in overall survival (OS) between mutRAS and wtRAS patients (p=0.326). Within the mutRAS patients treated with HDAC, cumulative OS was significantly higher than those treated with LDAC (p=0.001). This was not the case in the wtRAS group (p=0.285). There was no significant difference in disease The Prognostic Impact of K-RAS Mutations in Adult Acute Myeloid Leukemia Patients Treated with High Dose

  1. The prognostic impact of K-RAS mutations in adult acute myeloid leukemia patients treated with high-dose cytarabine

    Directory of Open Access Journals (Sweden)

    Ahmad EI

    2011-07-01

    Full Text Available Ebtesam I Ahmad, Heba H Gawish, Nashwa MA Al Azizi, Ashraf M ElhefniClinical Pathology Department, Hematology and Oncology Unit of Internal Medicine Department, Faculty of Medicine, Zagazig University, Sharkia, EgyptBackground: Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML. However, little is known about its clinical relevance in the treatment outcome for this leukemia.Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C used in postinduction consolidation chemotherapy in adult AML patients.Patients and methods: The study comprised of 71 de novo AML patients with male/female ratio 1.4:1; their ages ranged from 21–59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G-banding (Giemsa staining, and K-RAS mutation detection using real-time polymerase chain reaction. The patients were randomized into two groups according to the ara-C dose used in consolidation treatment, the high the dose ara-C (HDAC group receiving 400 mg ara-C and-low-dose ara-C (LDAC group receiving 100 mg ara-C; they were followed over a period of five years.Results: Mutations in the K-RAS gene (mutRAS were detected in 23 patients (32% with the remaining 48 patients (68% having wild-type RAS (wtRAS. The percent of blast cells was significantly lower in mutRAS compared to wtRAS patients (P ≤ 0.001 while M4 subtype of AML and Inv(16 frequencies were significantly higher in mutRAS compared to wtRAS patients (P = 0.015 and (P = 0.003, respectively. The patients were followed up for a median of 43 months (range 11–57 months. There was no significant difference in overall survival (OS between mutRAS and wtRAS (P = 0.326. Within the mutRAS

  2. Driver or passenger effects of augmented c-Myc and Cdc20 in gliomagenesis.

    Science.gov (United States)

    Ji, Ping; Zhou, Xinhui; Liu, Qun; Fuller, Gregory N; Phillips, Lynette M; Zhang, Wei

    2016-04-26

    Cdc20 and c-Myc are commonly overexpressed in a broad spectrum of cancers, including glioblastoma (GBM). Despite this clear association, whether c-Myc and Cdc20 overexpression is a driver or passenger event in gliomagenesis remains unclear. Both c-Myc and Cdc20 induced the proliferation of primary glial progenitor cells. c-Myc also promoted the formation of soft agar anchorage-independent colonies. In the RCAS/Ntv-a glia-specific transgenic mouse model, c-Myc increased the GBM incidence from 19.1% to 47.4% by 12 weeks of age when combined with kRas and Akt3 in Ntv-a INK4a-ARF (also known as CDKN2A)-null mice. In contrast, Cdc20 decreased the GBM incidence from 19.1% to 9.1%. Moreover, cell differentiation was modulated by c-Myc in kRas/Akt3-induced GBM on the basis of Nestin/GFAP expression (glial progenitor cell differentiation), while Cdc20 had no effect on primary glial progenitor cell differentiation. We used glial progenitor cells from Ntv-a newborn mice to evaluate the role of c-Myc and Cdc20 in the proliferation and transformation of GBM in vitro and in vivo. We further determined whether c-Myc and Cdc20 have a driver or passenger role in GBM development using kRas/Akt3 signals in a RCAS/Ntv-a mouse model. These results suggest that the driver or passenger of oncogene signaling is dependent on cellular status. c-Myc is a driver when combined with kRas/Akt3 oncogenic signals in gliomagenesis, whereas Cdc20 overexpression is a passenger. Inhibition of cell differentiation of c-Myc may be a target for anti-glioma therapy.

  3. RAS Initiative - Events

    Science.gov (United States)

    The NCI RAS Initiative has organized multiple events with outside experts to discuss how the latest scientific and technological breakthroughs can be applied to discover vulnerabilities in RAS-driven cancers.

  4. Cyclic AMP signalling in Dictyostelium : G-proteins activate separate Ras pathways using specific RasGEFs

    NARCIS (Netherlands)

    Kae, Helmut; Kortholt, Arjan; Rehmann, Holger; Insall, RobertH.; Van Haastert, Peter J. M.; Spiegelman, George B.; Weeks, Gerald

    In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium

  5. Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.

    Science.gov (United States)

    Blum, Roy; Jacob-Hirsch, Jasmine; Rechavi, Gideon; Kloog, Yoel

    2006-09-01

    The Ras inhibitor farnesylthiosalicylic acid (FTS) has been shown to induce apoptosis in glioblastoma multiforme, but its mechanism of action was unknown. We show that FTS or dominant-negative Ras, by deregulating extracellular signal-regulated kinase and Akt signaling, decreases survivin gene transcripts in U87 glioblastoma multiforme, leading to disappearance of survivin protein and cell death. FTS affected both Ras-controlled regulators of survivin transcription and Ras-regulated survival signals. Thus, Ras inhibition by FTS resulted in release of the survivin "brake" on apoptosis and in activation of the mitochondrial apoptotic pathway: dephosphorylation of Bad, activation of Bax, release of cytochrome c, and caspase activation. FTS-induced apoptosis of U87 cells was strongly attenuated by forced expression of survivin or by caspase inhibitors. These results show that resistance to apoptosis in glioblastoma multiforme can be abolished by a single Ras inhibitor, which targets both survivin, a critical inhibitor of apoptosis, and the intrinsic mitochondrial apoptotic machinery.

  6. Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway

    International Nuclear Information System (INIS)

    Tseng, Ya-Shih; Lee, Jenq-Chang; Huang, Chi-Ying F; Liu, Hsiao-Sheng

    2009-01-01

    Overexpression of Aurora-A and mutant Ras (Ras V12 ) together has been detected in human bladder cancer tissue. However, it is not clear whether this phenomenon is a general event or not. Although crosstalk between Aurora-A and Ras signaling pathways has been reported, the role of these two genes acting together in tumorigenesis remains unclear. Real-time PCR and sequence analysis were utilized to identify Ha- and Ki-ras mutation (Gly -> Val). Immunohistochemistry staining was used to measure the level of Aurora-A expression in bladder and colon cancer specimens. To reveal the effect of overexpression of the above two genes on cellular responses, mouse NIH3T3 fibroblast derived cell lines over-expressing either Ras V12 and wild-type Aurora-A (designated WT) or Ras V12 and kinase-inactivated Aurora-A (KD) were established. MTT and focus formation assays were conducted to measure proliferation rate and focus formation capability of the cells. Small interfering RNA, pharmacological inhibitors and dominant negative genes were used to dissect the signaling pathways involved. Overexpression of wild-type Aurora-A and mutation of Ras V12 were detected in human bladder and colon cancer tissues. Wild-type Aurora-A induces focus formation and aggregation of the Ras V12 transformants. Aurora-A activates Ral A and the phosphorylation of AKT as well as enhances the phosphorylation of MEK, ERK of WT cells. Finally, the Ras/MEK/ERK signaling pathway is responsible for Aurora-A induced aggregation of the Ras V12 transformants. Wild-type-Aurora-A enhances focus formation and aggregation of the Ras V12 transformants and the latter occurs through modulating the Ras/MEK/ERK signaling pathway

  7. Fetisisme Ras Kaukasoid dan Ras Mongoloid Sebagai Strategi Pemasaran dalam Sinetron Indonesia

    Directory of Open Access Journals (Sweden)

    Dimas Yudhistira

    2014-12-01

    ABSTRAK   Budaya populer yang tumbuh seiring dengan industrialisasi memengaruhi produksi per- filman di Indonesia. Salah satu genre perfilman di Indonesia adalah sinetron. Sinetron yang di- kategorikan sebagai produk seni kitsch memiliki dua kriteria yaitu sebagai komoditi seni yang populer dan sebagai komoditi dagang yang menghasilkan keuntungan ekonomis. Sebagai se- buah produk seni kitsch yang merupakan dasar pembuatan karyanya adalah selera masyarakat kebanyakan maka sinetron harus jeli dalam melihat keadaan dan latar belakang masyarakat. Penelitian ini menggunakan metode kualitatif. Hasil penelitian ini menggambarkan masyara- kat Indonesia yang merupakan ras Melayu telah dijajah oleh ras Kaukasoid dan Mongoloid sebelum tahun 1945 dan setelahnya. Efek dari penjajahan ini adalah ras Melayu telah ditanami fantasi yang menjadi stereotip mengenai ras Kaukasoid dan Mongoloid yang berakhir dengan fetisisme. Fetisisme ini dijadikan sebagai strategi pemasaran oleh produser dan sutradara un- tuk menarik antusiasme calon penonton sinetron. Caranya dengan menampilkan aktor dan aktris Melayu keturunan Kaukasoid dan Mongoloid sebagai pemeran utama.   Kata kunci: sinetron, seni kitsch, ras, fetisisme

  8. ArF Excimer Laser-induced Deposition of Ag/C Nanocomposite Thin Films in the Presence of n-Hexane

    Czech Academy of Sciences Publication Activity Database

    Gondal, M.A.; Fajgar, Radek; Chang, X.; Shen, K.; Xu, Q.

    2014-01-01

    Roč. 311, AUG 30 (2014), s. 95-100 ISSN 0169-4332 Grant - others:NNSFCH(CN) 51172044; NSFJP(CN) BK2011617; KFUPM(CN) RG 1311-1 Institutional support: RVO:67985858 Keywords : ArF excimer laser * nanocomposite * laser deposition Subject RIV: CH - Nuclear ; Quantum Chemistry Impact factor: 2.711, year: 2014

  9. Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

    Science.gov (United States)

    Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

    2017-10-01

    Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

  10. Epigenetic suppression of EGFR signaling in G-CIMP+ glioblastomas.

    Science.gov (United States)

    Li, Jie; Taich, Zachary J; Goyal, Amit; Gonda, David; Akers, Johnny; Adhikari, Bandita; Patel, Kunal; Vandenberg, Scott; Yan, Wei; Bao, Zhaoshi; Carter, Bob S; Wang, Renzhi; Mao, Ying; Jiang, Tao; Chen, Clark C

    2014-09-15

    The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood. Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406). Additionally, an independent collection of 25 fresh-frozen glioblastomas confirmed lowered pERK levels in G-CIMP+ specimens (pCIMP+ glioblastomas harbored lowered mRNA levels for EGFR and H-Ras. Induction of G-CIMP+ state by exogenous expression of a mutated isocitrate dehydrogenase 1, IDH1-R132H, suppressed EGFR and H-Ras protein expression as well as pERK accumulation in independent glioblastoma models. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. The IDH1-R132H expression-induced pERK suppression can be reversed by exogenous expression of H-RasG12V. Finally, the G-CIMP+ Ink4a-Arf-/- EGFRvIII glioblastoma line was more resistant to the EGFR inhibitor, Gefitinib, relative to its isogenic G-CIMP- counterpart. These results suggest that G-CIMP epigenetically regulates EGFR signaling and serves as a predictive biomarker for EGFR inhibitors in glioblastoma patients.

  11. Galectin-3 mediates cross-talk between K-Ras and Let-7c tumor suppressor microRNA.

    Directory of Open Access Journals (Sweden)

    Ran Levy

    Full Text Available BACKGROUND: Galectin-3 (Gal-3 and active (GTP-bound K-Ras contribute to the malignant phenotype of many human tumors by increasing the rate of cell proliferation, survival, and migration. These Gal-3-mediated effects result from a selective binding to K-Ras.GTP, causing increased nanoclustering in the cell membrane and leading to robust Ras signaling. Regulation of the interactions between Gal-3 and active K-Ras is not fully understood. METHODS AND FINDINGS: To gain a better understanding of what regulates the critical interactions between these two proteins, we examined the role of Gal-3 in the regulation of K-Ras by using Gal-3-knockout mouse embryonic-fibroblasts (Gal-3-/- MEFs and/or Gal-3/Gal-1 double-knockout MEFs. We found that knockout of Gal-3 induced strong downregulation (∼60% of K-Ras and K-Ras.GTP. The downregulation was somewhat more marked in the double-knockout MEFs, in which we also detected robust inhibition(∼50% of ERK and Akt activation. These additional effects are probably attributable to inhibition of the weak interactions of K-Ras.GTP with Gal-1. Re-expression of Gal-3 reversed the phenotype of the Gal-3-/- MEFs and dramatically reduced the disappearance of K-Ras in the presence of cycloheximide to the levels seen in wild-type MEFs. Furthermore, phosphorylation of Gal-3 by casein kinase-1 (CK-1 induced translocation of Gal-3 from the nucleus to the cytoplasm and the plasma membrane, leading to K-Ras stabilization accompanied by downregulation of the tumor suppressor miRNA let-7c, known to negatively control K-Ras transcription. CONCLUSIONS: Our results suggest a novel cross-talk between Gal-3-mediated downregulation of let 7c microRNA (which in turn negatively regulates K-Ras transcription and elucidates the association among Gal-3 let-7c and K-Ras transcription/translation, cellular compartmentalization and activity.

  12. The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

    International Nuclear Information System (INIS)

    Plowman, Sarah J.; Arends, Mark J.; Brownstein, David G.; Luo Feijun; Devenney, Paul S.; Rose, Lorraine; Ritchie, Ann-Marie; Berry, Rachel L.; Harrison, David J.; Hooper, Martin L.; Patek, Charles E.

    2006-01-01

    Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras tmΔ4A/tmΔ4A (exon 4A knock-out) ES cells which express K-ras4B only and K-ras -/- (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras tmΔ4A/tmΔ4A mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras tmΔ4A/tmΔ4A ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras tmΔ4A/tmΔ4A and K-ras -/- ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras -/- cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras tmΔ4A/tmΔ4A ES cells were more resistant to etoposide-induced apoptosis than K-ras -/- cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice

  13. Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway

    International Nuclear Information System (INIS)

    Tsubaki, Masanobu; Satou, Takao; Itoh, Tatsuki; Imano, Motohiro; Ogaki, Mitsuhiko; Yanae, Masashi; Nishida, Shozo

    2012-01-01

    Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Some patients continue to have a poor prognosis, because of the metastatic disease. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. YM529/ONO-5920 has recently been reported to induce apoptosis in various tumors including osteosarcoma. However, the mode of metastasis suppression in osteosarcoma by YM529/ONO-5920 is unclear. In the present study, we investigated whether YM529/ONO-5920 inhibited tumor cell migration, invasion, adhesion, or metastasis in the LM8 mouse osteosarcoma cell line. We found that YM529/ONO-5920 significantly inhibited metastasis, cell migration, invasion, and adhesion at concentrations that did not have antiproliferative effects on LM8 cells. YM529/ONO-5920 also inhibited the mRNA expression and protein activities of matrix metalloproteinases (MMPs). In addition, YM529/ONO-5920 suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine/threonine protein kinase B (Akt) by the inhibition of Ras prenylation. Moreover, U0126, a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, also inhibited LM8 cell migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP. The results indicated that YM529/ONO-5920 suppressed the Ras/MEK/ERK and Ras/PI3K/Akt pathways, thereby inhibiting LM8 cell migration, invasion, adhesion, and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumor cell metastasis in osteosarcoma. -- Highlights: ► We investigated whether YM529/ONO-5920 inhibited tumor metastasis in osteosarcoma. ► YM529/ONO-5920 inhibited metastasis, cell migration, invasion, and adhesion. ► YM529/ONO-5920 suppressed Ras signalings. ► YM529/ONO-5920

  14. Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

    OpenAIRE

    Baltus, E; Hanocq-Quertier, J; Hanocq, F; Brachet, J

    1988-01-01

    The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the ...

  15. Promoter methylation of RASSF1A and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers

    International Nuclear Information System (INIS)

    Liu, Yang; Gao, Weimin; Siegfried, Jill M; Weissfeld, Joel L; Luketich, James D; Keohavong, Phouthone

    2007-01-01

    Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors. Promoter methylation of the RASSF1A and DAPK genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-ras, p53, and EGFR were obtained from our previous studies. The RASSF1A gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The DAPK gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the RASSF1A or DAPK genes did not correlate with the frequency of mutations of the K-ras, p53, and EGFR gene. Our results showed that RASSF1A and DAPK genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-ras, p53 and EGFR genes, suggesting each of

  16. Tobacco TTG2 and ARF8 function concomitantly to control flower colouring by regulating anthocyanin synthesis genes.

    Science.gov (United States)

    Li, P; Chen, X; Sun, F; Dong, H

    2017-07-01

    Recently we elucidated that tobacco TTG2 cooperates with ARF8 to regulate the vegetative growth and seed production. Here we show that TTG2 and ARF8 control flower colouring by regulating expression of ANS and DFR genes, which function in anthocyanin biosynthesis. Genetic modifications that substantially altered expression levels of the TTG2 gene and production quantities of TTG2 protein were correlated with flower development and colouring. Degrees of flower colour were increased by TTG2 overexpression but decreased through TTG2 silencing, in coincidence with high and low concentrations of anthocyanins in flowers. Of five genes involved in the anthocyanin biosynthesis pathway, only ANS and DFR were TTG2-regulated and displayed enhancement and diminution of expression with TTG2 overexpression and silencing, respectively. The floral expression of ANS and DFR also needed a functional ARF8 gene, as ANS and DFR expression were attenuated by ARF8 silencing, which concomitantly diminished the role of TTG2 in anthocyanin production. While ARF8 required TTG2 to be expressed by itself and to regulate ANS and DFR expression, the concurrent presence of normally functional TTG2 and ARF8 was critical for floral production of anthocyanins and also for flower colouration. Our data suggest that TTG2 functions concomitantly with ARF8 to control degrees of flower colour by regulating expression of ANS and DFR, which are involved in the anthocyanin biosynthesis pathway. ARF8 depends on TTG2 to regulate floral expression of ANS and DFR with positive effects on anthocyanin production and flower colour. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

  17. Over-expression of mango (Mangifera indica L.) MiARF2 inhibits root and hypocotyl growth of Arabidopsis.

    Science.gov (United States)

    Wu, Bei; Li, Yun-He; Wu, Jian-Yong; Chen, Qi-Zhu; Huang, Xia; Chen, Yun-Feng; Huang, Xue-Lin

    2011-06-01

    An auxin response factor 2 gene, MiARF2, was cloned in our previous study [1] from the cotyledon section of mango (Mangifera indica L. cv. Zihua) during adventitious root formation, which shares an 84% amino acid sequence similarity to Arabidopsis ARF2. This study was to examine the effects of over-expression of the full-length MiARF2 open reading frame on the root and hypocotyl growth in Arabidopsis. Phenotype analysis showed that the T(3) transgenic lines had about 20-30% reduction in the length of hypocotyls and roots of the seedlings in comparison with the wild-type. The transcription levels of ANT and ARGOS genes which play a role in controlling organ size and cell proliferation in the transgenic seedlings also decreased. Therefore, the inhibited root and hypocotyl growth in the transgenic seedlings may be associated with the down-regulated transcription of ANT and ARGOS by the over-expression of MiARF2. This study also suggests that although MiARF2 only has a single DNA-binding domain (DBD), it can function as other ARF-like proteins containing complete DBD, middle region (MR) and carboxy-terminal dimerization domain (CTD).

  18. Molecular epidemiological study of human rectal cancer induced by radiotherapy

    International Nuclear Information System (INIS)

    Rytomaa, T.; Servomaa, K.; Kiuru, A.; Auvinen, A.; Makkonen, K.; Kosma, V.M.; Hirvikoski, P.

    1997-01-01

    In the present molecular epidemiological study we have examined possible presence of characteristic radiation-associated mutations in the p53 and K-ras genes in secondary rectal cancers in 67 female radiotherapy patients, compared with primary rectal cancers in 67 matched controls Exons 4-8 of the p53 and K-ras gen were amplified from histological sections, and screened for mutations by SSCP and direct sequencing. The results showed that p53 and K-ras gene mutations were very uncommon in apparent radiation-induced tumours compared with matched controls. This may, by itself, be a hallmark of high-dose radiation damage, but it also suggests that genes other than p53 and K-ras are critical in female rectal carcinogenesis associated with radiation exposure. (authors)

  19. Levels of H-ras codon 61 CAA to AAA mutation: response to 4-ABP-treatment and Pms2-deficiency.

    Science.gov (United States)

    Parsons, Barbara L; Delongchamp, Robert R; Beland, Frederick A; Heflich, Robert H

    2006-01-01

    DNA mismatch repair (MMR) deficiencies result in increased frequencies of spontaneous mutation and tumor formation. In the present study, we tested the hypothesis that a chemically-induced mutational response would be greater in a mouse with an MMR-deficiency than in the MMR-proficient mouse models commonly used to assay for chemical carcinogenicity. To accomplish this, the induction of H-ras codon 61 CAA-->AAA mutation was examined in Pms2 knockout mice (Pms2-/-, C57BL/6 background) and sibling wild-type mice (Pms2+/+). Groups of five or six neonatal male mice were treated with 0.3 micromol 4-aminobiphenyl (4-ABP) or the vehicle control, dimethylsulfoxide. Eight months after treatment, liver DNAs were isolated and analysed for levels of H-ras codon 61 CAA-->AAA mutation using allele-specific competitive blocker-PCR. In Pms2-proficient and Pms2-deficient mice, 4-ABP treatment caused an increase in mutant fraction (MF) from 1.65x10(-5) to 2.91x10(-5) and from 3.40x10(-5) to 4.70x10(-5), respectively. Pooling data from 4-ABP-treated and control mice, the approximately 2-fold increase in MF observed in Pms2-deficient as compared with Pms2-proficient mice was statistically significant (P=0.0207) and consistent with what has been reported previously in terms of induction of G:C-->T:A mutation in a Pms2-deficient background. Pooling data from both genotypes, the increase in H-ras MF in 4-ABP-treated mice, as compared with control mice, did not reach the 95% confidence level of statistical significance (P=0.0606). The 4-ABP treatment caused a 1.76-fold and 1.38-fold increase in average H-ras MF in Pms2-proficient and Pms2-deficient mice, respectively. Furthermore, the levels of induced mutation in Pms2-proficient and Pms2-deficient mice were nearly identical (1.26x10(-5) and 1.30x10(-5), respectively). We conclude that Pms2-deficiency does not result in an amplification of the H-ras codon 61 CAA-->AAA mutational response induced by 4-ABP.

  20. Interconversion of two GDP-bound conformations and their selection in an Arf-family small G protein.

    Science.gov (United States)

    Okamura, Hideyasu; Nishikiori, Masaki; Xiang, Hongyu; Ishikawa, Masayuki; Katoh, Etsuko

    2011-07-13

    ADP-ribosylation factor (Arf) and other Arf-family small G proteins participate in many cellular functions via their characteristic GTP/GDP conformational cycles, during which a nucleotide(∗)Mg(2+)-binding site communicates with a remote N-terminal helix. However, the conformational interplay between the nucleotides, the helix, the protein core, and Mg(2+) has not been fully delineated. Herein, we report a study of the dynamics of an Arf-family protein, Arl8, under various conditions by means of NMR relaxation spectroscopy. The data indicated that, when GDP is bound, the protein core, which does not include the N-terminal helix, reversibly transition between an Arf-family GDP form and another conformation that resembles the Arf-family GTP form. Additionally, we found that the N-terminal helix and Mg(2+), respectively, stabilize the aforementioned former and latter conformations in a population-shift manner. Given the dynamics of the conformational changes, we can describe the Arl8 GTP/GDP cycle in terms of an energy diagram. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Active Erk Regulates Microtubule Stability in H-ras-Transformed Cells

    Directory of Open Access Journals (Sweden)

    Rene E. Harrison

    2001-01-01

    Full Text Available Increasing evidence suggests that activated erk regulates cell functions, at least in part, by mechanisms that do not require gene transcription. Here we show that the map kinase, erk, decorates microtubules (MTs and mitotic spindles in both parental and mutant active rastransfected 10T1 /2 fibroblasts and MCF10A breast epithelial cells. Approximately 20% of total cellular erk decorated MTs in both cell lines. A greater proportion of activated erk was associated with MTs in the presence of mutant active H-ras than in parental cells. Activation of erk by the ras pathway coincided with a decrease in the stability of MT, as detected by a stability marker. The MKK1 inhibitor, PD98059 and transfection of a dominant negative MKK1 blocked ras-induced instability of MTs but did not modify the association of erk with MTs or affect MT stability of the parental cells. These results indicate that the subset of active erk kinase that associates with MTs contributes to their instability in the presence of a mutant active ras. The MT-associated subset of active erk likely contributes to the enhanced invasive and proliferative abilities of cells containing mutant active H-ras.

  2. Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

    Science.gov (United States)

    Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

    2013-01-01

    Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

  3. Biological aspects and tumorigenic activity of the Ras proto-oncogenic family Aspectos biológicos e atividade tumorigênica da família proto-oncogênica Ras

    Directory of Open Access Journals (Sweden)

    Juliano André Boquett

    2010-12-01

    Full Text Available Proto-oncogenes play an important role in the regulation of the cellular cycle, being critical to the tumorigenesis. In this category we can find the RAS family. Due to the high transformation potential of these genes, this family is the best described and most studied one. It is formed by the H-, K- and the N-RAS genes, that codify highly related proteins expressed in several types of cells, denominated p21.These proteins act in the sign transduction from the membrane to the nucleus, as well as in the control of proliferation, differentiation and cellular death, and they are regulated by the interaction with GDP (inactive and GTP (active. These proteins show variation in only 10 - 15% of the primary structure, in the C-terminal portion denominated hyper-variant region. When in the oncogenic form, the p21 proteins remain active, providing continuous stimuli to the cellular proliferation. Among the RAS genes, K-RAS ones have been the most studied for presenting more frequent mutations and for being present in more aggressive tumors, implying the patients’ shorter survival time. Due to these facts and relative bibliography lack in the Portuguese language on this family, we presented in this work a systematized and updated review on the RAS genes. Os proto-oncogenes desempenham importante papel na regulação do ciclo celular, e são críticos à tumorigênese. Nessa categoria se encontra a família RAS, que, devido ao elevado potencial transformante dos genes que a compõem, é uma das mais bem descritas e estudadas. É formada pelos genes H-, K- e N-RAS, que codificam proteínas altamente relacionadas expressas em vários tipos de células, denominadas p21. Estas atuam na transdução de sinal da membrana ao núcleo, estão envolvidas no controle da proliferação, diferenciação e morte celular e são reguladas pela interação com GDP (inativa e GTP (ativa. As proteínas p21 diferem em apenas 10-15% da sua estrutura primária, na porção C

  4. Gibberellin Application at Pre-Bloom in Grapevines Down-Regulates the Expressions of VvIAA9 and VvARF7, Negative Regulators of Fruit Set Initiation, during Parthenocarpic Fruit Development

    Science.gov (United States)

    Jung, Chan Jin; Hur, Youn Young; Yu, Hee-Ju; Noh, Jung-Ho; Park, Kyo-Sun; Lee, Hee Jae

    2014-01-01

    Fruit set is initiated only after fertilization and is tightly regulated primarily by gibberellins (GAs) and auxins. The application of either of these hormones induces parthenocarpy, fruit set without fertilization, but the molecular mechanism underlying this induction is poorly understood. In the present study, we have shown that the parthenocarpic fruits induced by GA application at pre-bloom result from the interaction of GA with auxin signaling. The transcriptional levels of the putative negative regulators of fruit set initiation, including Vitis auxin/indole-3-acetic acid transcription factor 9 (VvIAA9), Vitis auxin response factor 7 (VvARF7), and VvARF8 were monitored during inflorescence development in seeded diploid ‘Tamnara’ grapevines with or without GA application. Without GA application, VvIAA9, VvARF7, and VvARF8 were expressed at a relatively high level before full bloom, but decreased thereafter following pollination. After GA application at 14 days before full bloom (DBF); however, the expression levels of VvIAA9 and VvARF7 declined at 5 DBF prior to pollination. The effects of GA application on auxin levels or auxin signaling were also analyzed by monitoring the expression patterns of auxin biosynthesis genes and auxin-responsive genes with or without GA application. Transcription levels of the auxin biosynthesis genes Vitis anthranilate synthase β subunit (VvASB1-like), Vitis YUCCA2 (VvYUC2), and VvYUC6 were not significantly changed by GA application. However, the expressions of Vitis Gretchen Hagen3.2 (VvGH3.2) and VvGH3.3, auxin-responsive genes, were up-regulated from 2 DBF to full bloom with GA application. Furthermore, the Vitis GA signaling gene, VvDELLA was up-regulated by GA application during 12 DBF to 7 DBF, prior to down-regulation of VvIAA9 and VvARF7. These results suggest that VvIAA9 and VvARF7 are negative regulators of fruit set initiation in grapevines, and GA signaling is integrated with auxin signaling via VvDELLA during

  5. Depletion of Pokemon gene inhibits hepatocellular carcinoma cell growth through inhibition of H-ras.

    Science.gov (United States)

    Zhang, Quan-Le; Tian, De-An; Xu, Xiang-Jiang

    2011-01-01

    Pokemon is a transcription repressor which plays a critical role in cell transformation and malignancy. However, little is known about its effect on the development and progression of hepatocellular carcinoma (HCC). The aim of this study was to investigate the expression of Pokemon in human HCC tissues and the biological behavior of Pokemon in HCC cells in which it is overexpressed. We also explored the expression of potential downstream cofactors of Pokemon. Reverse transcription polymerase chain reaction and Western blot analysis were used to investigate the expression of Pokemon in tissues of 30 HCC patients. We then examined cell proliferation or apoptosis and β-catenin or H-ras expression in Pokemon-depleted HepG(2) cells using DNA vector-based RNA interference technology. Pokemon was markedly expressed in 22/30 (73.3%) HCC tissues, with expression levels higher than in adjacent normal liver tissues (p Pokemon inhibited proliferation of HepG(2) or induced apoptosis. Also, H-ras expression decreased to a large extent. Pokemon exerts its oncogenic activity in the development of HCC by promoting cancer cell growth and reducing apoptosis, and the effect may be mediated by H-ras. Copyright © 2011 S. Karger AG, Basel.

  6. About the RAS Initiative

    Science.gov (United States)

    The RAS Initiative, a "hub and spoke" model, connects researchers to better understand and target the more than 30% of cancers driven by mutations in RAS genes. Includes oversight and contact information.

  7. The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23

    Science.gov (United States)

    Weinhold, Mario; Eisenblätter, Martin; Jasny, Edith; Fehlings, Michael; Finke, Antje; Gayum, Hermine; Rüschendorf, Ursula; Renner Viveros, Pablo; Moos, Verena; Allers, Kristina; Schneider, Thomas; Schaible, Ulrich E.; Schumann, Ralf R.; Mielke, Martin E.; Ignatius, Ralf

    2013-01-01

    Background Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4+ and CD8+ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S) 19, which has previously been employed successfully to vaccinate cattle. Methodology/Principal findings We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR)2. Conclusions/Significance Thus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2) human DCs to produce Th1-promoting cytokines. PMID:23805193

  8. The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23.

    Directory of Open Access Journals (Sweden)

    Mario Weinhold

    Full Text Available Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4(+ and CD8(+ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs, which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S 19, which has previously been employed successfully to vaccinate cattle.We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR2.Thus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2 human DCs to produce Th1-promoting cytokines.

  9. PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers.

    Directory of Open Access Journals (Sweden)

    Filip Janku

    Full Text Available Oncogenic mutations of PIK3CA, RAS (KRAS, NRAS, and BRAF have been identified in various malignancies, and activate the PI3K/AKT/mTOR and RAS/RAF/MEK pathways, respectively. Both pathways are critical drivers of tumorigenesis.Tumor tissues from 504 patients with diverse cancers referred to the Clinical Center for Targeted Therapy at MD Anderson Cancer Center starting in October 2008 were analyzed for PIK3CA, RAS (KRAS, NRAS, and BRAF mutations using polymerase chain reaction-based DNA sequencing.PIK3CA mutations were found in 54 (11% of 504 patients tested; KRAS in 69 (19% of 367; NRAS in 19 (8% of 225; and BRAF in 31 (9% of 361 patients. PIK3CA mutations were most frequent in squamous cervical (5/14, 36%, uterine (7/28, 25%, breast (6/29, 21%, and colorectal cancers (18/105, 17%; KRAS in pancreatic (5/9, 56%, colorectal (49/97, 51%, and uterine cancers (3/20, 15%; NRAS in melanoma (12/40, 30%, and uterine cancer (2/11, 18%; BRAF in melanoma (23/52, 44%, and colorectal cancer (5/88, 6%. Regardless of histology, KRAS mutations were found in 38% of patients with PIK3CA mutations compared to 16% of patients with wild-type (wtPIK3CA (p = 0.001. In total, RAS (KRAS, NRAS or BRAF mutations were found in 47% of patients with PIK3CA mutations vs. 24% of patients wtPIK3CA (p = 0.001. PIK3CA mutations were found in 28% of patients with KRAS mutations compared to 10% with wtKRAS (p = 0.001 and in 20% of patients with RAS (KRAS, NRAS or BRAF mutations compared to 8% with wtRAS (KRAS, NRAS or wtBRAF (p = 0.001.PIK3CA, RAS (KRAS, NRAS, and BRAF mutations are frequent in diverse tumors. In a wide variety of tumors, PIK3CA mutations coexist with RAS (KRAS, NRAS and BRAF mutations.

  10. What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

    Science.gov (United States)

    Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

    2010-06-11

    Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd

  11. Which downstream signal transduction pathway(s) of H-ras are necessary for the cellular response(s) to ionizing radiation? (Results of an astro research fellowship year)

    International Nuclear Information System (INIS)

    Rudoltz, Marc S.; Muschel, Ruth J.; McKenna, W. Gillies

    1996-01-01

    Purpose/Background: The H-ras oncogene encodes a protein which is an essential component of multiple downstream effector pathways required for induction of proliferation and differentiation. Ras plays a role in the control some of these signal transduction pathways, such as the MAP kinase pathway which controls gene expression and the Rac-Rho pathway which controls cell morphology. Previous work from our laboratory has associated H-ras expression with radiation resistance, a prolonged delay in G2 following exposure to ionizing radiation, and suppression of radiation-induced apoptosis. In addition, H-ras cooperates with myc in transformation. Recent work by White et al. (Cell 80:533-541, 1995) and Joneson et al. (Science 271: 810-812, 1996) describes three mutations in H-ras which were engineered to eliminate different downstream signal transduction pathways of H-ras. T35S contains a serine in place of threonine at amino acid 35 and is defective for ras-induced cytoskeletal changes and initiation of DNA synthesis. E37G contains a glutamic acid in place of glycine at amino acid 37 which eliminates interaction of H-ras with a GDP/GTP exchange factor. C40 contains a substitution of cysteine for tyrosine at amino acid 40 and is defective for H-ras induction of the MAP kinase pathway. We propose that by expressing these mutant H-ras proteins in immortalized cells the downstream pathways of H-ras which regulate the cellular response(s) to ionizing radiation may be determined. Materials and Methods: pHP-5 plasmids encoding these H-ras mutant genes (see White et al.) were transfected by calcium phosphate precipitation into MR4 cells, rat embryo fibroblasts immortalized by expression of v-myc. In this vector, the cDNA for H-ras is placed under the control of a CMV constitutive promoter, and selection is provided by hygromycin. The transfections performed were as follows: V12Ras (no mutation), T35S, E37G, C40, T35S + E37G, and T35S + C40. Twenty four hours after transfection

  12. ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

    International Nuclear Information System (INIS)

    Vieira da Silva, Claudio; Alves da Silva, Erika; Costa Cruz, Mario; Chavrier, Philippe; Arruda Mortara, Renato

    2009-01-01

    Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP 2 and PIP 3 to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

  13. Bypass of cell cycle arrest induced by transient DNMT1 post-transcriptional silencing triggers aneuploidy in human cells

    Directory of Open Access Journals (Sweden)

    Barra Viviana

    2012-02-01

    Full Text Available Abstract Background Aneuploidy has been acknowledged as a major source of genomic instability in cancer, and it is often considered the result of chromosome segregation errors including those caused by defects in genes controlling the mitotic spindle assembly, centrosome duplication and cell-cycle checkpoints. Aneuploidy and chromosomal instability has been also correlated with epigenetic alteration, however the molecular basis of this correlation is poorly understood. Results To address the functional connection existing between epigenetic changes and aneuploidy, we used RNA-interference to silence the DNMT1 gene, encoding for a highly conserved member of the DNA methyl-transferases. DNMT1 depletion slowed down proliferation of near-diploid human tumor cells (HCT116 and triggered G1 arrest in primary human fibroblasts (IMR90, by inducing p53 stabilization and, in turn, p21waf1 transactivation. Remarkably, p53 increase was not caused by DNA damage and was not observed after p14-ARF post-transcriptional silencing. Interestingly, DNMT1 silenced cells with p53 or p14-ARF depleted did not arrest in G1 but, instead, underwent DNA hypomethylation and became aneuploid. Conclusion Our results suggest that DNMT1 depletion triggers a p14ARF/p53 dependent cell cycle arrest to counteract the aneuploidy induced by changes in DNA methylation.

  14. RAS Initiative - Community Outreach

    Science.gov (United States)

    Through community and technical collaborations, workshops and symposia, and the distribution of reference reagents, the RAS Initiative seeks to increase the sharing of knowledge and resources essential to defeating cancers caused by mutant RAS genes.

  15. 19-Hydroxyeicosatetraenoic acid and isoniazid protect against angiotensin II-induced cardiac hypertrophy

    Energy Technology Data Exchange (ETDEWEB)

    Elkhatali, Samya; El-Sherbeni, Ahmed A.; Elshenawy, Osama H. [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1 (Canada); Abdelhamid, Ghada [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1 (Canada); Department of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Helwan (Egypt); El-Kadi, Ayman O.S., E-mail: aelkadi@ualberta.ca [Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1 (Canada)

    2015-12-15

    We have recently demonstrated that 19-hydroxyeicosatetraenoic acid (19-HETE) is the major subterminal-HETE formed in the heart tissue, and its formation was decreased during cardiac hypertrophy. In the current study, we examined whether 19-HETE confers cardioprotection against angiotensin II (Ang II)-induced cardiac hypertrophy. The effect of Ang II, with and without 19-HETE (20 μM), on the development of cellular hypertrophy in cardiomyocyte RL-14 cells was assessed by real-time PCR. Also, cardiac hypertrophy was induced in Sprague–Dawley rats by Ang II, and the effect of increasing 19-HETE by isoniazid (INH; 200 mg/kg/day) was assessed by heart weight and echocardiography. Also, alterations in cardiac cytochrome P450 (CYP) and their associated arachidonic acid (AA) metabolites were determined by real-time PCR, Western blotting and liquid-chromatography–mass-spectrometry. Our results demonstrated that 19-HETE conferred a cardioprotective effect against Ang II-induced cellular hypertrophy in vitro, as indicated by the significant reduction in β/α-myosin heavy chain ratio. In vivo, INH improved heart dimensions, and reversed the increase in heart weight to tibia length ratio caused by Ang II. We found a significant increase in cardiac 19-HETE, as well as a significant reduction in AA and its metabolite, 20-HETE. In conclusion, 19-HETE, incubated with cardiomyocytes in vitro or induced in the heart by INH in vivo, provides cardioprotection against Ang II-induced hypertrophy. This further confirms the role of CYP, and their associated AA metabolites in the development of cardiac hypertrophy. - Highlights: • We found 19-hydroxy arachidonic acid to protect cardiomyocytes from hypertrophy. • We validated the use of isoniazid as a cardiac 19-hydroxy arachidonic acid inducer. • We found isoniazid to increase protective and inhibit toxic eicosanoides. • We found isoniazid to protect against angiotensin-induced cardiac hypertrophy. • This will help to

  16. Structural Insights into Arl1-Mediated Targeting of the Arf-GEF BIG1 to the trans-Golgi

    Directory of Open Access Journals (Sweden)

    Antonio Galindo

    2016-07-01

    Full Text Available The GTPase Arf1 is the major regulator of vesicle traffic at both the cis- and trans-Golgi. Arf1 is activated at the cis-Golgi by the guanine nucleotide exchange factor (GEF GBF1 and at the trans-Golgi by the related GEF BIG1 or its paralog, BIG2. The trans-Golgi-specific targeting of BIG1 and BIG2 depends on the Arf-like GTPase Arl1. We find that Arl1 binds to the dimerization and cyclophilin binding (DCB domain in BIG1 and report a crystal structure of human Arl1 bound to this domain. Residues in the DCB domain that bind Arl1 are required for BIG1 to locate to the Golgi in vivo. DCB domain-binding residues in Arl1 have a distinct conformation from those in known Arl1-effector complexes, and this plasticity allows Arl1 to interact with different effectors of unrelated structure. The findings provide structural insight into how Arf1 GEFs, and hence active Arf1, achieve their correct subcellular distribution.

  17. Inhibition of p38 MAPK attenuates renal atrophy and fibrosis in a murine renal artery stenosis model.

    Science.gov (United States)

    Wang, Diping; Warner, Gina M; Yin, Ping; Knudsen, Bruce E; Cheng, Jingfei; Butters, Kim A; Lien, Karen R; Gray, Catherine E; Garovic, Vesna D; Lerman, Lilach O; Textor, Stephen C; Nath, Karl A; Simari, Robert D; Grande, Joseph P

    2013-04-01

    Renal artery stenosis (RAS) is an important cause of chronic renal dysfunction. Recent studies have underscored a critical role for CCL2 (MCP-1)-mediated inflammation in the progression of chronic renal damage in RAS and other chronic renal diseases. In vitro studies have implicated p38 MAPK as a critical intermediate for the production of CCL2. However, a potential role of p38 signaling in the development and progression of chronic renal disease in RAS has not been previously defined. We sought to test the hypothesis that inhibition of p38 MAPK ameliorates chronic renal injury in mice with RAS. We established a murine RAS model by placing a cuff on the right renal artery and treated mice with the p38 inhibitor SB203580 or vehicle for 2 wk. In mice treated with vehicle, the cuffed kidney developed interstitial fibrosis, tubular atrophy, and interstitial inflammation. In mice treated with SB203580, the RAS-induced renal atrophy was reduced (70% vs. 39%, P < 0.05). SB203580 also reduced interstitial inflammation and extracellular matrix deposition but had no effect on the development of hypertension. SB203580 partially blocked the induction of CCL2, CCL7 (MCP-3), CC chemokine receptor 2 (CCR2), and collagen 4 mRNA expression in the cuffed kidneys. In vitro, blockade of p38 hindered both TNF-α and TGF-β-induced CCL2 upregulation. Based on these observations, we conclude that p38 MAPK plays a critical role in the induction of CCL2/CCL7/CCR2 system and the development of interstitial inflammation in RAS.

  18. The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis

    DEFF Research Database (Denmark)

    Evangelou, K.; Bartkova, J.; Kotsinas, A.

    2013-01-01

    oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides...

  19. High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

    Science.gov (United States)

    Chavan, Tanmay S.; Jang, Hyunbum; Khavrutskii, Lyuba; Abraham, Sherwin J.; Banerjee, Avik; Freed, Benjamin C.; Johannessen, Liv; Tarasov, Sergey G.; Gaponenko, Vadim; Nussinov, Ruth; Tarasova, Nadya I.

    2015-01-01

    Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible. PMID:26682817

  20. P120-GAP associated with syndecan-2 to function as an active switch signal for Src upon transformation with oncogenic ras

    International Nuclear Information System (INIS)

    Huang, J.-W.; Chen, C.-L.; Chuang, N.-N.

    2005-01-01

    BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q 61 K)] of shrimp Penaeus japonicus were applied to reveal a complex of p120-GAP/syndecan-2 being highly expressed upon transformation. Of interest, most of the p120-GAP/syndecan-2 complex was localized at caveolae, a membrane microdomain enriched with caveolin-1. To confirm the molecular interaction between syndecan-2 and p120-GAP, we further purified p120-GAP protein from mouse brains by using an affinity column of HiTrap-RACK1 and expressed mouse RACK1-encoded fusion protein and mouse syndecan-2-encoded fusion protein in bacteria. We report molecular affinities exist between p120-GAP and RACK1, syndecan-2 and RACK1 as well as p120-GAP and syndecan-2. The selective affinity between p120-GAP and syndecan-2 was found to be sufficient to detach RACK1. The p120-GAP/syndecan-2 complex was demonstrated to keep Src tyrosine kinase in an activated form. On the other hand, the syndecan-2/RACK1 complex was found to have Src in an inactivated form. These data indicate that the p120-GAP/syndecan-2 complex at caveolae could provide a docking site for Src to transmit tyrosine signaling, implying that syndecan-2/p120-GAP functions as a tumor promoter upon transformation with oncogenic ras of shrimp P. japonicus

  1. Ras proteins have multiple functions in vegetative cells of Dictyostelium.

    Science.gov (United States)

    Bolourani, Parvin; Spiegelman, George; Weeks, Gerald

    2010-11-01

    During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.

  2. Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Meng; He, Hong-wei; Sun, Huan-xing; Ren, Kai-huan [Department of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100050 (China); Shao, Rong-guang, E-mail: shaor@bbn.cn [Department of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100050 (China)

    2009-09-18

    Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.

  3. Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

    Science.gov (United States)

    Pazman, C; Mayes, C A; Fanto, M; Haynes, S R; Mlodzik, M

    2000-04-01

    The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

  4. Association of folate intake, dietary habits, smoking and COX-2 promotor -765G>C polymorphism with K-ras mutation in patients with colorectal cancer.

    Science.gov (United States)

    Kamal, Manal M; Youssef, Omar Z; Lotfy, Ahmed N; Elsaed, Eman T; Fawzy, May M T

    2012-09-01

    Understanding the role of environmental and molecular influences on the nature and rate of K-ras mutations in colorectal neoplasms is crucial. COX-2 polymorphisms -765G>C may play a role in carcinogenic processes in combination with specific life-style conditions or dependent on the racial composition of a particular population. If mutational events play an important role in colorectal carcinogenesis sequence, one can hypothesize that modification of these events by life-style or other factors would be a useful prevention strategy. To explore the association between K-ras mutation and potential variables known or suspected to be related to the risk of colorectal cancer (CRC) as well as determining the possible modulating effect of the COX-2 polymorphism, -765G>C. The study was conducted on 80 patients with colorectal cancer from Tropical Medicine and Gastrointestinal Tract endoscopy Departments and those attending clinic of the National Cancer Institute, Cairo University during the period extending from April 2009 to March 2010. Full history taking with emphasis on the risk factors of interest, namely age, sex, family history, smoking and dietary history. Serum CEA and CA19-9, RBCs folic acid and occult blood in stool were done to all samples. K-ras protooncogene mutation at codon 12 (exon 1) and cyclooxygenase 2 (COX-2) -765G>C polymorphism were determined by PCR-RFLP. The K-ras mutation was positive in 23 (28.7%) patients. COX-2 polymorphism revealed GG in 62.5%, GC in 26.2 % and CC genotype was found in 11.3 % of cases. The mean red blood cell folic acid level was lower in the K-ras positive group (100.96±51.3 ng/ml) than the negative group (216.6±166.4 ng/ml), (P<0.01). Higher folate levels were found in males than females (median=173 ng/ml and 85 ng/ml; respectively, P=0.002) with adjusted odds ratio (OR) of 0.984. Only, the RBCs folate (P=0.0018) followed by gender (P=0.036) contributed significantly in the discrimination between patients prone to develop K-ras

  5. Association of folate intake, dietary habits, smoking and COX-2 promotor-765G > C polymorphism with K-ras mutation in patients with colorectal cancer

    International Nuclear Information System (INIS)

    Kamal, M.M.; Youssef, O.Z.; Lotfy, A.N.; Elsaed, E.T.; Fawzy, M.M.T.

    2012-01-01

    Background: Understanding the role of environmental and molecular influences on the nature and rate of K-ras mutations in colorectal neoplasms is crucial. COX-2 polymorphisms -765G > C may play a role in carcinogenic processes in combination with specific life-style conditions or dependent on the racial composition of a particular population. If mutational events play an important role in colorectal carcinogenesis sequence, one can hypothesize that modification of these events by life-style or other factors would be a useful prevention strategy. Aim of work: To explore the association between K-ras mutation and potential variables known or suspected to be related to the risk of colorectal cancer (CRC) as well as determining the possible modulating effect of the COX-2 polymorphism, —765G > C. Subjects and methods: The study was conducted on 80 patients with colorectal cancer from Tropical Medicine and Gastrointestinal Tract endoscopy Departments and those attending clinic of the National Cancer Institute, Cairo University during the period extending from April 2009 to March 2010. Full history taking with emphasis on the risk factors of interest, namely age, sex, family history, smoking and dietary history. Serum CEA and CA19-9, RBCs folic acid and occult blood in stool were done to all samples. K-ras protooncogene mutation at codon 12 (exon 1) and cyclooxygenase 2 (COX-2) —765G > C polymorphism were determined by PCR-RFLP. Results: The K-ras mutation was positive in 23 (28.7%) patients. COX-2 polymorphism revealed GG in 62.5%, GC in 26.2 % and CC genotype was found in 11.3 % of cases. The mean red blood cell folic acid level was lower in the K-ras positive group (100.96 ± 51.3 ng/ml) than the negative group (216.6 ± 166.4 ng/ml), (P < 0.01). Higher folate levels were found in males than females (median = 173 ng/ml and 85 ng/ml; respectively, P = 0.002) with adjusted odds ratio (OR) of 0.984. Only, the RBCs folate (P = 0.0018) followed by gender (P = 0

  6. Mechanisms of bee venom-induced acute renal failure.

    Science.gov (United States)

    Grisotto, Luciana S D; Mendes, Glória E; Castro, Isac; Baptista, Maria A S F; Alves, Venancio A; Yu, Luis; Burdmann, Emmanuel A

    2006-07-01

    The spread of Africanized bees in the American continent has increased the number of severe envenomation after swarm attacks. Acute renal failure (ARF) is one of the major hazards in surviving patients. To assess the mechanisms of bee venom-induced ARF, rats were evaluated before, up to 70 min and 24h after 0.5mg/kg of venom injection. Control rats received saline. Bee venom caused an early and significant reduction in glomerular filtration rate (GFR, inulin clearance, 0.84+/-0.05 to 0.40+/-0.08 ml/min/100g, pbee venom-induced ARF that may occur even without hemolysis or hypotension.

  7. Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains

    DEFF Research Database (Denmark)

    Anders, Nadine; Nielsen, Michael M.; Keicher, Jutta

    2008-01-01

    vesicle formation by activating ARF GTPases on specific membranes in animals, plants, and fungi. However, apart from the catalytic exchange activity of the SEC7 domain, the functional significance of other conserved domains is virtually unknown. Here, we show that a distinct N-terminal domain of GNOM......The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate...... mediates dimerization and in addition interacts heterotypically with two other conserved domains in vivo. In contrast with N-terminal dimerization, the heterotypic interaction is essential for GNOM function, as mutations abolishing this interaction inactivate the GNOM protein and compromise its membrane...

  8. Genomewide identification and expression analysis of the ARF gene ...

    Indian Academy of Sciences (India)

    2014-12-26

    Dec 26, 2014 ... In this study, 31 putative apple ARF genes have been identified and located within the apple genome. ... including growth and development of the root and stem, for- ..... Script 1st Strand cDNA Synthesis Kit (Takara, Dalian,.

  9. INDUCTION OF DNA ADDUCTS, TUMORS, AND KI-RAS ONCOGENE MUTATIONS IN STRAIN A/J MOUSE LUNG BY IP. ADMINISTRATION OF DIBENZ[A,H]ANTHRACENE

    Science.gov (United States)

    Induction of DNA adducts, tumors, and Ki-ras oncogene mutations in strain AlJ mouse lung by ip. administration of dibenz[a,h]anthracene Previous studies of polycyclic aromatic hydrocarbon (P AH) induced lung tumors in the strain NJ mouse model system have demonstrated qua...

  10. Bypass of senescence by the polycomb group protein CBX8 through direct binding to the INK4A-ARF locus

    DEFF Research Database (Denmark)

    Dietrich, Nikolaj; Bracken, Adrian P; Trinh, Emmanuelle

    2007-01-01

    -ARF, and that ectopic expression of CBX8 leads to repression of the Ink4a-Arf locus and bypass of senescence, leading to cellular immortalization. Gene expression and location analysis demonstrate that besides the INK4A-ARF locus, CBX8 also regulates a number of other genes important for cell growth and survival...

  11. Ras signaling in aging and metabolic regulation.

    Science.gov (United States)

    Slack, Cathy

    2017-12-07

    Aberrant signal transduction downstream of the Ras GTPase has a well-established role in tumorigenesis. Mutations that result in hyperactivation of Ras are responsible for a third of all human cancers. Hence, small molecule inhibitors of the Ras signal transduction cascade have been under intense focus as potential cancer treatments. In both invertebrate and mammalian models, emerging evidence has also implicated components of the Ras signaling pathway in aging and metabolic regulation. Here, I review the current evidence for Ras signaling in these newly discovered roles highlighting the interactions between the Ras pathway and other longevity assurance mechanisms. Defining the role of Ras signaling in maintaining age-related health may have important implications for the development of interventions that could not only increase lifespan but also delay the onset and/or progression of age-related functional decline.

  12. Effect of Cuscuta chinensis on renal function in ischemia/reperfusion-induced acute renal failure rats.

    Science.gov (United States)

    Shin, Sun; Lee, Yun Jung; Kim, Eun Ju; Lee, An Sook; Kang, Dae Gill; Lee, Ho Sub

    2011-01-01

    The kidneys play a central role in regulating water, ion composition and excretion of metabolic waste products in the urine. Cuscuta chinensis has been known as an important traditional Oriental medicine for the treatment of liver and kidney disorders. Thus, we studied whether an aqueous extract of Cuscuta chinensis (ACC) seeds has an effect on renal function parameters in ischemia/reperfusion-induced acute renal failure (ARF) rats. Administration of 250 mg/kg/day ACC showed that renal functional parameters including urinary excretion rate, osmolality, Na(+), K(+), Cl(-), creatinine clearance, solute-free water reabsorption were significantly recovered in ischemia/reperfusion-induced ARF. Periodic acid Schiff staining showed that administration of ACC improved tubular damage in ischemia/reperfusion-induced ARF. In immunoblot and immunohistological examinations, ischemia/reperfusion-induced ARF decreased the expressions of water channel AQP 2, 3 and sodium potassium pump Na,K-ATPase in the renal medulla. However, administration of ACC markedly incremented AQP 2, 3 and Na,K-ATPase expressions. Therefore, these data indicate that administration of ACC ameliorates regulation of the urine concentration and renal functions in rats with ischemia/reperfusion-induced ARF.

  13. Identification and expression analysis of two interleukin-23α (p19) isoforms, in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar.

    Science.gov (United States)

    Jiang, Yousheng; Husain, Mansourah; Qi, Zhitao; Bird, Steve; Wang, Tiehui

    2015-08-01

    Interleukin (IL)-23 is a heterodimeric IL-12 family cytokine composed of a p19 α-chain, linked to a p40 β-chain that is shared with IL-12. IL-23 is distinguished functionally from IL-12 by its ability to induce the production of IL-17, and differentiation of Th17 cells in mammals. Three isoforms of p40 (p40a, p40b and p40c) have been found in some 3R teleosts. Salmonids also possess three p40 isoforms (p40b1, p40b2 and p40c) although p40a is missing, and two copies (paralogues) of p40b are present that have presumably been retained following the 4R duplication in this fish lineage. Teleost p19 has been discovered recently in zebrafish, but to date there is limited information on expression and modulation of this molecule. In this report we have cloned two p19 paralogues (p19a and p19b) in salmonids, suggesting that a salmonid can possess six potential IL-23 isoforms. Whilst Atlantic salmon has two active p19 genes, the rainbow trout p19b gene may have been pseudogenized. The salmonid p19 translations share moderate identities (22.8-29.9%) to zebrafish and mammalian p19 molecules, but their identity was supported by structural features, a conserved 4 exon/3 intron gene organisation, and phylogenetic tree analysis. The active salmonid p19 genes are highly expressed in blood and gonad. Bacterial (Yersinia ruckeri) and viral infection in rainbow trout induces the expression of p19a, suggesting pathogen-specific induction of IL-23 isoforms. Trout p19a expression was also induced by PAMPs (poly IC and peptidoglycan) and the proinflammatory cytokine IL-1β in primary head kidney macrophages. These data may indicate diverse functional roles of trout IL-23 isoforms in regulating the immune response in fish. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Transcriptional upregulation of p19INK4d upon diverse genotoxic stress is critical for optimal DNA damage response.

    Science.gov (United States)

    Ceruti, Julieta M; Scassa, María E; Marazita, Mariela C; Carcagno, Abel C; Sirkin, Pablo F; Cánepa, Eduardo T

    2009-06-01

    p19INK4d promotes survival of several cell lines after UV irradiation due to enhanced DNA repair, independently of CDK4 inhibition. To further understand the action of p19INK4d in the cellular response to DNA damage, we aimed to elucidate whether this novel regulator plays a role only in mechanisms triggered by UV or participates in diverse mechanisms initiated by different genotoxics. We found that p19INK4d is induced in cells injured with cisplatin or beta-amyloid peptide as robustly as with UV. The mentioned genotoxics transcriptionally activate p19INK4d expression as demonstrated by run-on assay without influencing its mRNA stability and with partial requirement of protein synthesis. It is not currently known whether DNA damage-inducible genes are turned on by the DNA damage itself or by the consequences of that damage. Experiments carried out in cells transfected with distinct damaged DNA structures revealed that the damage itself is not responsible for the observed up-regulation. It is also not known whether the increased expression of DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. We found that ectopic expression of p19INK4d improves DNA repair ability and protects neuroblastoma cells from apoptosis caused by cisplatin or beta-amyloid peptide. Using clonal cell lines where p19INK4d levels can be modified at will, we show that p19INK4d expression correlates with increased survival and clonogenicity. The results presented here, prompted us to suggest that p19INK4d displays an important role in an early stage of cellular DNA damage response.

  15. Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages

    Directory of Open Access Journals (Sweden)

    Youhei Egami

    2015-01-01

    Full Text Available Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.

  16. TW-01, a piperazinedione-derived compound, inhibits Ras-mediated cell proliferation and angioplasty-induced vascular restenosis

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Chao-Feng [The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan (China); Department of Medicine, MacKay Medical College, New Taipei City, Taiwan (China); Division of Cardiology, Department of Internal Medicine, MacKay Memorial Hospital, Taipei, Taiwan (China); Division of Cardiology, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan (China); Huang, Han-Li [The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan (China); Peng, Chieh-Yu [Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan (China); School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan (China); Lee, Yu-Ching [The Center of Translational Medicine, Taipei Medical University, Taipei, Taiwan (China); Ph.D. Program for Biotechnology in Medicine, Taipei Medical University, Taipei, Taiwan (China); Wang, Hui-Po [College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan (China); Teng, Che-Ming [College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan (China); Pharmacological Institute, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Pan, Shiow-Lin, E-mail: slpan@tmu.edu.tw [The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan (China); Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei 10031, Taiwan (China)

    2016-08-15

    Purpose: Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the pathogenesis of atherosclerosis and restenosis. This study investigated piperazinedione derived compound TW-01-mediated inhibitory effects on VSMC proliferation and intimal hyperplasia. Methods: Cell proliferation was determined using [{sup 3}H]-thymidine incorporation and MTT assay; cell cycle distribution was measured using flow cytometry; proteins and mRNA expression were determined using western blotting and RT-PCR analyses; DNA binding activity of nuclear factor-κB (NF-κB), as measured using enzyme-linked immunosorbent assays (ELISA); in vivo effects of TW-01 were determined using balloon angioplasty in the rat. Results: TW-01 significantly inhibited cell proliferation. At the concentrations used, no cytotoxic effects were observed. Three predominant signaling pathways were inhibited by TW-01: (a) extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) activation and its downstream effectors of c-fos, c-jun, and c-myc; (b) DNA binding activity of nuclear factor-κB (NF-κB); and, (c) Akt/protein kinase B (PKB) and cell cycle progression. Furthermore, TW-01 also inhibited Ras activation, a shared upstream event of each of these signaling cascades. In vascular injury studies, oral administration of TW-01 significantly suppressed intimal hyperplasia induced by balloon angioplasty. Conclusion: The present study suggests that TW-01 might be a potential candidate for atherosclerosis treatment. - Highlights: • TW-01significantly inhibits vascular smooth muscle cell proliferation. • TW-01 inhibits ERK, Akt and Ras pathway and DNA binding activity of NF-κB. • TW-01 significantly suppresses intimal hyperplasia induced by balloon angioplasty. • TW-01 might be a potential candidate for atherosclerosis treatment.

  17. TW-01, a piperazinedione-derived compound, inhibits Ras-mediated cell proliferation and angioplasty-induced vascular restenosis

    International Nuclear Information System (INIS)

    Lin, Chao-Feng; Huang, Han-Li; Peng, Chieh-Yu; Lee, Yu-Ching; Wang, Hui-Po; Teng, Che-Ming; Pan, Shiow-Lin

    2016-01-01

    Purpose: Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the pathogenesis of atherosclerosis and restenosis. This study investigated piperazinedione derived compound TW-01-mediated inhibitory effects on VSMC proliferation and intimal hyperplasia. Methods: Cell proliferation was determined using [ 3 H]-thymidine incorporation and MTT assay; cell cycle distribution was measured using flow cytometry; proteins and mRNA expression were determined using western blotting and RT-PCR analyses; DNA binding activity of nuclear factor-κB (NF-κB), as measured using enzyme-linked immunosorbent assays (ELISA); in vivo effects of TW-01 were determined using balloon angioplasty in the rat. Results: TW-01 significantly inhibited cell proliferation. At the concentrations used, no cytotoxic effects were observed. Three predominant signaling pathways were inhibited by TW-01: (a) extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) activation and its downstream effectors of c-fos, c-jun, and c-myc; (b) DNA binding activity of nuclear factor-κB (NF-κB); and, (c) Akt/protein kinase B (PKB) and cell cycle progression. Furthermore, TW-01 also inhibited Ras activation, a shared upstream event of each of these signaling cascades. In vascular injury studies, oral administration of TW-01 significantly suppressed intimal hyperplasia induced by balloon angioplasty. Conclusion: The present study suggests that TW-01 might be a potential candidate for atherosclerosis treatment. - Highlights: • TW-01significantly inhibits vascular smooth muscle cell proliferation. • TW-01 inhibits ERK, Akt and Ras pathway and DNA binding activity of NF-κB. • TW-01 significantly suppresses intimal hyperplasia induced by balloon angioplasty. • TW-01 might be a potential candidate for atherosclerosis treatment.

  18. Product (P1) from project 0-6738 : performance studies and future directions for mixes containing RAP and RAS.

    Science.gov (United States)

    2013-01-01

    In recent years both reclaimed asphalt pavement (RAP) and recycled asphalt shingles (RAS) have been widely used in asphalt mixes by the asphalt paving industry in Texas. The use of RAP and RAS can save tax payers money, and it is also good for the...

  19. Complex formation of p65/RelA with nuclear Akt1 for enhanced transcriptional activation of NF-κB

    International Nuclear Information System (INIS)

    Kwon, Osong; Kim, Kyung A; He, Long; Jung, Mira; Jeong, Sook Jung; Ahn, Jong Seog; Kim, Bo Yeon

    2008-01-01

    Akt1 was revealed to interact with Ki-Ras in the cytoplasm of Ki-Ras-transformed human prostate epithelial cells, 267B1/K-ras. Moreover, p65/RelA in the nucleus was found to interact with both Ki-Ras and Akt1, suggesting the nuclear translocation of Akt1:Ki-Ras complex for NF- κB activation. In support of this, compared with wild type Akt1, the dominant negative Akt1 mutant was decreased in its nuclear expression, reducing the Ki-Ras-induced NF-κB transcriptional activation. Moreover, inhibitors of Ras (sulindac sulfide and farnesyltransferase inhibitor I) or PI3K/Akt (wortmannin), reduced the amounts of Akt1 and Ki-Ras in the nucleus as well as partial NF-κB activity. The complete inhibition of Ki-Ras-induced NF-κB activation, however, could only be obtained by combined treatment with wortmannin and proteasome inhibitor-1. Accordingly, clonogenic assay showed Akt1 contribution to IκBα-mediated NF-κB activation for oncogenic cell growth by Ki-Ras. Our data suggest a crucial role of Ki-Ras:Akt1 complex in NF-κB transcriptional activation and enhancement of cell survival

  20. Endocytosis of HERG is clathrin-independent and involves arf6.

    Directory of Open Access Journals (Sweden)

    Rucha Karnik

    Full Text Available The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.

  1. S3226, a novel NHE3 inhibitor, attenuates ischemia-induced acute renal failure in rats.

    Science.gov (United States)

    Hropot, M; Juretschke, H P; Langer, K H; Schwark, J R

    2001-12-01

    Acute renal failure (ARF) remains a major problem in clinical nephrology characterized by sudden loss of the kidney function due to ischemia, trauma, and/or nephrotoxic drugs. The current therapy of ARF is symptomatic with mortality rates exceeding 50%. The aim of this study was to investigate the effects of an intravenous infusion of S3226 (3-[2-(3-guanidino-2-methyl-3-oxopropenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride), a selective Na+/H+ exchange subtype 3 (NHE3) blocker, in ischemia-induced ARF in rats. In a second series of experiments cytosolic pH (pHi) changes in the kidney during ARF were continuously measured by means of nuclear magnetic resonance spectroscopy (MRS). ARF was induced by bilateral occlusion of renal arteries for 40 minutes in three groups of anaesthetized Wistar rats. Control rats (N = 12) were infused with saline (6.25 mL/kg over 30 min) before occlusion and the compound groups (each N = 12) were infused with S3226 at a dose of 20 mg/kg over 30 minutes either before initiation of ischemia or immediately after release of clamps. Plasma creatinine (PCr), creatinine clearance (CCr), urine volume, sodium, and potassium excretion were determined up to seven days after release of clamps. In the second series of experiments in anaesthetized rats the left kidney was exposed by flank incision and fixed in a non-magnetic device. An inflatable cuff was positioned around the pedicle to induce ischemia without removing animals from the magnet. A double-tuned 1H-31P home-built surface coil was placed above the exposed kidney for the detection of pHi. At day 1 after ischemia CCr in the control group was significantly lower as compared to S3226-treated animals (control 0.30 +/- 0.05 vs. before 0.90 +/- 0.26 and reperfusion 0.83 +/- 0.15 mL/min/kg, respectively). PCr increased from 18 +/- 0.1 micromol/L before occlusion to 245 +/- 7 micromol/L in the control. The increase in PCr was significantly lower in the S3226 treated

  2. High Intra- and Inter-Tumoral Heterogeneity of RAS Mutations in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Marion Jeantet

    2016-12-01

    Full Text Available Approximately 30% of patients with wild type RAS metastatic colorectal cancer are non-responders to anti-epidermal growth factor receptor monoclonal antibodies (anti-EGFR mAbs, possibly due to undetected tumoral subclones harboring RAS mutations. The aim of this study was to analyze the distribution of RAS mutations in different areas of the primary tumor, metastatic lymph nodes and distant metastasis. A retrospective cohort of 18 patients with a colorectal cancer (CRC was included in the study. Multiregion analysis was performed in 60 spatially separated tumor areas according to the pathological tumor node metastasis (pTNM staging and KRAS, NRAS and BRAF mutations were tested using pyrosequencing. In primary tumors, intra-tumoral heterogeneity for RAS mutation was found in 33% of cases. Inter-tumoral heterogeneity for RAS mutation between primary tumors and metastatic lymph nodes or distant metastasis was found in 36% of cases. Moreover, 28% of tumors had multiple RAS mutated subclones in the same tumor. A high proportion of CRCs presented intra- and/or inter-tumoral heterogeneity, which has relevant clinical implications for anti-EGFR mAbs prescription. These results suggest the need for multiple RAS testing in different parts of the same tumor and/or more sensitive techniques.

  3. The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP

    International Nuclear Information System (INIS)

    Ganzenmueller, Tina; Matthaei, Markus; Muench, Peter; Scheible, Michael; Iftner, Angelika; Hiller, Thomas; Leiprecht, Natalie; Probst, Sonja; Stubenrauch, Frank; Iftner, Thomas

    2008-01-01

    Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19 ARF induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53

  4. Defined spatiotemporal features of RAS-ERK signals dictate cell fate in MCF-7 mammary epithelial cells.

    Science.gov (United States)

    Herrero, Ana; Casar, Berta; Colón-Bolea, Paula; Agudo-Ibáñez, Lorena; Crespo, Piero

    2016-06-15

    Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane-disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals. © 2016 Herrero et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  5. WAVE regulatory complex activation by cooperating GTPases Arf and Rac1

    DEFF Research Database (Denmark)

    Koronakis, Vassilis; Hume, Peter J; Humphreys, Daniel

    2011-01-01

    The WAVE regulatory complex (WRC) is a critical element in the control of actin polymerization at the eukaryotic cell membrane, but how WRC is activated remains uncertain. While Rho GTPase Rac1 can bind and activate WRC in vitro, this interaction is of low affinity, suggesting other factors may...... be important. By reconstituting WAVE-dependent actin assembly on membrane-coated beads in mammalian cell extracts, we found that Rac1 was not sufficient to engender bead motility, and we uncovered a key requirement for Arf GTPases. In vitro, Rac1 and Arf1 were individually able to bind weakly to recombinant...... be central components in WAVE signalling, acting directly, alongside Rac1....

  6. Epidermal Growth Factor Receptor and K-RAS status in two cohorts of squamous cell carcinomas

    Directory of Open Access Journals (Sweden)

    Van Laethem Jean-Luc

    2010-05-01

    Full Text Available Abstract Background With the availability of effective anti-EGFR therapies for various solid malignancies, such as non-cell small lung cancer, colorectal cancer and squamous cell carcinoma of the head and neck, the knowledge of EGFR and K-RAS status becomes clinically important. The aim of this study was to analyse EGFR expression, EGFR gene copy number and EGFR and K-RAS mutations in two cohorts of squamous cell carcinomas, specifically anal canal and tonsil carcinomas. Methods Formalin fixed, paraffin-embedded tissues from anal and tonsil carcinoma were used. EGFR protein expression and EGFR gene copy number were analysed by means of immunohistochemistry and fluorescence in situ hybridisation. The somatic status of the EGFR gene was investigated by PCR using primers specific for exons 18 through 21. For the K-RAS gene, PCR was performed using exon 2 specific primers. Results EGFR immunoreactivity was present in 36/43 (83.7% of anal canal and in 20/24 (83.3% of tonsil squamous cell carcinomas. EGFR amplification was absent in anal canal tumours (0/23, but could be identified in 4 of 24 tonsil tumours. From 38 anal canal specimens, 26 specimens were successfully analysed for exon 18, 30 for exon 19, 34 for exon 20 and 30 for exon 21. No EGFR mutations were found in the investigated samples. Thirty samples were sequenced for K-RAS exon 2 and no mutation was identified. From 24 tonsil specimens, 22 were successfully analysed for exon 18 and all 24 specimens for exon 19, 20 and 21. No EGFR mutations were found. Twenty-two samples were sequenced for K-RAS exon 2 and one mutation c.53C > A was identified. Conclusion EGFR mutations were absent from squamous cell carcinoma of the anus and tonsils, but EGFR protein expression was detected in the majority of the cases. EGFR amplification was seen in tonsil but not in anal canal carcinomas. In our investigated panel, only one mutation in the K-RAS gene of a tonsil squamous cell carcinoma was identified

  7. Epidermal Growth Factor Receptor and K-RAS status in two cohorts of squamous cell carcinomas

    International Nuclear Information System (INIS)

    Van Damme, Nancy; Pauwels, Patrick; Peeters, Marc; Deron, Philippe; Van Roy, Nadine; Demetter, Pieter; Bols, Alain; Dorpe, Jo Van; Baert, Filip; Van Laethem, Jean-Luc; Speleman, Franki

    2010-01-01

    With the availability of effective anti-EGFR therapies for various solid malignancies, such as non-cell small lung cancer, colorectal cancer and squamous cell carcinoma of the head and neck, the knowledge of EGFR and K-RAS status becomes clinically important. The aim of this study was to analyse EGFR expression, EGFR gene copy number and EGFR and K-RAS mutations in two cohorts of squamous cell carcinomas, specifically anal canal and tonsil carcinomas. Formalin fixed, paraffin-embedded tissues from anal and tonsil carcinoma were used. EGFR protein expression and EGFR gene copy number were analysed by means of immunohistochemistry and fluorescence in situ hybridisation. The somatic status of the EGFR gene was investigated by PCR using primers specific for exons 18 through 21. For the K-RAS gene, PCR was performed using exon 2 specific primers. EGFR immunoreactivity was present in 36/43 (83.7%) of anal canal and in 20/24 (83.3%) of tonsil squamous cell carcinomas. EGFR amplification was absent in anal canal tumours (0/23), but could be identified in 4 of 24 tonsil tumours. From 38 anal canal specimens, 26 specimens were successfully analysed for exon 18, 30 for exon 19, 34 for exon 20 and 30 for exon 21. No EGFR mutations were found in the investigated samples. Thirty samples were sequenced for K-RAS exon 2 and no mutation was identified. From 24 tonsil specimens, 22 were successfully analysed for exon 18 and all 24 specimens for exon 19, 20 and 21. No EGFR mutations were found. Twenty-two samples were sequenced for K-RAS exon 2 and one mutation c.53C > A was identified. EGFR mutations were absent from squamous cell carcinoma of the anus and tonsils, but EGFR protein expression was detected in the majority of the cases. EGFR amplification was seen in tonsil but not in anal canal carcinomas. In our investigated panel, only one mutation in the K-RAS gene of a tonsil squamous cell carcinoma was identified. This indicates that EGFR and K-RAS mutation analysis is not

  8. miR-654-5p Targets GRAP to Promote Proliferation, Metastasis, and Chemoresistance of Oral Squamous Cell Carcinoma Through Ras/MAPK Signaling.

    Science.gov (United States)

    Lu, Meng; Wang, Chengyong; Chen, Weihui; Mao, Chuanqing; Wang, Jin

    2018-04-01

    Oral squamous cell carcinoma (OSCC) is characterized by rapid local migration and invasion. This study was aimed at clarifying the effect of miR-654-5p on progression of OSCC. miR-654-5p promoted proliferation, metastasis, and chemoresistance of OSCC in vitro and in vivo. Consistently, miR-654-5p was upregulated in late-stage OSCC and was correlated with poor prognosis of OSCC patients. Furthermore, miR-654-5p was mechanistically verified to target Grb-2-related adaptor protein (GRAP), accompanied by the activation of Ras/MAPK signaling and the facilitation of epithelial-mesenchymal transition in OSCC cells. GRAP was downregulated in T1-2 stage versus T3-4 stage head and neck squamous cell carcinoma (HNSC) and was negatively correlated with tumor-node-metastases (TNM) stage in HNSC patients based on The Cancer Genome Atlas (TCGA) analysis. In addition, GRAP was positively correlated with good prognosis in HNSC patients. Our findings suggest that the miR-654-5p/GRAP/Ras/Erk signaling pathway in OSCC cells might contribute to the underlying mechanism through which miR-654-5p participates in the regulation of OSCC progression. miR-654-5p, as a potential biomarker for the clinical diagnosis and prognosis of OSCC, may be an effective anticancer target for the treatment of OSCC.

  9. Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Yogi, Alvaro; Callera, Glaucia E. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Mecawi, André S. [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Batalhão, Marcelo E.; Carnio, Evelin C. [Department of General and Specialized Nursing, College of Nursing of Ribeirão Preto, USP, São Paulo (Brazil); Antunes-Rodrigues, José [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Queiroz, Regina H. [Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences, USP, São Paulo (Brazil); Touyz, Rhian M. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Tirapelli, Carlos R., E-mail: crtirapelli@eerp.usp.br [Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP (Brazil)

    2012-11-01

    Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT{sub 1} receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT{sub 1}-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT{sub 1} receptor activation.

  10. Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

    Science.gov (United States)

    Miller, M J; Maher, V M; McCormick, J J

    1992-11-01

    Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Fujiyama, A.; Tamanoi, F.

    1986-01-01

    The authors demonstrate the pathway for the biosynthesis of RAS1 and RAS2 gene products of Saccharomyces cerevisiae leading to their localization in membranes. The primary translation products of these genes are detected in a soluble fraction. Shortly after synthesis, these precursor molecules are converted to forms that migrate slightly faster than the precursor forms on a NaDodSO 4 /polyacrylamide gel. These processed proteins are further modified by fatty acid acylation, which is detected by [ 3 H]palmitic acid labeling. The acylated derivatives are found exclusively in cell membranes, indicating the translocation of the RAS proteins from cytosol to membranes during maturation process. The attached fatty acids can be released by mild alkaline hydrolysis, suggesting that the linkage between the fatty acid and the protein is an ester bond. The site of the modification by fatty acid is presumably localized to the COOH-terminal portion of the RAS proteins. Fraction of the membranes by sucrose gradient demonstrates that a majority of the fatty-acylated RAS proteins are localized in plasma membrane

  12. Genome-Wide Characterization and Expression Profiling of the AUXIN RESPONSE FACTOR (ARF) Gene Family in Eucalyptus grandis

    Science.gov (United States)

    Yu, Hong; Soler, Marçal; Mila, Isabelle; San Clemente, Hélène; Savelli, Bruno; Dunand, Christophe; Paiva, Jorge A. P.; Myburg, Alexander A.; Bouzayen, Mondher; Grima-Pettenati, Jacqueline; Cassan-Wang, Hua

    2014-01-01

    Auxin is a central hormone involved in a wide range of developmental processes including the specification of vascular stem cells. Auxin Response Factors (ARF) are important actors of the auxin signalling pathway, regulating the transcription of auxin-responsive genes through direct binding to their promoters. The recent availability of the Eucalyptus grandis genome sequence allowed us to examine the characteristics and evolutionary history of this gene family in a woody plant of high economic importance. With 17 members, the E. grandis ARF gene family is slightly contracted, as compared to those of most angiosperms studied hitherto, lacking traces of duplication events. In silico analysis of alternative transcripts and gene truncation suggested that these two mechanisms were preeminent in shaping the functional diversity of the ARF family in Eucalyptus. Comparative phylogenetic analyses with genomes of other taxonomic lineages revealed the presence of a new ARF clade found preferentially in woody and/or perennial plants. High-throughput expression profiling among different organs and tissues and in response to environmental cues highlighted genes expressed in vascular cambium and/or developing xylem, responding dynamically to various environmental stimuli. Finally, this study allowed identification of three ARF candidates potentially involved in the auxin-regulated transcriptional program underlying wood formation. PMID:25269088

  13. Genome-wide characterization and expression profiling of the AUXIN RESPONSE FACTOR (ARF gene family in Eucalyptus grandis.

    Directory of Open Access Journals (Sweden)

    Hong Yu

    Full Text Available Auxin is a central hormone involved in a wide range of developmental processes including the specification of vascular stem cells. Auxin Response Factors (ARF are important actors of the auxin signalling pathway, regulating the transcription of auxin-responsive genes through direct binding to their promoters. The recent availability of the Eucalyptus grandis genome sequence allowed us to examine the characteristics and evolutionary history of this gene family in a woody plant of high economic importance. With 17 members, the E. grandis ARF gene family is slightly contracted, as compared to those of most angiosperms studied hitherto, lacking traces of duplication events. In silico analysis of alternative transcripts and gene truncation suggested that these two mechanisms were preeminent in shaping the functional diversity of the ARF family in Eucalyptus. Comparative phylogenetic analyses with genomes of other taxonomic lineages revealed the presence of a new ARF clade found preferentially in woody and/or perennial plants. High-throughput expression profiling among different organs and tissues and in response to environmental cues highlighted genes expressed in vascular cambium and/or developing xylem, responding dynamically to various environmental stimuli. Finally, this study allowed identification of three ARF candidates potentially involved in the auxin-regulated transcriptional program underlying wood formation.

  14. The dimethylthiourea-induced attenuation of cisplatin nephrotoxicity is associated with the augmented induction of heat shock proteins

    International Nuclear Information System (INIS)

    Tsuji, Takayuki; Kato, Akihiko; Yasuda, Hideo; Miyaji, Takehiko; Luo, Jinghui; Sakao, Yukitoshi; Ito, Hideaki; Fujigaki, Yoshihide; Hishida, Akira

    2009-01-01

    Dimethylthiourea (DMTU), a potent hydroxyl radical scavenger, affords protection against cisplatin (CDDP)-induced acute renal failure (ARF). Since the suppression of oxidative stress and the enhancement of heat shock proteins (HSPs) are both reported to protect against CDDP-induced renal damage, we tested whether increased HSP expression is involved in the underlying mechanisms of the DMTU-induced renal protection. We examined the effect of DMTU treatment on the expression of HSPs in the kidney until day 5 following a single injection of CDDP (5 mg/kg BW). DMTU significantly inhibited the CDDP-induced increments of serum creatinine, the number of 8-hydroxyl-2'-deoxyguanosine (8-OHdG)- and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive tubular cells, and tubular damage score (p < 0.05). CDDP significantly increased renal abundances of HO-1, HSP60, HSP72 and HSP90 at days 1, 3, and 5. DMTU significantly augmented only the expression of HSP60 expression mainly in the cytoplasm of the proximal tubular cells at days 1 and 3 in CDDP-induced ARF. DMTU also inhibited the CDDP-induced increment of Bax, a pro-apoptotic protein, in the fraction of organelles/membranes at day 3. The findings suggest that DMTU may afford protection against CDDP-induced ARF, partially through the early induction of cytoplasmic HSP60, thereby preventing the Bax-mediated apoptosis in renal tubular cells

  15. Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

    Science.gov (United States)

    Barbakadze, Tamar; Goloshvili, Galina; Narmania, Nana; Zhuravliova, Elene; Mikeladze, David

    2017-10-01

    Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. According to the results, it may be proposed that hypoxia can decrease S

  16. S1P/S1PR3 signaling mediated proliferation of pericytes via Ras/pERK pathway and CAY10444 had beneficial effects on spinal cord injury.

    Science.gov (United States)

    Tang, Hai-Bin; Jiang, Xiao-Jian; Wang, Chen; Liu, Shi-Chang

    2018-04-15

    Pericytes have long been regarded merely to maintain structural and functional integrity of blood-brain barrier (BBB). Nevertheless, it has also been identified as a component of scar-forming stromal cells after spinal cord injury (SCI). In process of enlargement of spinal cavity after SCI, the number of pericytes increased and outnumbered astrocytes. However, the mechanism of proliferation of pericytes remains unclear. Sphingosine-1-phosphate (S1P) has been reported to play important roles in the formation of glia scar, but previous studies had paid more attention to the astrocytes. The present study aimed to observe the effects of S1P and S1P receptors (S1PRs) on proliferation of pericytes and investigate the underlying mechanism. By double immunostaining, we found that the number of PDGFRβ-positive pericytes was gradually increased and sealed the cavity, which surrounded by reactive astrocytes. Moreover, the subtype of S1PR3 was found to be induced by SCI and mainly expressed on pericytes. Further, by use of CAY10444, an inhibitor of S1PR3, we showed that S1P/S1PR3 mediated the proliferation of pericytes through Ras/pERK pathway. Moreover, CAY10444 was found to have the effects of enhancing neuronal survival, alleviating glial scar formation, and improving locomotion recovery after SCI. The results suggested that S1P/S1PR3 might be a promising target for clinical therapy for SCI. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. An orthosteric inhibitor of the RAS-SOS interaction.

    Science.gov (United States)

    Nickerson, Seth; Joy, Stephen T; Arora, Paramjit S; Bar-Sagi, Dafna

    2013-01-01

    Rat sarcoma (RAS) proteins are signaling nodes that transduce extracellular cues into precise alterations in cellular physiology by engaging effector pathways. RAS signaling thus regulates diverse cell processes including proliferation, migration, differentiation, and survival. Owing to this central role in governing mitogenic signals, RAS pathway components are often dysregulated in human diseases. Targeted therapy of RAS pathways has generally not been successful, largely because of the robust biochemistry of the targets and their multifaceted network of molecular regulators. The rate-limiting step of RAS activation is Son of Sevenless (SOS)-mediated nucleotide exchange involving a single evolutionarily conserved catalytic helix from SOS. Structure function data of this mechanism provided a strong platform to design an SOS-derived, helically constrained peptide mimic as an inhibitor of the RAS-SOS interaction. In this chapter, we review RAS-SOS signaling dynamics and present evidence supporting the novel paradigm of inhibiting their interaction as a therapeutic strategy. We then describe a method of generating helically constrained peptide mimics of protein surfaces, which we have employed to inhibit the RAS-SOS active site interaction. The biochemical and functional properties of this SOS mimic support the premise that inhibition of RAS-nucleotide exchange can effectively block RAS activation and downstream signaling. © 2013 Elsevier Inc. All rights reserved.

  18. Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

    Science.gov (United States)

    Kim, Tae Won; Peeters, Marc; Thomas, Anne L; Gibbs, Peter; Hool, Kristina; Zhang, Jianqi; Ang, Agnes; Bach, Bruce Allen; Price, Timothy

    2018-06-13

    The accumulation of emergent RAS mutations during anti-epidermal growth factor receptor (EGFR) therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status. This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the Plasma Select -R™ 64-gene panel at 0.1% sensitivity. Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival (OS) for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval: 11.5-15.4) and 7.9 months (6.4-9.6), respectively ( P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations. Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Copyright ©2018, American Association for Cancer Research.

  19. RAS - Screens & Assays - Drug Discovery

    Science.gov (United States)

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  20. Code verification and Y2K testing, calibration, testing, and installation of the radionuclide assay system-photon (RAS-P) at multiple sites for the Savannah River Site

    International Nuclear Information System (INIS)

    Hodge, C.A.

    2000-01-01

    The Radionuclide Assay System - Photon (RAS-P) is a near-field, transmission-corrected assay system developed for measurement of the actinide content of relatively homogeneous waste generated by facility operations. It is intended for use by facility operations personnel, and has features to enhance its usefulness and efficiency. These include multinuclide assay capability, automatic (off-shift) collection of background and straight-through transmission source data, enforcement of measurement control requirements, Go-NoGo or Assay modes, password protection, and reporting of total fissile gram equivalent values. System hardware consists of a shielded high-resolution germanium detector, a turntable, a shielded transmission source and shutter assembly, and a desktop computer and laser printer mounted on a compact frame. RAS-P was designed to assay the contents of cylindrical containers up to 30 inches diameter by 32 inches high, boxes up to 30 inches diagonal by 32 inches high, and HE PA filters up to 2 x 2 x 1 feet. Prior to installation at the Savannah River Site (SRS), code validation, system performance, and assurance against Y2K effects all were confirmed. Code validation was accomplished using spreadsheet calculations that were independent of the original code to calculate intermediate and final result produced by RAS-P. System testing was performed by repeated operation of the instrument under all required circumstances. Y2K testing was performed simultaneously with code validation following a protocol prescribed by the SRS Y2K subcommittee that required assays with dates varying throughout the expected useful life of the RAS-P, particularly those bracketing Y2K boundaries. Performance history has been compiled demonstrating reliability (system availability), diversability (the ability to alter assay parameters and obtain results quickly), and measurement control characteristics

  1. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    International Nuclear Information System (INIS)

    Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

    2012-01-01

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ∼ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ∼ 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  2. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Naumov, Inna [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Kazanov, Dina [Integrated Cancer Prevention Center, Tel Aviv (Israel); Lisiansky, Victoria [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Starr, Alex [Lung and Allergy Institute, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Aroch, Ilan; Shapira, Shiran; Kraus, Sarah [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Arber, Nadir, E-mail: narber@post.tau.ac.il [Integrated Cancer Prevention Center, Tel Aviv (Israel); Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel)

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  3. β2-adrenoceptor blockage induces G1/S phase arrest and apoptosis in pancreatic cancer cells via Ras/Akt/NFκB pathway

    Directory of Open Access Journals (Sweden)

    Zhang Dong

    2011-11-01

    Full Text Available Abstract Background Smoking and stress, pancreatic cancer (PanCa risk factors, stimulate nitrosamine 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK and catecholamines production respectively. NNK and catecholamine bind the β-adrenoceptors and induce PanCa cell proliferation; and we have previously suggested that β-adrenergic antagonists may suppress proliferation and invasion and stimulate apoptosis in PanCa. To clarify the mechanism of apoptosis induced by β2-adrenergic antagonist, we hypothesize that blockage of the β2-adrenoceptor could induce G1/S phase arrest and apoptosis and Ras may be a key player in PanCa cells. Results The β1 and β2-adrenoceptor proteins were detected on the cell surface of PanCa cells from pancreatic carcinoma specimen samples by immunohistochemistry. The β2-adrenergic antagonist ICI118,551 significantly induced G1/S phase arrest and apoptosis compared with the β1-adrenergic antagonist metoprolol, which was determined by the flow cytometry assay. β2-adrenergic antagonist therapy significantly suppressed the expression of extracellular signal-regulated kinase, Akt, Bcl-2, cyclin D1, and cyclin E and induced the activation of caspase-3, caspase-9 and Bax by Western blotting. Additionally, the β2-adrenergic antagonist reduced the activation of NFκB in vitro cultured PanCa cells. Conclusions The blockage of β2-adrenoceptor markedly induced PanCa cells to arrest at G1/S phase and consequently resulted in cell death, which is possibly due to that the blockage of β2-adrenoceptor inhibited NFκB, extracellular signal-regulated kinase, and Akt pathways. Therefore, their upstream molecule Ras may be a key factor in the β2-adrenoceptor antagonist induced G1/S phase arrest and apoptosis in PanCa cells. The new pathway discovered in this study may provide an effective therapeutic strategy for PanCa.

  4. DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

    Science.gov (United States)

    Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation AnalysisPhouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.<...

  5. CDK5-mediated phosphorylation of p19INK4d avoids DNA damage-induced neurodegeneration in mouse hippocampus and prevents loss of cognitive functions.

    Science.gov (United States)

    Ogara, María Florencia; Belluscio, Laura M; de la Fuente, Verónica; Berardino, Bruno G; Sonzogni, Silvina V; Byk, Laura; Marazita, Mariela; Cánepa, Eduardo T

    2014-07-01

    DNA damage, which perturbs genomic stability, has been linked to cognitive decline in the aging human brain, and mutations in DNA repair genes have neurological implications. Several studies have suggested that DNA damage is also increased in brain disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise mechanisms connecting DNA damage with neurodegeneration remain poorly understood. CDK5, a critical enzyme in the development of the central nervous system, phosphorylates a number of synaptic proteins and regulates dendritic spine morphogenesis, synaptic plasticity and learning. In addition to these physiological roles, CDK5 has been involved in the neuronal death initiated by DNA damage. We hypothesized that p19INK4d, a member of the cell cycle inhibitor family INK4, is involved in a neuroprotective mechanism activated in response to DNA damage. We found that in response to genotoxic injury or increased levels of intracellular calcium, p19INK4d is transcriptionally induced and phosphorylated by CDK5 which provides it with greater stability in postmitotic neurons. p19INK4d expression improves DNA repair, decreases apoptosis and increases neuronal survival under conditions of genotoxic stress. Our in vivo experiments showed that decreased levels of p19INK4d rendered hippocampal neurons more sensitive to genotoxic insult resulting in the loss of cognitive abilities that rely on the integrity of this brain structure. We propose a feedback mechanism by which the neurotoxic effects of CDK5-p25 activated by genotoxic stress or abnormal intracellular calcium levels are counteracted by the induction and stabilization of p19INK4d protein reducing the adverse consequences on brain functions. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. FGFR3, PIK3CA and RAS mutations in benign lichenoid keratosis.

    Science.gov (United States)

    Groesser, L; Herschberger, E; Landthaler, M; Hafner, C

    2012-04-01

    Benign lichenoid keratoses (BLKs) are solitary skin lesions which have been proposed to represent a regressive form of pre-existent epidermal tumours such as solar lentigo or seborrhoeic keratosis. However, the genetic basis of BLK is unknown. FGFR3, PIK3CA and RAS mutations have been shown to be involved in the pathogenesis of seborrhoeic keratosis and solar lentigo. We thus investigated whether these mutations are also present in BLK. After manual microdissection and DNA isolation, 52 BLKs were screened for FGFR3, PIK3CA and RAS hotspot mutations using SNaPshot(®) multiplex assays. We identified 6/52 (12%) FGFR3 mutations, 10/52 (19%) PIK3CA mutations, 6/52 (12%) HRAS mutations and 2/52 (4%) KRAS mutations. FGFR3 and RAS mutations were mutually exclusive. One BLK showed a simultaneous PIK3CA and HRAS mutation. In nine BLKs with a mutation, nonlesional control tissue from the epidermal margin and the dermal lymphocytic infiltrate were wild-type, indicating that these mutations are somatic. To demonstrate that these findings are specific, 10 samples of lichen planus were analysed without evidence for FGFR3, PIK3CA or RAS mutations. Our results indicate that FGFR3, PIK3CA and RAS mutations are present in approximately 50% of BLKs. These findings support the concept on the molecular genetic level that at least a proportion of BLKs represents regressive variants resulting from former benign epidermal tumours such as seborrhoeic keratosis and solar lentigo. © 2011 The Authors. BJD © 2011 British Association of Dermatologists 2011.

  7. Qingfei Xiaoyan Wan, a traditional Chinese medicine formula, ameliorates Pseudomonas aeruginosa–induced acute lung inflammation by regulation of PI3K/AKT and Ras/MAPK pathways

    Directory of Open Access Journals (Sweden)

    Yuanyuan Hou

    2016-05-01

    Full Text Available Gram-negative pathogen–induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF, a traditional Chinese medicine (TCM formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosa–induced acute pneumonia in mice were evaluated. The mechanisms by which four typical anti-inflammatory ingredients from QF, arctigenin (ATG, cholic acid (CLA, chlorogenic acid (CGA and sinapic acid (SPA, regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6 and chemokines (IL-8 and RANTES, reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In addition, ATG was identified as the primary anti-inflammatory agent with action on the PI3K/AKT and Ras/MAPK pathways. CLA and CGA enhanced the actions of ATG and exhibited synergistic NF-κB inactivation effects possibly via the Ras/MAPK signaling pathway. Moreover, CLA is speculated to target FGFR and MEK firstly. Overall, QF regulated the PI3K/AKT and Ras/MAPK pathways to inhibit pathogenic bacterial infections effectively.

  8. Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization.

    Science.gov (United States)

    Taylor, Lisa M; James, Alexander; Schuller, Christine E; Brce, Jesena; Lock, Richard B; Mackenzie, Karen L

    2004-10-15

    Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16INK4a-positive, but not p16INK4a-negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.

  9. [miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

    Science.gov (United States)

    Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

    2016-12-23

    Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P HeLa cells through targeted regulating the expression of K-ras gene. In human cervical cancer tissues of a small sample set, the expression of miR-143 is downregulated, and the expression of K-ras is upregulated.

  10. Endocytosis of hERG Is Clathrin-Independent and Involves Arf6

    Science.gov (United States)

    Abuarab, Nada; Smith, Andrew J.; Hardy, Matthew E. L.; Elliott, David J. S.; Sivaprasadarao, Asipu

    2013-01-01

    The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6. PMID:24392021

  11. 9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

    Science.gov (United States)

    Dodonova, Svetlana O; Aderhold, Patrick; Kopp, Juergen; Ganeva, Iva; Röhling, Simone; Hagen, Wim J H; Sinning, Irmgard; Wieland, Felix; Briggs, John A G

    2017-06-16

    COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.

  12. Analog reactor simulator RAS; Reaktorski analogni simulator RAS

    Energy Technology Data Exchange (ETDEWEB)

    Radanovic, Lj; Bingulac, S; Popovic, D [The Institute of Nuclear Sciences Boris Kidric, Vinca, Beograd (Yugoslavia)

    1961-07-01

    Analog computer RAS was designed as a nuclear reactor simulator, but it can be simultaneously used for solving a number of other problems. This paper contains a brief description of the simulator parts and their principal characteristics.

  13. Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

    Science.gov (United States)

    Wasylyk, Christine; Zheng, Hong; Castell, Christelle; Debussche, Laurent; Multon, Marie-Christine; Wasylyk, Bohdan

    2008-03-01

    Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

  14. Gene expression in skin tumors induced in hairless mice by chronic exposure to ultraviolet B irradiation

    International Nuclear Information System (INIS)

    Sato, Hiromi; Tanaka, Misao; Kobayashi, Shizuko; Suzuki, Junko S.; Ogiso, Manabu; Tohyama, Chiharu

    1997-01-01

    We investigated the expressions of c-Ha-ras, c-jun, c-fos, c-myc genes and p53 protein in the development of skin tumours induced by chronic exposure to UVB without a photosensitizer using hairless mice. When mice were exposed to UVB at a dose of 2 kJ/m 2 three times a week, increased c-Ha-ras and c-myc transcripts were detected after only 5 weeks of exposure, while no tumour appeared on the exposed skin. The increase in gene expression continued until 25 weeks, when tumours, identified pathologically as mainly squamous cell carcinomas (SCC), developed in the dorsal skin. In these SCC, overexpression of c-fos mRNA was also observed along with the increases in c-Ha-ras and c-myc. A single dose of UVB (2 kJ/m 2 ) applied to the backs of hairless mice transiently induced overexpression of the early event genes c-fos, c-jun and c-myc, but not c-Ha-ras, in the exposed area of skin. Accumulation of p53 protein was determined by Western blotting analysis of immunohistochemistry using monoclonal antibodies PAb 240 or 246, which recognize mutant or wide type, respectively. In the SCC, a mutant p53 protein accumulated in the cytoplasm and nucleus. After single-dose irradiation, the increased wild-type p53 protein was observed in the nuclei of epidermal cells. The present results suggest that overexpression of the c-fos, c-myc and c-Ha-ras genes, and the mutational changes in p53 protein might be associated with skin photocarcinogenesis. Moreover, overexpression of the c-Ha-ras and c-myc genes might be an early event in the development of UVB-induced skin tumors in mice. (author)

  15. Using the capital markets in Ras Gas

    International Nuclear Information System (INIS)

    Voge, B.; Penzer, M.

    1997-01-01

    In December 1996, Ras Laffan Liquefied Natural Gas Company Ltd (Ras Gas) closed a multi-source financing that included an offering of US$1.2bn of bonds. The sponsors of the Ras Gas project overcame a number of obstacles on the road to closing the capital markets offering. This article provides a general overview of capital markets offerings in international project financings and discusses how Ras Gas was able to successfully integrate a capital markets offering into a financing plan which included a commercial bank facility and several export-credit agency facilities. (Author)

  16. Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Shenyuan; Long, Brian N.; Boris, Gabriel H.; Chen, Anqi; Ni, Shuisong; Kennedy, Michael A.

    2017-11-10

    <p>K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.p>

  17. Effects of mutant human Ki-rasG12C gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

    International Nuclear Information System (INIS)

    Dance-Barnes, Stephanie T.; Kock, Nancy D.; Floyd, Heather S.; Moore, Joseph E.; Mosley, Libyadda J.; D'Agostino, Ralph B.; Pettenati, Mark J.; Miller, Mark Steven

    2008-01-01

    Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras G12C allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 μg/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras G12C allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 μg/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 μg/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 μg/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras G12C expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models

  18. Ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding

    International Nuclear Information System (INIS)

    Comerford, J.G.; Gibson, J.R.; Dawson, A.P.; Gibson, I.

    1989-01-01

    The presence of guanine nucleotide binding proteins in mouse and human cell lines was investigated using [gamma- 35 S]GTP gamma S and [gamma-32P]GTP. Cell lysate polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma- 35 S]GTP gamma S identified 9 distinct GTP-binding polypeptides in all lysates. One of these is the ras oncogene product, p21, as demonstrated by subsequent immunochemical staining of the nitrocellulose blots. We have shown that this procedure provides a sensitive method for detection of p21 in culture cell lines

  19. The human T-lymphotropic virus type I tax gene can cooperate with the ras oncogene to induce neoplastic transformation of cells.

    Science.gov (United States)

    Pozzatti, R; Vogel, J; Jay, G

    1990-01-01

    Epidemiologic studies have linked infection by the human T-lymphotropic virus type I (HTLV-I) with the development of adult T-cell leukemia. The low penetrance of the virus and the long latency for disease manifestation are factors that obscure the role of HTLV-I infection in oncogenesis. We have used an in vitro transformation assay system to determine directly whether the HTLV-I tax gene has transformation potential. Transfection of the tax gene alone into early-passage rat embryo fibroblasts did not induce morphological alterations. However, cotransfection of tax with the selectable marker plasmid pRSVneo gave rise to G418-resistant colonies that could be established as immortalized cell lines. Cotransfection of tax with the ras oncogene into rat embryo fibroblasts gave rise to foci of transformed cells that were highly tumorigenic in nude mice. These data represent a direct demonstration of the oncogenic potential of the tax gene in nonlymphoid cells and establish HTLV-I as a transforming virus.

  20. In situ monitoring of the surface reconstructions on InP(001) prepared by molecular beam epitaxy

    Science.gov (United States)

    Ozanyan, K. B.; Parbrook, P. J.; Hopkinson, M.; Whitehouse, C. R.; Sobiesierski, Z.; Westwood, D. I.

    1997-07-01

    Reflection anisotropy spectroscopy (RAS) and reflection high-energy electron diffraction (RHEED) were applied to study clean InP(001) surfaces prepared by molecular beam epitaxy (MBE). At phosphorus beam equivalent pressures (BEPs) between 3.5×10-7 and 3.5×10-6 mbar and substrate temperature (Ts) falling from 590 to 150 °C, (2×4), (2×1), (2×2), and c(4×4) RHEED patterns are observed. The main RAS features, observed at 1.7-1.9 and 2.6-2.9 eV are assigned to In and P dimers, respectively. The above reconstruction sequence is associated closely with transformations identified in RAS signatures that are induced by progressively increasing the P surface coverage. The RAS results also imply the existence of (2×4)α and (2×4)β phases. A surface-phase diagram for MBE-grown (001) InP, in the whole range of Ts and phosphorus BEPs is proposed.

  1. Inhibition of RAS in diabetic nephropathy

    Directory of Open Access Journals (Sweden)

    Yacoub R

    2015-04-01

    Full Text Available Rabi Yacoub, Kirk N Campbell Department of Medicine, Division of Nephrology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Abstract: Diabetic kidney disease (DKD is a progressive proteinuric renal disorder in patients with type 1 or type 2 diabetes mellitus. It is a common cause of end-stage kidney disease worldwide, particularly in developed countries. Therapeutic targeting of the renin–angiotensin system (RAS is the most validated clinical strategy for slowing disease progression. DKD is paradoxically a low systematic renin state with an increased intrarenal RAS activity implicated in its pathogenesis. Angiotensin II (AngII, the main peptide of RAS, is not only a vasoactive peptide but functions as a growth factor, activating interstitial fibroblasts and mesangial and tubular cells, while promoting the synthesis of extracellular matrix proteins. AngII also promotes podocyte injury through increased calcium influx and the generation of reactive oxygen species. Blockade of the RAS using either angiotensin converting enzyme inhibitors, or angiotensin receptor blockers can attenuate progressive glomerulosclerosis in animal models, and slows disease progression in humans with DKD. In this review, we summarize the role of intrarenal RAS activation in the pathogenesis and progression of DKD and the rationale for RAS inhibition in this population. Keywords: renin–angiotensin system, diabetic kidney disease, angiotensin II, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers

  2. Urine RAS components in mice and people with type 1 diabetes and chronic kidney disease.

    Science.gov (United States)

    Wysocki, Jan; Goodling, Anne; Burgaya, Mar; Whitlock, Kathryn; Ruzinski, John; Batlle, Daniel; Afkarian, Maryam

    2017-08-01

    The pathways implicated in diabetic kidney disease (DKD) are largely derived from animal models. To examine if alterations in renin-angiotensin system (RAS) in humans are concordant with those in rodent models, we measured concentration of angiotensinogen (AOG), cathepsin D (CTSD), angiotensin-converting enzyme (ACE), and ACE2 and enzymatic activities of ACE, ACE2, and aminopeptidase-A in FVB mice 13-20 wk after treatment with streptozotocin ( n = 9) or vehicle ( n = 15) and people with long-standing type 1 diabetes, with ( n = 37) or without ( n = 81) DKD. In streptozotocin-treated mice, urine AOG and CTSD were 10.4- and 3.0-fold higher than in controls, respectively ( P animals ( P animals ( P = 0.017). Compared with people without DKD, those with DKD had higher urine AOG (170 vs. 15 μg/g) and CTSD (147 vs. 31 μg/g). In people with DKD, urine ACE concentration was 1.8-fold higher (1.4 vs. 0.8 μg/g in those without DKD), while its enzymatic activity was 0.6-fold lower (1.0 vs. 1.6 × 10 9 RFU/g in those without DKD). Lower ACE activity, but not ACE protein concentration, was associated with ACE inhibitor (ACEI) treatment. After adjustment for clinical covariates, AOG, CTSD, ACE concentration, and ACE activity remained associated with DKD. In conclusion, in mice with streptozotocin-induced diabetes and in humans with DKD, urine concentrations and enzymatic activities of several RAS components are concordantly increased, consistent with enhanced RAS activity and greater angiotensin II formation. ACEI use was associated with a specific reduction in urine ACE activity, not ACE protein concentration, suggesting that it may be a marker of exposure to this widely-used therapy. Copyright © 2017 the American Physiological Society.

  3. Pharmacological investigations of Punica granatum in glycerol-induced acute renal failure in rats.

    Science.gov (United States)

    Singh, Amrit Pal; Singh, Amteshwar Jaggi; Singh, Nirmal

    2011-09-01

    The present study was designed to investigate the ameliorative potential and possible mechanism of hydroalcoholic extract of flowers of P. granatum in glycerol-induced acute renal failure (ARF) in rats. The rats were subjected to rhabdomyolytic ARF by single intramuscular injection of hypertonic glycerol (50% v/v; 8 ml/kg) and the animals were sacrificed after 24 hours of glycerol injection. The plasma creatinine, blood urea nitrogen, creatinine clearance, and histopathological studies were performed to assess the degree of renal injury. Pretreatment with hydroalcoholic extract of flowers of P. granatum (125 and 250 mg/kg p.o. twice daily for 3 days) significantly attenuated hypertonic glycerol-induced renal dysfunction in a dose-dependent manner. BADGE (Bisphenol-A-diglycidyl ether) (30 mg/kg), a peroxisome proliferator-activated receptor (PPAR)-γ antagonist, and N(omega)-nitro-l-arginine-methyl ester (L-NAME) (10, 20, and 40 mg/kg), nitric oxide synthase inhibitor, were employed to explore the mechanism of renoprotective effects of Punica granatum. Administration of BADGE (30 mg/kg) and L-NAME (40 mg/kg) abolished the beneficial effects of P. granatum in glycerol-induced renal dysfunction. Hydroalcoholic extract of flowers of P. granatum has ameliorative potential in attenuating myoglobinuric renal failure and its renoprotective effects involve activation of PPAR-γ and nitric oxide-dependent signaling pathway.

  4. P53, K-RAS, β-CATENIN, C-KIT and BAK mutations in the lung cancer of Chinese and Japanese patients

    International Nuclear Information System (INIS)

    Shuo Xing; Nobotoshi Nawa; Kazuhiro Tanabe; Tadashi Hongyo; Li- Ya Li; Jing-Tian Tang; Mitsunori Ohta

    2005-01-01

    Seventeen Chinese (Beijing) and 24 Japanese (Osaka) lung cancer cases were analyzed for mutations of p53, K-ras, β-catenin, c-kit and bak genes by PCR-SSCP analysis followed by direct sequencing. Significantly higher mutation frequency of p53 gene, one of key genes for radiation sensitivity, was found in Chinese cases (11/17; 64.7 %) than Japanese cases (8/24; 33.3 %) (p< O.O5). Fourteen of the 16 mutations found in the Chinese cases were transitions at exon 4,5 and intron 4. In the Japanese cases, of the total of 11 mutations, 5 were transitions and 5 were transversions and one was deletion. Six β-catenin mutations were found in 6 Chinese cases (35.3 % ) at codon 53 and 58, and 4 were found in 3 Japanese cases (12.5 %). C-kit mutations were detected in 5 Chinese cases (29.4 %), while no mutations were found in Japanese cases (p< O.O5). No K-ras mutation was found in both Chinese and Japanese cases. For the first time, we report on bak mutation in human lung cancer in Chinese (2/17; 11.8% ) and Japanese cases (2/24; 8.3% ). C-kit and bak genes are also definitive factors to radiosensitivity. These data thus suggest that there were apparent differences in frequency and/or mutational types of p53, β-catenin and c-kit? genes between Chinese and Japanese cases. The differences can be attributed to factors such as lifestyles including smoking and racial and/or environmental factors, and also to the prediction of the response to radiotherapy. (author)

  5. Renal Artery Stenosis (RAS) Case study

    International Nuclear Information System (INIS)

    Zaater, M.K.

    2012-01-01

    Renal Artery Stenosis (RAS), is one of the causes of secondary hypertension; there are many causes of renal artery stenosis, as atherosclerosis of the renal artery which account for 90% of cases of RAS; fibromuscular dysplasia accounts for 10% of RAS. Various causes of thrombophilia either due congenital causes or acquired causes and can lead to RAS. Our patient was presented by acute attack of epistaxis and hypertension. Angiography of the Renal Arteries,are showed no sign of renal artery stenosis. However, the right kidney showed upper pole infarction, and the left kidney showed evidence of functional lower pole renal artery stenosis, although there is no anatomical stenosis detected in angiography. Work up for the cause of thrombophilia did not help in the diagnosis, which may be due to an undiscovered cause of thrombophilia

  6. Differences in K-ras and mitochondrial DNA mutations and microsatellite instability between colorectal cancers of Vietnamese and Japanese patients.

    Science.gov (United States)

    Miwata, Tomohiro; Hiyama, Toru; Quach, Duc Trong; Le, Huy Minh; Hua, Ha Ngoc Thi; Oka, Shiro; Tanaka, Shinji; Arihiro, Koji; Chayama, Kazuaki

    2014-11-30

    The incidence of early-onset (under 50 years of age) colorectal cancer (CRC) in the Vietnamese has been reported to be quite higher than that in the Japanese. To clarify the differences in genetic alterations between Vietnamese and Japanese CRCs, we investigated mutations in K-ras and mitochondrial DNA (mtDNA) and high-frequency microsatellite instability (MSI-H) in the CRCs of Vietnamese and Japanese patients. We enrolled 60 Vietnamese and 233 Japanese patients with invasive CRCs. DNA was extracted from formalin-fixed, paraffin-embedded tissue sections. K-ras mutations were examined with PCR-single-strand conformation polymorphism analysis. mtDNA mutations and MSI-H were examined with microsatellite analysis using D310 and BAT-26, respectively. K-ras mutations were examined in 60 Vietnamese and 45 Japanese CRCs. The frequency of the mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (8 of 24 [33%] vs 5 of 45 [11%], p =0.048). MSI-H was examined in 60 Vietnamese and 130 Japanese CRCs. The frequency of MSI-H in the Vietnamese CRCs was also significantly higher than that in the Japanese CRCs (6 of 27 [22%] vs 10 of 130 [8%], p =0.030). mtDNA mutations were examined in 60 Vietnamese and 138 Japanese CRCs. The frequency of mtDNA mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (19 of 44 [43%] vs 11 of 133 [9%], p Vietnamese and Japanese patients. These results indicate that the developmental pathways of CRCs in the Vietnamese may differ from those of CRCs in the Japanese.

  7. Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

    Directory of Open Access Journals (Sweden)

    Tracy P. M. Chong

    2011-03-01

    Full Text Available The potent mitogenic toxin from Pasteurella multocida (PMT is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH4Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn and cholera toxin (CT, the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity.

  8. Role of mTOR, Bad, and Survivin in RasGAP Fragment N-Mediated Cell Protection

    Science.gov (United States)

    Yang, Jiang-Yan; Widmann, Christian

    2013-01-01

    Partial cleavage of p120 RasGAP by caspase-3 in stressed cells generates an N-terminal fragment, called fragment N, which activates an anti-apoptotic Akt-dependent survival response. Akt regulates several effectors but which of these mediate fragment N-dependent cell protection has not been defined yet. Here we have investigated the role of mTORC1, Bad, and survivin in the capacity of fragment N to protect cells from apoptosis. Neither rapamycin, an inhibitor of mTORC1, nor silencing of raptor, a subunit of the mTORC1 complex, altered the ability of fragment N from inhibiting cisplatin- and Fas ligand-induced death. Cells lacking Bad, despite displaying a stronger resistance to apoptosis, were still protected by fragment N against cisplatin-induced death. Fragment N was also able to protect cells from Fas ligand-induced death in conditions where Bad plays no role in apoptosis regulation. Fragment N expression in cells did neither modulate survivin mRNA nor its protein expression. Moreover, the expression of cytoplasmic survivin, known to exert anti-apoptotic actions in cells, still occurred in UV-B-irradiated epidermis of mouse expressing a caspase-3-resistant RasGAP mutant that cannot produce fragment N. Additionally, survivin function in cell cycle progression was not affected by fragment N. These results indicate that, taken individually, mTOR, Bad, or Survivin are not required for fragment N to protect cells from cell death. We conclude that downstream targets of Akt other than mTORC1, Bad, or survivin mediate fragment N-induced protection or that several Akt effectors can compensate for each other to induce the pro-survival fragment N-dependent response. PMID:23826368

  9. Flavopiridol Synergizes with Sorafenib to Induce Cytotoxicity and Potentiate Antitumorigenic Activity in EGFR/HER-2 and Mutant RAS/RAF Breast Cancer Model Systems

    Directory of Open Access Journals (Sweden)

    Teddy S Nagaria

    2013-08-01

    Full Text Available Oncogenic receptor tyrosine kinase (RTK signaling through the Ras-Raf-Mek-Erk (Ras-MAPK pathway is implicated in a wide array of carcinomas, including those of the breast. The cyclin-dependent kinases (CDKs are implicated in regulating proliferative and survival signaling downstream of this pathway. Here, we show that CDK inhibitors exhibit an order of magnitude greater cytotoxic potency than a suite of inhibitors targeting RTK and Ras-MAPK signaling in cell lines representative of clinically recognized breast cancer (BC subtypes. Drug combination studies show that the pan-CDK inhibitor, flavopiridol (FPD, synergistically potentiated cytotoxicity induced by the Raf inhibitor, sorafenib (SFN. This synergy was most pronounced at sub-EC50 SFN concentrations in MDA-MB-231 (KRAS-G13D and BRAF-G464V mutations, MDA-MB-468 [epidermal growth factor receptor (EGFR overexpression], and SKBR3 [ErbB2/EGFR2 (HER-2 overexpression] cells but not in hormone-dependent MCF-7 and T47D cells. Potentiation of SFN cytotoxicity by FPD correlated with enhanced apoptosis, suppression of retinoblastoma (Rb signaling, and reduced Mcl-1 expression. SFN and FPD were also tested in an MDA-MB-231 mammary fat pad engraftment model of tumorigenesis. Mice treated with both drugs exhibited reduced primary tumor growth rates and metastatic tumor load in the lungs compared to treatment with either drug alone, and this correlated with greater reductions in Rb signaling and Mcl-1 expression in resected tumors. These findings support the development of CDK and Raf co-targeting strategies in EGFR/HER-2-overexpressing or RAS/RAF mutant BCs.

  10. CREBBP knockdown enhances RAS/RAF/MEK/ERK signaling in Ras pathway mutated acute lymphoblastic leukemia but does not modulate chemotherapeutic response.

    Science.gov (United States)

    Dixon, Zach A; Nicholson, Lindsay; Zeppetzauer, Martin; Matheson, Elizabeth; Sinclair, Paul; Harrison, Christine J; Irving, Julie A E

    2017-04-01

    Relapsed acute lymphoblastic leukemia is the most common cause of cancer-related mortality in young people and new therapeutic strategies are needed to improve outcome. Recent studies have shown that heterozygous inactivating mutations in the histone acetyl transferase, CREBBP , are particularly frequent in relapsed childhood acute lymphoblastic leukemia and associated with a hyperdiploid karyotype and KRAS mutations. To study the functional impact of CREBBP haploinsufficiency in acute lymphoblastic leukemia, RNA interference was used to knock down expression of CREBBP in acute lymphoblastic leukemia cell lines and various primagraft acute lymphoblastic leukemia cells. We demonstrate that attenuation of CREBBP results in reduced acetylation of histone 3 lysine 18, but has no significant impact on cAMP-dependent target gene expression. Impaired induction of glucocorticoid receptor targets was only seen in 1 of 4 CREBBP knockdown models, and there was no significant difference in glucocorticoid-induced apoptosis, sensitivity to other acute lymphoblastic leukemia chemotherapeutics or histone deacetylase inhibitors. Importantly, we show that CREBBP directly acetylates KRAS and that CREBBP knockdown enhances signaling of the RAS/RAF/MEK/ERK pathway in Ras pathway mutated acute lymphoblastic leukemia cells, which are still sensitive to MEK inhibitors. Thus, CREBBP mutations might assist in enhancing oncogenic RAS signaling in acute lymphoblastic leukemia but do not alter response to MEK inhibitors. Copyright© Ferrata Storti Foundation.

  11. Exploiting the bad eating habits of Ras-driven cancers.

    Science.gov (United States)

    White, Eileen

    2013-10-01

    Oncogenic Ras promotes glucose fermentation and glutamine use to supply central carbon metabolism, but how and why have only emerged recently. Ras-mediated metabolic reprogramming generates building blocks for growth and promotes antioxidant defense. To fuel metabolic pathways, Ras scavenges extracellular proteins and lipids. To bolster metabolism and mitigate stress, Ras activates cellular self-cannibalization and recycling of proteins and organelles by autophagy. Targeting these distinct features of Ras-driven cancers provides novel approaches to cancer therapy.

  12. Inhibition of proliferation and differentiation and promotion of apoptosis by cyclin L2 in mouse embryonic carcinoma P19 cells

    International Nuclear Information System (INIS)

    Zhuo, Lili; Gong, Jie; Yang, Rong; Sheng, Yanhui; Zhou, Lei; Kong, Xiangqing; Cao, Kejiang

    2009-01-01

    Cyclin L2 (CCNL2) is a novel member of the cyclin gene family. In a previous study, we demonstrated that CCNL2 expression was upregulated in ventricular septum tissues from patients with ventricular septal defect compared to healthy controls. In the present study, we established a stable CCNL2-overexpressing P19 cell line that can differentiate to myocardial cells when treated with 1% dimethyl sulfoxide (DMSO). Our data showed that stable CCNL2-overexpressing P19 cells were less differentiated after treatment with 1% DMSO and that expression of myocardial cell differentiation-related genes (such as cardiac actin, GATA4, Mef2C, Nkx2.5, and BNP) were reduced compared to vector-only transfected P19. Moreover, P19 cells overexpressing the CCNL2 gene had a reduced growth rate and a remarkably decreased S phase. We also found that these cells underwent apoptosis, as detected by two different apoptosis assays. The anti-apoptotic Bcl-2 protein was also downregulated in these cells. In addition, real-time PCR analysis revealed that expression of Wnt and β-catenin was suppressed and GSK3β was induced in the CCNL2-overexpressing P19 cells. These data suggest that overexpression of CCNL2 inhibited proliferation and differentiation of mouse embryonic carcinoma P19 cells and induced them to undergo apoptosis, possibly through the Wnt signal transduction pathway.

  13. Codon 61 mutations in the c-Harvey-ras gene in mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene plus okadaic acid class tumor promoters.

    Science.gov (United States)

    Fujiki, H; Suganuma, M; Yoshizawa, S; Kanazawa, H; Sugimura, T; Manam, S; Kahn, S M; Jiang, W; Hoshina, S; Weinstein, I B

    1989-01-01

    Three okadaic acid class tumor promoters, okadaic acid, dinophysistoxin-1, and calyculin A, have potent tumor-promoting activity in two-stage carcinogenesis experiments on mouse skin. DNA isolated from tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and each of these tumor promoters revealed the same mutation at the second nucleotide of codon 61 (CAA----CTA) in the c-Ha-ras gene, determined by the polymerase chain reaction procedure and DNA sequencing. Three potent 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, TPA, teleocidin, and aplysiatoxin, showed the same effects. These results provide strong evidence that this mutation in the c-Ha-ras gene is due to a direct effect of DMBA rather than a selective effect of specific tumor promoters.

  14. RasGRP1, but not RasGRP3, is required for efficient thymic β-selection and ERK activation downstream of CXCR4.

    Directory of Open Access Journals (Sweden)

    Dominic P Golec

    Full Text Available T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4(-CD8(- 'double negative' (DN thymocytes, pass through a checkpoint termed "β-selection" before maturing into CD4(+CD8(+ 'double positive' (DP thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ(+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

  15. Chronic Renin-Angiotensin System (RAS) Blockade May Not Induce Hypotension During Anaesthesia for Bariatric Surgery.

    Science.gov (United States)

    Salvetti, Guido; Di Salvo, Claudio; Ceccarini, Giovanni; Abramo, Antonio; Fierabracci, Paola; Magno, Silvia; Piaggi, Paolo; Vitti, Paolo; Santini, Ferruccio

    2016-06-01

    The use of angiotensin-converting enzyme inhibitors (ACE-I) and angiotensin II receptor blockers (ARB) for the treatment of hypertensive obese patients is steadily increasing. Some studies have reported that the use of these drugs was associated with an increased risk of hypotensive episodes, during general anaesthesia. The number of bariatric procedures is also increasing worldwide, but there is a lack of studies investigating the hypotensive effect of renin-angiotensin system (RAS) blockers in severely obese patients during general anaesthesia for bariatric surgery. The aim of this pilot study was to evaluate hemodynamic changes induced by general anaesthesia in obese patients chronically treated with ACE-I or ARB compared to a control group not treated with antihypertensive therapy. Fourteen obese subjects (mean body mass index (BMI) 47.5 kg/m(2)) treated with ACE-I or ARB and twelve obese (mean BMI 45.7 kg/m2) controls not treated with antihypertensive therapy underwent general anaesthesia to perform laparoscopic bariatric surgery. Systolic blood pressure, diastolic blood pressure, and heart rate were monitored continuously and registered at different time points: T0 before induction, then at 2, 5, 7, 10, 15, 20, 30, 60, 90, 120, and 150 min after induction, and the last time point taken following recovery from anaesthesia. A progressive reduction of both systolic and diastolic blood pressure values was observed without significant differences between the two groups. A similar trend of heart rate values was observed. In conclusion, our pilot study suggests that RAS blockers may be continued during the perioperative period in patients undergoing bariatric surgery, without increasing the risk of hypotensive episodes.

  16. Cell cycle inhibitor, p19INK4d, promotes cell survival and decreases chromosomal aberrations after genotoxic insult due to enhanced DNA repair.

    Science.gov (United States)

    Scassa, María E; Marazita, Mariela C; Ceruti, Julieta M; Carcagno, Abel L; Sirkin, Pablo F; González-Cid, Marcela; Pignataro, Omar P; Cánepa, Eduardo T

    2007-05-01

    Genome integrity and cell proliferation and survival are regulated by an intricate network of pathways that includes cell cycle checkpoints, DNA repair and recombination, and programmed cell death. It makes sense that there should be a coordinated regulation of these different processes, but the components of such mechanisms remain unknown. In this report, we demonstrate that p19INK4d expression enhances cell survival under genotoxic conditions. By using p19INK4d-overexpressing clones, we demonstrated that p19INK4d expression correlates with the cellular resistance to UV treatment with increased DNA repair activity against UV-induced lesions. On the contrary, cells transfected with p19INK4d antisense cDNA show reduced ability to repair DNA damage and increased sensitivity to genotoxic insult when compared with their p19INK4d-overexpressing counterparts. Consistent with these findings, our studies also show that p19INK4d-overexpressing cells present not only a minor accumulation of UV-induced chromosomal aberrations but a lower frequency of spontaneous chromosome abnormalities than p19INK4d-antisense cells. Lastly, we suggest that p19INK4d effects are dissociated from its role as CDK4/6 inhibitor. The results presented herein support a crucial role for p19INK4d in regulating genomic stability and overall cell viability under conditions of genotoxic stress. We propose that p19INK4d would belong to a protein network that would integrate DNA repair, apoptotic and checkpoint mechanisms in order to maintain the genomic integrity.

  17. Electrostatic Interactions Positively Regulate K-Ras Nanocluster Formation and Function▿

    Science.gov (United States)

    Plowman, Sarah J.; Ariotti, Nicholas; Goodall, Andrew; Parton, Robert G.; Hancock, John F.

    2008-01-01

    The organization of Ras proteins into plasma membrane nanoclusters is essential for high-fidelity signal transmission, but whether the nanoscale enviroments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras-GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras-GTP nanoclusters. Similarly, upon epidermal growth factor receptor activation, Raf-1 is preferentially recruited to K-Ras-GTP and not H-Ras-GTP nanoclusters. The formation of K-Ras-GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not, however, regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together, these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades. PMID:18458061

  18. Molecular markers for diagnostic cytology of neoplasms in the head region of the pancreas: mutation of K-ras and overexpression of the p53 protein product

    NARCIS (Netherlands)

    van Es, J. M.; Polak, M. M.; van den Berg, F. M.; Ramsoekh, T. B.; Craanen, M. E.; Hruban, R. H.; Offerhaus, G. J.

    1995-01-01

    To determine the potential efficiency of molecular markers specific for neoplastic change--mutations of the K-ras oncogene and the p53 tumour suppressor gene--in diagnosing pancreatic carcinoma. Archival cytology samples obtained from 17 patients with established pancreatic carcinoma were assayed

  19. Investigation of pUC19 DNA damage induced by direct and indirect effect of 7Li ions radiation

    International Nuclear Information System (INIS)

    Sui Li; Zhao Kui; Guo Jiyu; Ni Meinan; Kong Fuquan; Cai Minghui; Yang Mingjian

    2006-01-01

    The effect of direct and indirect action on DNA damage in 7 Li ions radiation is investigated. Using 7 Li ions generated by HI-13 tandem accelerator, three conditions of pUC19 plasmid DNA samples including dry, with or without mannitol are irradiated at different doses in air. These irradiated DNA samples are analyzed with atomic force microscopy (AFM) in nanometer-scale. The changes of DNA forms as the dose increases are observed. The results show that free radical is the main factor in DNA strand breaks induced by 7 Li ions radiation under condition of aqueous solution. The mannitol can effectively scavenge free radical and reduce the yields of DNA strand breaks. The experimental results of this report can offered valuable basal data for cancer therapy by boron neutron capture therapy (BNCT) or heavy ion radiation method, etc. (author)

  20. Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

    International Nuclear Information System (INIS)

    Dominguez, J.M.; Lanoix, J.; Paiement, J.

    1991-01-01

    We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha- 32 P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha- 32 P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations

  1. Circulating forms of immunoreactive parathyroid hormone-related protein for identifying patients with humoral hypercalcemia of malignancy. A comparative study with C-terminal (109-141)- and N-terminal (1-86)-region-specific PTHrP radioassay

    International Nuclear Information System (INIS)

    Suehiro, Mitsuko; Murakami, Minoru; Fukuchi, Minoru

    1994-01-01

    We evaluated the circulating forms of immunoreactive parathyroid hormone-related protein(PTHrP) in 115 healthy subjects and 122 patients with malignant diseases by using radioassay systems (RAS) specific for the C-terminal (109-141) fragment of PTHrP (C-RAS) and for the N-terminal(1-86) (N-RAS). PTHrP levels in healthy controls ranged from 1.5 to 38.2 (mean: 24.5) pmol/L with the C-RAS and from 0.9 to 2.5 (mean: 1.7) pmol/L with the N-RAS. The ratio of circulating N-terminal fragment (N) to C-terminal fragment (C) of PTHrP was calculated to be about 1 : 14.4 in the healthy subjects. Of the 122 patients with malignant diseases, 40 (32.8%) had circulating PTHrP levels undetectable with the N-RAS, but only 11 (9.0%) patients had levels undetectable with the C-RAS. Of the former 122 patients, 41 (33.6%) had high PTHrP as determined with the C-RAS, and 10 (8.2%) had high PTHrP as determined with the N-RAS. The former of these included only 8 (19.5%) humoral hypercalcemia malignancy(HHM) patients, while the latter included 8 (80.0%) HHM patients. The circulating N to C ratio was about 1 : 70.7 in the HHM patients. The N and C obtained with the different RASs showed a close correlation (r=0.86). The values also showed a close correlation with serum Ca; r=0.75 for C-RAS and r=0.81 for N-RAS. In addition, the correlation between the PTHrP reading obtained with the different RASs and serum Cr were: r=0.42 with C-RAS and r=0.26 with N-RAS. The circulating form of immunoreactive PTHrP fragments is therefore comprised mainly of PTHrP (109-141). In contrast, circulating concentrations of the PTHrP (1-86) fragment are very low, but detection of the PTHrP (1-86) fragment with the N-RAS is a more useful indicator of HHM with fewer false positive results and is less likely to be influenced by renal function than the detection of the PHPrP (109-141) fragment with C-RAS. (author)

  2. A Novel Ras Effector Pathway Found to Play Significant Role in Tumor Suppression | Poster

    Science.gov (United States)

    By Nancy Parrish, Staff Writer; photo by Richard Frederickson, Staff Photographer Normal cells have mechanisms to prevent the development of cancer. Among these is a type of tumor suppressor mechanism known as oncogene-induced senescence, or OIS, which halts the uncontrolled growth of cells caused by mutations in oncogenes. The oncogene Ras plays a crucial role in inducing OIS

  3. Atorvastatin inhibits insulin synthesis by inhibiting the Ras/Raf/ERK/CREB pathway in INS-1 cells

    Science.gov (United States)

    Sun, Hongxi; Li, Yu; Sun, Bei; Hou, Ningning; Yang, Juhong; Zheng, Miaoyan; Xu, Jie; Wang, Jingyu; Zhang, Yi; Zeng, Xianwei; Shan, Chunyan; Chang, Bai; Chen, Liming; Chang, Baocheng

    2016-01-01

    Abstract Backround: Type 2 diabetes has become a global epidemic disease. Atorvastatin has become a cornerstone in the prevention and treatment of atherosclerosis. However, increasing evidence showed that statins can dose-dependently increase the risk of diabetes mellitus. The mechanism is not clear. Objective: The Ras complex pathway (Ras/Raf/extracellular signal-regulated kinase [ERK]/cAMP response element-binding protein [CREB]) is the major pathway that regulates the gene transcription. Except for the inhibition of cholesterol synthesis by inhibiting the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-COA) reductase, statins can also downregulate the phosphorylation of a series of downstream substrates including the key proteins of the Ras complex pathway, therefore may inhibit the insulin syntheses in pancreatic beta cells. In our study, we investigated the inhibitory effect and the underlying mechanism of atorvastatin on insulin synthesis in rat islets. Methods: Islets were isolated from Wistar rats and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. The insulin content in the medium was measured by radioimmunoassay before and after the treatment of 50 μM atorvastatin. Effect of atorvastatin on the expression of insulin message Ribonucleic acid (mRNA) in pancreatic islet beta cells was also detected using quantitative real-time polymerase chain reaction. Western blotting was used to explore the possible role of the Ras complex pathway (Ras/Raf/ERK/CREB) in atorvastatin-inhibited insulin synthesis. The effects of atorvastatin on the binding of nuclear transcription factor p-CREB with CRE in INS-1 cells were examined via chromatin immunoprecipitation assay. Results: Compared with the control group, the insulin level decreased by 27.1% at 24 hours after atorvastatin treatment. Atorvastatin inhibited insulin synthesis by decreasing insulin mRNA expression of pancreatic islet beta cells. The activities of Ras, Raf-1, and p-CREB in the Ras complex

  4. The Frequency and Type of K-RAS Mutations in Mexican Patients With Colorectal Cancer: A National Study.

    Science.gov (United States)

    Cárdenas-Ramos, Susana G; Alcázar-González, Gregorio; Reyes-Cortés, Luisa M; Torres-Grimaldo, Abdiel A; Calderón-Garcidueñas, Ana L; Morales-Casas, José; Flores-Sánchez, Patricia; De León-Escobedo, Raúl; Gómez-Díaz, Antonio; Moreno-Bringas, Carmen; Sánchez-Guillén, Jorge; Ramos-Salazar, Pedro; González-de León, César; Barrera-Saldaña, Hugo A

    2017-06-01

    Current metastatic colorectal cancer (mCRC) therapy uses monoclonal antibodies against the epidermal growth factor receptor. This treatment is only useful in the absence of K-RAS gene mutations; therefore the study of such mutations is part of a personalized treatment. The aim of this work is to determine the frequency and type of the most common K-RAS mutations in Mexican patients with metastatic disease by nucleotide sequencing. We studied 888 patients with mCRC from different regions of Mexico. The presence of mutations in exon 2, codons 12 and 13, of the K-RAS gene was determined by nucleotide sequencing. Patients exhibited K-RAS gene mutations in 35% (310/888) of cases. Mutation frequency of codons 12 and 13 was 71% (221/310) and 29% (89/310), respectively. The most common mutation (45.7%) in codon 12 was c.35G>A (p.G12D), whereas the one in codon 13 was c.38G>A (p.G13D) (78.7%). Given the frequency of K-RAS mutations in Mexicans, making a genetic study before deciding to treat mCRC patients with monoclonal antibodies is indispensable.

  5. The Arf-GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1 and HDS2 and a Golgi-localized protein receptor.

    Science.gov (United States)

    Quilty, Douglas; Chan, Calvin J; Yurkiw, Katherine; Bain, Alexandra; Babolmorad, Ghazal; Melançon, Paul

    2018-04-19

    We previously proposed a novel mechanism by which the enzyme Golgi-specific Brefeldin A resistance factor 1 (GBF1) is recruited to the membranes of the cis -Golgi, based on in vivo experiments. Here, we extended our in vivo analysis on the production of regulatory Arf-GDP and observed that ArfGAP2 and ArfGAP3 do not play a role in GBF1 recruitment. We confirm that Arf-GDP localization is critical, as a TGN-localized Arf-GDP mutant protein fails to promote GBF1 recruitment. We also reported the establishment of an in vitro GBF1 recruitment assay that supports the regulation of GBF1 recruitment by Arf-GDP. This in vitro assay yielded further evidence for the requirement of a Golgi-localized protein because heat denaturation or protease treatment of Golgi membranes abrogated GBF1 recruitment. Finally, combined in vivo and in vitro measurements indicated that the recruitment to Golgi membranes via a putative receptor requires only the HDS1 and HDS2 domains in the C-terminal half of GBF1. © 2018. Published by The Company of Biologists Ltd.

  6. Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

    Directory of Open Access Journals (Sweden)

    Yu Jing

    2009-12-01

    Full Text Available Abstract Background Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma. Methods We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation. Results Promoter methylation of RASSF1A could be detected in 71.05% (27/38 of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p p p p Conclusion Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated

  7. Acquired resistance to cetuximab is associated with the overexpression of Ras family members and the loss of radiosensitization in head and neck cancer cells

    International Nuclear Information System (INIS)

    Saki, Mohammad; Toulany, Mahmoud; Rodemann, H. Peter

    2013-01-01

    Purpose: Cetuximab in combination with radiation therapy is used to treat patients with head and neck squamous cell carcinoma (HNSCC). In the present study, the mechanism of acquired resistance to cetuximab in HNSCC cells was investigated in vitro. Material and methods: The HNSCC cell lines UT5 and SAS and UT5 cells with acquired resistance to cetuximab (UT5R9) were used. The radiotoxicity potentials of cetuximab and inhibitors of PI3K, MAPK and farnesylation were tested using a clonogenic survival assay. Western blotting was used to evaluate protein expression. The levels of EGFR ligands were detected by ELISA. Results: Cetuximab inhibited proliferation and induced radiosensitization in UT5 cells but not in SAS cells. In comparison with UT5 cells, cetuximab-resistant SAS cells markedly overexpressed the K-Ras, H-Ras and N-Ras proteins, as detected by Western blotting. Resistance in UT5R9 cells was associated with the overexpression of the K-Ras, H-Ras and N-Ras proteins as well as an increase in the autocrine production of the EGFR ligands amphiregulin and transforming growth factor α (TGFα). UT5R9 cells were significantly more radioresistant than UT5 cells. Radioresistant UT5R9 cells were not radiosensitized by cetuximab, but knocking down H-RAS and N-RAS with siRNA and targeting Ras farnesylation using the farnesyltransferase inhibitor lonafarnib induced radiosensitization in these cells. Targeting PI3K and MEK revealed that the activation of the PI3K/Akt pathway but not the MAPK/ERK pathway is associated with radioresistance in UT5R9 cells. Conclusion: Targeting Ras and PI3K activity improves the outcome of irradiation in cetuximab-resistant HNSCC cell lines in vitro

  8. Expression of ras oncogene and major histocompatibility complex (MHC) antigen in carcinomas of the uterine cervix

    International Nuclear Information System (INIS)

    Cho, Kyung Ja; Jang, Ja June; Kim, Yong Dae; Ha, Chang Won; Koh, Jae Soo

    1993-01-01

    Consecutive 50 cases of squamous cell carcinomas of the uterine cervix diagnosed in 1992 were subjected to immunohistochemical study for ras oncogene product (p21) and MHC class II (DR) antigen using a microprobe immunostainer. Activated ras and aberrant DR expression were noted in 26 cases (52%) and 11 cases (22%) of cervical squamous cell carcinomas, respectively, without difference among histologic types. The reaction was mainly intracytoplasmic, with granular staining pattern and diffuse distribution. No direct histologic correlation between ras and DR expression was found. Four cases with HPV 16/18 DNA in superficial koilocytotic cells, revealed by in situ hybridization, showed various expression of ras and DR, and these 3 factors histologically did not seem to be affected one another. (Author)

  9. Ribosomopathy-like properties of murine and human cancers.

    Directory of Open Access Journals (Sweden)

    Sucheta Kulkarni

    Full Text Available Ribosomopathies comprise a heterogeneous group of hematologic and developmental disorders, often characterized by bone marrow failure, skeletal and other developmental abnormalities and cancer predisposition. They are associated with mutations and/or haplo-insufficiencies of ribosomal proteins (RPs and inefficient ribosomal RNA (rRNA processing. The resulting ribosomal stress induces the canonical p19ARF/Mdm2/p53 tumor suppressor pathway leading to proliferative arrest and/or apoptosis. It has been proposed that this pathway is then inactivated during subsequent neoplastic evolution. We show here that two murine models of hepatoblastoma (HB and hepatocellular carcinoma (HCC unexpectedly possess features that mimic the ribosomopathies. These include loss of the normal stoichiometry of RP transcripts and proteins and the accumulation of unprocessed rRNA precursors. Silencing of p19ARF, cytoplasmic sequestration of p53, binding to and inactivation of Mdm2 by free RPs, and up-regulation of the pro-survival protein Bcl-2 may further cooperate to drive tumor growth and survival. Consistent with this notion, re-instatement of constitutive p19ARF expression in the HB model completely suppressed tumorigenesis. In >2000 cases of human HCC, colorectal, breast, and prostate cancer, RP transcript deregulation was a frequent finding. In HCC and breast cancer, the severity of this dysregulation was associated with inferior survival. In HCC, the presence of RP gene mutations, some of which were identical to those previously reported in ribosomopathies, were similarly negatively correlated with long-term survival. Taken together, our results indicate that many if not all cancers possess ribosomopathy-like features that may affect their biological behaviors.

  10. Novel 19F MRS/I nanoprobe based on pH-responsive PEGylated nanogel. pH-dependent 19F magnetic resonance studies

    International Nuclear Information System (INIS)

    Oishi, Motoi; Sumitani, Shogo; Nagasaki, Yukio; Bronich, Tatiana K.; Kabanov, Alexander V.; Boska, Michael D.

    2009-01-01

    The pH-responsive PEGylated nanogels composed of the cross-linked poly[2-(N,N-diethylamino)ethyl methacrylate]-co-poly(2,2,2-trifluoroethyl methacrylate) gel core showed a remarkable on-off regulation of 19 F magnetic resonance signal intensity (T 2 values) as well as signal-to-noise ratios in response to extracellular pH 6.5 of tumor environment under 19 F magnetic resonance spectroscopic imaging (MRS/I), demonstrating the utility of the PEGylated nanogels as solid tumor-specific 19 F MRI/S nanoprobes. (author)

  11. Long term protection after immunization with P. berghei sporozoites correlates with sustained IFNγ responses of hepatic CD8+ memory T cells.

    Directory of Open Access Journals (Sweden)

    Krystelle Nganou-Makamdop

    Full Text Available Protection against P. berghei malaria can successfully be induced in mice by immunization with both radiation attenuated sporozoites (RAS arresting early during liver stage development, or sporozoites combined with chloroquine chemoprophylaxis (CPS, resulting in complete intra-hepatic parasite development before killing of blood-stages by chloroquine takes place. We assessed the longevity of protective cellular immune responses by RAS and CPS P. berghei immunization of C57BL/6j mice. Strong effector and memory (T(EM CD8+ T cell responses were induced predominantly in the liver of both RAS and CPS immunized mice while CD4+ T cells with memory phenotype remained at base line levels. Compared to unprotected naïve mice, we found high sporozoite-specific IFNγ ex vivo responses that associated with induced levels of in vivo CD8+ T(EM cells in the liver but not spleen. Long term evaluation over a period of 9 months showed a decline of malaria-specific IFNγ responses in RAS and CPS mice that significantly correlated with loss of protection (r(2 = 0.60, p<0.0001. The reducing IFNγ response by hepatic memory CD8+ T cells could be boosted by re-exposure to wild-type sporozoites. Our data show that sustainable protection against malaria associates with distinct intra-hepatic immune responses characterized by strong IFNγ producing CD8+ memory T cells.

  12. Lipoprotein-biomimetic nanostructure enables efficient targeting delivery of siRNA to Ras-activated glioblastoma cells via macropinocytosis

    Science.gov (United States)

    Huang, Jia-Lin; Jiang, Gan; Song, Qing-Xiang; Gu, Xiao; Hu, Meng; Wang, Xiao-Lin; Song, Hua-Hua; Chen, Le-Pei; Lin, Ying-Ying; Jiang, Di; Chen, Jun; Feng, Jun-Feng; Qiu, Yong-Ming; Jiang, Ji-Yao; Jiang, Xin-Guo; Chen, Hong-Zhuan; Gao, Xiao-Ling

    2017-05-01

    Hyperactivated Ras regulates many oncogenic pathways in several malignant human cancers including glioblastoma and it is an attractive target for cancer therapies. Ras activation in cancer cells drives protein internalization via macropinocytosis as a key nutrient-gaining process. By utilizing this unique endocytosis pathway, here we create a biologically inspired nanostructure that can induce cancer cells to `drink drugs' for targeting activating transcription factor-5 (ATF5), an overexpressed anti-apoptotic transcription factor in glioblastoma. Apolipoprotein E3-reconstituted high-density lipoprotein is used to encapsulate the siRNA-loaded calcium phosphate core and facilitate it to penetrate the blood-brain barrier, thus targeting the glioblastoma cells in a macropinocytosis-dependent manner. The nanostructure carrying ATF5 siRNA exerts remarkable RNA-interfering efficiency, increases glioblastoma cell apoptosis and inhibits tumour cell growth both in vitro and in xenograft tumour models. This strategy of targeting the macropinocytosis caused by Ras activation provides a nanoparticle-based approach for precision therapy in glioblastoma and other Ras-activated cancers.

  13. Genetic analysis of Ras genes in epidermal development and tumorigenesis

    Science.gov (United States)

    Drosten, Matthias; Lechuga, Carmen G; Barbacid, Mariano

    2013-01-01

    Proliferation and differentiation of epidermal keratinocytes are tightly controlled to ensure proper development and homeostasis of the epidermis. The Ras family of small GTPases has emerged as a central node in the coordination of cell proliferation in the epidermis. Recent genetic evidence from mouse models has revealed that the intensity of Ras signaling modulates the proliferative capacity of epidermal keratinocytes. Interfering with Ras signaling either by combined elimination of the 3 Ras genes from the basal layer of the epidermis or by overexpression of dominant-negative Ras isoforms caused epidermal thinning due to hypoproliferation of keratinocytes. In contrast, overexpression of oncogenic Ras mutants in different epidermal cell layers led to hyperproliferative phenotypes including the development of papillomas and squamous cell carcinomas. Here, we discuss the value of loss- and gain-of-function studies in mouse models to assess the role of Ras signaling in the control of epidermal proliferation. PMID:24150175

  14. The absence of an association between Interleukin 1β gene polymorphisms and recurrent aphthous stomatitis (RAS).

    Science.gov (United States)

    Ślebioda, Zuzanna; Kowalska, Anna; Rozmiarek, Marta; Krawiecka, Ewa; Szponar, Elżbieta; Dorocka-Bobkowska, Barbara

    2017-12-01

    Recurrent aphthous stomatitis (RAS) is a chronic, ulcerative disease with a probable polygenic mode of inheritance and complex etiology with a strong immunological background. The aim of the present study was to determine the possible association between two single nucleotide polymorphisms (SNPs) of the IL-1β gene: IL-1β-511 T>C (rs16944) and IL-1β+3954C>T (rs1143634) and RAS susceptibility in a moderately large group of patients. One hundred and four patients with minor, major and herpetiform RAS and 75 healthy volunteers were genotyped at IL-1β-511 T>C (rs16944) and IL-1β+3954C>T (rs1143634) using the PCR-RFLP approach. The results were statistically analysed with chi-square test and test of difference between two rates of structure, with p<0.05 assumed to be a statistically significance level (Statistica 10, StatSoft ® , Kraków, Poland). There were no statistically significant differences in the genotype distribution for the IL-1β C[+3954]T polymorphism between the RAS and control groups. The frequency of IL-1β*T[-511]/*T[-511] homozygotes among the patients was significantly higher when compared to our study control (p<0.0347). The results after stratification into carriers and non-carriers of C and T alleles did not clearly indicate which SNP may be considered a risk factor for RAS. The genetic association between the studied SNPs of the IL-1β gene and RAS remains controversial and requires further investigation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Differential changes in functional activity of organic cation transporters in rats with uranyl nitrate-induced acute renal failure.

    Science.gov (United States)

    Maeng, Han-Joo; Shim, Won-Sik; Ahn, Sun-Joo; Yu, Sang-Soo; Kim, Dae-Duk; Shim, Chang-Koo; Chung, Suk-Jae

    2012-08-01

    We studied the impact of experimental kidney failure on the pharmacokinetics of a model organic cation and investigated the underlying mechanism(s) of the organic cation transporters. The systemic pharmacokinetics and tissue distribution of triethylmethylammonium (TEMA), a model organic cation, were characterized after intravenous doses of 0.3-30 μmol/kg in rats with or without uranyl nitrate-induced acute renal failure (UN-ARF). To study the effect of endogenous substrates in plasma from UN-ARF rats on organic cation transport, rOCT- or rOCT2-dependent uptake of tetraethylammonium (TEA) was studied in rOCT1-transfected or rOCT2-transfected LLC-PK1 cells, respectively. As a result, the AUC for TEMA was increased, probably because of decreased total clearance, and the tissue-to-plasma concentration ratio (T/P ratio) of TEMA was unchanged in the liver but decreased significantly in the kidneys of UN-ARF rats. In vitro, the uptake of TEA was decreased significantly by adding UN-ARF plasma, compared with control plasma, in rOCT2-overexpressing LLC-PK1 cells, but not in rOCT1-overexpressing LLC-PK1 cells. These observations suggest that the induction of UN-ARF leads to an accumulation of endogenous organic cation(s), probably rOCT2 substrate(s), in the plasma, thereby affecting the TEMA pharmacokinetics and distribution to the kidneys in rats.

  16. Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice

    NARCIS (Netherlands)

    Bruggeman, SWM; Valk-Lingbeek, ME; van der Stoop, PPM; Jacobs, JJL; Kieboom, K; Tanger, E; Hulsman, D; Leung, C; Arsenijevic, Y; Marino, S; van Lohuizen, M

    2005-01-01

    The Polycomb group (PcG) gene Bmi1 promotes cell proliferation and stem cell self-renewal by repressing the Ink4a/Arf locus. We used a genetic approach to investigate whether Ink4a or Arf is more critical for relaying Bmi1 function in lymphoid cells, neural progenitors, and neural stem cells. We

  17. Evolution of AF6-RAS association and its implications in mixed-lineage leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Matthew J.; Ottoni, Elizabeth; Ishiyama, Noboru; Goudreault, Marilyn; Haman, André; Meyer, Claus; Tucholska, Monika; Gasmi-Seabrook, Genevieve; Menezes, Serena; Laister, Rob C.; Minden, Mark D.; Marschalek, Rolf; Gingras, Anne-Claude; Hoang, Trang; Ikura, Mitsuhiko

    2017-10-23

    Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding of MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements.

  18. RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells.

    Science.gov (United States)

    Saito, Suguru; Kawamura, Toshihiko; Higuchi, Masaya; Kobayashi, Takahiro; Yoshita-Takahashi, Manami; Yamazaki, Maya; Abe, Manabu; Sakimura, Kenji; Kanda, Yasuhiro; Kawamura, Hiroki; Jiang, Shuying; Naito, Makoto; Yoshizaki, Takumi; Takahashi, Masahiko; Fujii, Masahiro

    2015-05-01

    Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Radiosensitivity and ras oncogene expression in preneoplastic rat tracheal epithelial cells

    International Nuclear Information System (INIS)

    Thomassen, D.G.; Wuensch, S.A.; Kelly, G.

    1988-01-01

    The sensitivity of preneoplastic rat tracheal epithelial (RTE) cells to the cytotoxic effects of high- and low-LET radiation, and the modulating effect of the viral ras oncogene on this sensitivity were determined. Two lines of preneoplastic RTE cells have the same responsiveness to high-LET radiation, but differ in their responsiveness to a transfected ras oncogene and in their sensitivities to low-LET radiation. Cells that respond to ras by becoming neoplastic are more resistant to the cytotoxic effects of low-LET radiation than cells that are not transformable by ras. The radiosensitivity of ras-responsive cells was not altered by transfection with ras. However, transfection of ras-non responsive cells with ras decreased their sensitivity to low-LET radiation. These data suggest that the ability of cells to repair radiation damage changes as they progress to neoplasia. (author)

  20. Mutational analysis of a ras catalytic domain

    DEFF Research Database (Denmark)

    Willumsen, B M; Papageorge, A G; Kung, H F

    1986-01-01

    localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target....

  1. Influence of a ras oncogene on platelet-derived growth factor (PDGF)-stimulated phosphoinositide hydrolysis in murine fibroblasts

    International Nuclear Information System (INIS)

    Parries, G.; Racker, E.

    1986-01-01

    The authors have examined the effects of transfection of rat-1 fibroblasts with the ras oncogene on the metabolism of phosphatidylinositol (PI). Incubation of [ 3 H]inositol-labeled rat-1 cells with PDGF resulted in a 2- to 3-fold increase in [ 3 H]IP3 levels within 90 s. In the presence of 25 mM Li+, [ 3 H]IP1 levels were increased 8-fold after 30 min. In contrast, incubation of ras-transfected fibroblasts (EJ-2 line) with PDGF had little or no effect on the level of either [ 3 H]IP3 or [ 3 H]IP1. Similar stimulations by PDGF were observed in NIH 3T3 cells, but not in Kirsten virus-transformed or Harvey ras-transfected cell lines. On the other hand, NIH 3T3 cells transfected with v-src responded to PDGF by stimulation of PI turnover similar to the parent cell line. In NIH 3T3 cells transfected with an expression vector containing the v-Ha-ras gene under transcriptional control of the glucocorticoid-inducible mouse mammary tumor virus promoter, the PDGF stimulation of [ 3 H]inositol incorporation into PI was reduced from 10-fold in the absence of dexamethasone to 1.8-fold when the cells were pretreated for 26 h with 2 μM dexamethasone. In the parental 3T3 cells PDGF stimulation was reduced by about 40% in the presence of dexamethasone. In the absence of PDGF the rate of PI turnover (i.e., the kinetics of [ 3 H]IP1 accumulation in the presence of Li+) in EJ-2 cells was similar to that in rat-1 cells. Thus, in the presence of PDGF, the rate of PI turnover in rat-1 cells was several fold higher than in the transfected cells. These results suggest that the ras gene product (p21) may exert an inhibitory effect on PDGF-stimulated phosphoinositide metabolism

  2. Valproic acid exposure decreases Cbp/p300 protein expression and histone acetyltransferase activity in P19 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lamparter, Christina L. [Department of Biomedical and Molecular Sciences, Graduate Program in Pharmacology and Toxicology, Queen' s University, Kingston, Ontario K7L 3N6 (Canada); Winn, Louise M., E-mail: winnl@queensu.ca [Department of Biomedical and Molecular Sciences, Graduate Program in Pharmacology and Toxicology, Queen' s University, Kingston, Ontario K7L 3N6 (Canada); School of Environmental Studies, Queen' s University, Kingston, Ontario K7L 3N6 (Canada)

    2016-09-01

    The teratogenicity of the antiepileptic drug valproic acid (VPA) is well established and its inhibition of histone deacetylases (HDAC) is proposed as an initiating factor. Recently, VPA-mediated HDAC inhibition was demonstrated to involve transcriptional downregulation of histone acetyltransferases (HATs), which was proposed to compensate for the increased acetylation resulting from HDAC inhibition. Cbp and p300 are HATs required for embryonic development and deficiencies in either are associated with congenital malformations and embryolethality. The objective of the present study was to characterize Cbp/p300 following VPA exposure in P19 cells. Consistent with previous studies, exposure to 5 mM VPA over 24 h induced a moderate decrease in Cbp/p300 mRNA, which preceded a strong decrease in total cellular protein mediated by ubiquitin-proteasome degradation. Nuclear Cbp/p300 protein was also decreased following VPA exposure, although to a lesser extent. Total cellular and nuclear p300 HAT activity was reduced proportionately to p300 protein levels, however while total cellular HAT activity also decreased, nuclear HAT activity was unaffected. Using the Cbp/p300 HAT inhibitor C646, we demonstrated that HAT inhibition similarly affected many of the same endpoints as VPA, including increased reactive oxygen species and caspase-3 cleavage, the latter of which could be attenuated by pre-treatment with the antioxidant catalase. C646 exposure also decreased NF-κB/p65 protein, which was not due to reduced mRNA and was not attenuated with catalase pre-treatment. This study provides support for an adaptive HAT response following VPA exposure and suggests that reduced Cbp/p300 HAT activity could contribute to VPA-mediated alterations. - Highlights: • VPA exposure in vitro downregulates Cbp/p300 mRNA and induces protein degradation. • Cbp/p300 histone acetyltransferase activity is similarly reduced with VPA exposure. • Inhibition of Cbp/p300 acetyltransferase activity

  3. Circumvention and reactivation of the p53 oncogene checkpoint in mouse colon tumors.

    Science.gov (United States)

    Aizu, Wataru; Belinsky, Glenn S; Flynn, Christopher; Noonan, Emily J; Boes, Colleen C; Godman, Cassandra A; Doshi, Bindi; Nambiar, Prashant R; Rosenberg, Daniel W; Giardina, Charles

    2006-10-16

    The p53 tumor suppressor protein is sequence-normal in azoxymethane (AOM)-induced mouse colon tumors, making them a good model for human colon cancers that retain a wild type p53 gene. Cellular localization and co-immunoprecipitation experiments using a cell line derived from an AOM-induced colon tumor (AJ02-NM(0) cells) pointed to constitutively expressed Mdm2 as being an important negative regulator of p53 in these cells. Although the Mdm2 inhibitory protein p19/ARF was expressed in AJ02-NM(0) cells, its level of expression was not sufficient for p53 activation. We tested the response of AJ02-NM(0) cells to the recently developed Mdm2 inhibitor, Nutlin-3. Nutlin-3 was found to activate p53 DNA binding in AJ02-NM(0) cells, to a level comparable to doxorubicin and 5-fluorouracil (5-FU). In addition, Nutlin-3 increased expression of the p53 target genes Bax and PERP to a greater extent than doxorubicin or 5-FU, and triggered a G2/M phase arrest in these cells, compared to a G1 arrest triggered by doxorubicin and 5-FU. The differences in the cellular response may be related to differences in the kinetics of p53 activation and/or its post-translational modification status. In an ex vivo experiment, Nutlin-3 was found to activate p53 target gene expression and apoptosis in AOM-induced tumor tissue, but not in normal adjacent mucosa. Our data indicate that Mdm2 inhibitors may be an effective means of selectively targeting colon cancers that retain a sequence-normal p53 gene while sparing normal tissue and that the AOM model is an appropriate model for the preclinical development of these drugs.

  4. RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies.

    Science.gov (United States)

    Fang, Bingliang

    2016-01-01

    Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology

  5. RAS1, a quantitative trait locus for salt tolerance and ABA sensitivity in Arabidopsis

    KAUST Repository

    Ren, Zhonghai

    2010-03-08

    Soil salinity limits agricultural production and is a major obstacle for feeding the growing world population. We used natural genetic variation in salt tolerance among different Arabidopsis accessions to map a major quantitative trait locus (QTL) for salt tolerance and abscisic acid (ABA) sensitivity during seed germination and early seedling growth. A recombinant inbred population derived from Landsberg erecta (Ler; salt and ABA sensitive) x Shakdara (Sha; salt and ABA resistant) was used for QTL mapping. High-resolution mapping and cloning of this QTL, Response to ABA and Salt 1 (RAS1), revealed that it is an ABA- and salt stress-inducible gene and encodes a previously undescribed plant-specific protein. A premature stop codon results in a truncated RAS1 protein in Sha. Reducing the expression of RAS1 by transfer-DNA insertion in Col or RNA interference in Ler leads to decreased salt and ABA sensitivity, whereas overexpression of the Ler allele but not the Sha allele causes increased salt and ABA sensitivity. Our results suggest that RAS1 functions as a negative regulator of salt tolerance during seed germination and early seedling growth by enhancing ABA sensitivity and that its loss of function contributes to the increased salt tolerance of Sha.

  6. Differential p53 engagement in response to oxidative and oncogenic stresses in Fanconi anemia mice

    Science.gov (United States)

    Rani, Reena; Li, Jie; Pang, Qishen

    2008-01-01

    Members of the Fanconi anemia (FA) protein family are involved in repair of genetic damage caused by DNA cross-linkers. It is not clear whether the FA proteins function in oxidative DNA damage and oncogenic stress response. Here we report that deficiency in the Fanca gene in mice elicits a p53-dependent growth arrest and DNA damage response to oxidative DNA damage and oncogenic stress. Using a Fanca-/- Trp53-/- double knockout model and a functionally switchable p53 retrovirus, we define the kinetics, dependence, and persistence of p53-mediated response to oxidative and oncogenic stresses in Fanca-/- cells. Notably, oxidative stress induces persistent p53 response in Fanca-/- cells, likely due to accumulation of unrepaired DNA damage. On the other hand, whereas WT cells exhibit prolonged response to oncogene activation, the p53-activating signals induced by oncogenic ras are short-lived in Fanca-/- cells, suggesting that Fanca may be required for the cell to engage p53 during constitutive ras activation. We propose that the FA proteins protect cells from stress-induced proliferative arrest and tumor evolution by acting as a modulator of the signaling pathways that link FA to p53. PMID:19047147

  7. Differential p53 engagement in response to oxidative and oncogenic stresses in Fanconi anemia mice.

    Science.gov (United States)

    Rani, Reena; Li, Jie; Pang, Qishen

    2008-12-01

    Members of the Fanconi anemia (FA) protein family are involved in repair of genetic damage caused by DNA cross-linkers. It is not clear whether the FA proteins function in oxidative DNA damage and oncogenic stress response. Here, we report that deficiency in the Fanca gene in mice elicits a p53-dependent growth arrest and DNA damage response to oxidative DNA damage and oncogenic stress. Using a Fanca-/-Trp53-/- double knockout model and a functionally switchable p53 retrovirus, we define the kinetics, dependence, and persistence of p53-mediated response to oxidative and oncogenic stresses in Fanca-/- cells. Notably, oxidative stress induces persistent p53 response in Fanca-/- cells, likely due to accumulation of unrepaired DNA damage. On the other hand, whereas wild-type cells exhibit prolonged response to oncogene activation, the p53-activating signals induced by oncogenic ras are short-lived in Fanca-/- cells, suggesting that Fanca may be required for the cell to engage p53 during constitutive ras activation. We propose that the FA proteins protect cells from stress-induced proliferative arrest and tumor evolution by acting as a modulator of the signaling pathways that link FA to p53.

  8. Comparison of HEC-RAS with FLDWAV and DAMBRK models for dam break analysis

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, R.D.; Judge, D.G.; Donnelly, C.R. [Acres International Ltd., Niagara Falls, ON (Canada)

    2004-09-01

    Dam break analysis is conducted as a routine dam safety assessment in determining the incremental hazard potential (IHP) that would occur in the event of a dam failure. Dam safety analysis also provides the information needed to make flood inundation maps and emergency preparedness plans (EPP) for dams that present a risk to human safety. For several years, the standard programs that have been used by the national weather service (NWS) for dam break flood simulation were the FLDWAV and DAMBRK models. However, another popular hydraulic model has been introduced. The HEC-RAS is an upgraded version of the former HEC-2 model used for the assessment of river floodplain inundation. A dam break analysis module has been added to HEC-2 and renamed the HEC-RAS which offers similar modeling capabilities to the DAMBRK and FLDWAV models for simulating flood hydrographs and flood wave propagation in river channels located downstream of a breached dam. The use of the alternative HEC-RAS could save time and resources. As such, it has the potential to replace the FLDWAV and DAMBRK. This paper compares the HEC-RAS dam break module with the FLDWAV and DAMBRK to determine if the new module accurately represents the dam break flood process. The comparison focuses on the theoretical background of the models, numerical solution techniques, ease of use of the module, modeling accuracy, practical aspects in performing dam break simulations, and capability for coupling the models with GIS for inundation mapping. The advantages and disadvantages of each model were summarized. 19 refs., 5 figs.

  9. E2F1 induces p19INK4d, a protein involved in the DNA damage response, following UV irradiation.

    Science.gov (United States)

    Carcagno, Abel L; Giono, Luciana E; Marazita, Mariela C; Castillo, Daniela S; Pregi, Nicolás; Cánepa, Eduardo T

    2012-07-01

    Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.

  10. Arf6, Rab11 and transferrin receptor define distinct populations of recycling endosomes.

    Science.gov (United States)

    Kobayashi, Hotaka; Fukuda, Mitsunori

    2013-09-01

    Recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane. To determine how recycling endosomes perform their functions, searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers. However, it remains unclear whether all the conventional markers have identical localizations. Here we report finding that three well-known recycling endosome markers, i.e., Arf6, Rab11 and transferrin receptor (TfR), have different intracellular localizations in PC12 cells. The results of immunofluorescence analyses showed that the signals of endogenous Arf6, Rab11 and TfR in nerve growth factor-stimulated PC12 cells generally differed, although there was some overlapping. Our findings provide new information about recycling endosome markers, and they highlight the heterogeneity of recycling endosomes.

  11. Spatial and temporal expression patterns of auxin response transcription factors in the syncytium induced by the beet cyst nematode Heterodera schachtii in Arabidopsis.

    Science.gov (United States)

    Hewezi, Tarek; Piya, Sarbottam; Richard, Geoffrey; Rice, J Hollis

    2014-09-01

    Plant-parasitic cyst nematodes induce the formation of a multinucleated feeding site in the infected root, termed the syncytium. Recent studies point to key roles of the phytohormone auxin in the regulation of gene expression and establishment of the syncytium. Nevertheless, information about the spatiotemporal expression patterns of the transcription factors that mediate auxin transcriptional responses during syncytium formation is limited. Here, we provide a gene expression map of 22 auxin response factors (ARFs) during the initiation, formation and maintenance stages of the syncytium induced by the cyst nematode Heterodera schachtii in Arabidopsis. We observed distinct and overlapping expression patterns of ARFs throughout syncytium development phases. We identified a set of ARFs whose expression is predominantly located inside the developing syncytium, whereas others are expressed in the neighbouring cells, presumably to initiate specific transcriptional programmes required for their incorporation within the developing syncytium. Our analyses also point to a role of certain ARFs in determining the maximum size of the syncytium. In addition, several ARFs were found to be highly expressed in fully developed syncytia, suggesting a role in maintaining the functional phenotype of mature syncytia. The dynamic distribution and overlapping expression patterns of various ARFs seem to be essential characteristics of ARF activity during syncytium development. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  12. The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells.

    Science.gov (United States)

    Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

    2015-01-01

    The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival.

  13. NMR 1H,13C, 15N backbone and 13C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP.

    Science.gov (United States)

    Sharma, Alok K; Lee, Seung-Joo; Rigby, Alan C; Townson, Sharon A

    2018-05-02

    K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1 H N, 15 N, and 13 C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS G12C-GDP ), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1 H- 15 N correlations have been assigned for all non-proline residues, except for the first methionine residue.

  14. Association between H-RAS T81C genetic polymorphism and gastrointestinal cancer risk: A population based case-control study in China

    Directory of Open Access Journals (Sweden)

    Li Qilong

    2008-09-01

    Full Text Available Abstract Background Gastrointestinal cancer, such as gastric, colon and rectal cancer, is a major medical and economic burden worldwide. However, the exact mechanism of gastrointestinal cancer development still remains unclear. RAS genes have been elucidated as major participants in the development and progression of a series of human tumours and the single nucleotide polymorphism at H-RAS cDNA position 81 was demonstrated to contribute to the risks of bladder, oral and thyroid carcinoma. Therefore, we hypothesized that this polymorphisms in H-RAS could influence susceptibility to gastrointestinal cancer as well, and we conducted this study to test the hypothesis in Chinese population. Methods A population based case-control study, including 296 cases with gastrointestinal cancer and 448 healthy controls selected from a Chinese population was conducted. H-RAS T81C polymorphism was genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP assay. Results In the healthy controls, the TT, TC and CC genotypes frequencies of H-RAS T81C polymorphism, were 79.24%, 19.87% and 0.89%, respectively, and the C allele frequency was 10.83%. Compared with TT genotype, the TC genotype was significantly associated with an increased risk of gastric cancer (adjusted OR = 3.67, 95%CI = 2.21–6.08, while the CC genotype showed an increased risk as well (adjusted OR = 3.29, 95%CI = 0.54–19.86, but it was not statistically significant. In contrast, the frequency of TC genotype was not significantly increased in colon cancer and rectal cancer patients. Further analysis was performed by combining TC and CC genotypes compared against TT genotype. As a result, a statistically significant risk with adjusted OR of 3.65 (95%CI, 2.22–6.00 was found in gastric cancer, while no significant association of H-RAS T81C polymorphism with colon cancer and rectal cancer was observed. Conclusion These findings indicate, for the first time, that there

  15. Association between H-RAS T81C genetic polymorphism and gastrointestinal cancer risk: A population based case-control study in China

    International Nuclear Information System (INIS)

    Zhang, Yongjing; Jin, Mingjuan; Liu, Bing; Ma, Xinyuan; Yao, Kaiyan; Li, Qilong; Chen, Kun

    2008-01-01

    Gastrointestinal cancer, such as gastric, colon and rectal cancer, is a major medical and economic burden worldwide. However, the exact mechanism of gastrointestinal cancer development still remains unclear. RAS genes have been elucidated as major participants in the development and progression of a series of human tumours and the single nucleotide polymorphism at H-RAS cDNA position 81 was demonstrated to contribute to the risks of bladder, oral and thyroid carcinoma. Therefore, we hypothesized that this polymorphisms in H-RAS could influence susceptibility to gastrointestinal cancer as well, and we conducted this study to test the hypothesis in Chinese population. A population based case-control study, including 296 cases with gastrointestinal cancer and 448 healthy controls selected from a Chinese population was conducted. H-RAS T81C polymorphism was genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay. In the healthy controls, the TT, TC and CC genotypes frequencies of H-RAS T81C polymorphism, were 79.24%, 19.87% and 0.89%, respectively, and the C allele frequency was 10.83%. Compared with TT genotype, the TC genotype was significantly associated with an increased risk of gastric cancer (adjusted OR = 3.67, 95%CI = 2.21–6.08), while the CC genotype showed an increased risk as well (adjusted OR = 3.29, 95%CI = 0.54–19.86), but it was not statistically significant. In contrast, the frequency of TC genotype was not significantly increased in colon cancer and rectal cancer patients. Further analysis was performed by combining TC and CC genotypes compared against TT genotype. As a result, a statistically significant risk with adjusted OR of 3.65 (95%CI, 2.22–6.00) was found in gastric cancer, while no significant association of H-RAS T81C polymorphism with colon cancer and rectal cancer was observed. These findings indicate, for the first time, that there is an H-RAS T81C polymorphism existing in Chinese population

  16. The oncogenic RAS2 val19 mutation locks respiration, independently of PKA, in a mode prone to generate ROS

    Czech Academy of Sciences Publication Activity Database

    Hlavatá, Lydie; Aguilaniu, H.; Pichová, Alena; Nystrom, T.

    2003-01-01

    Roč. 22, č. 13 (2003), s. 3337-3345 ISSN 0261-4189 R&D Projects: GA ČR GA301/03/0289 Institutional research plan: CEZ:AV0Z5020903 Keywords : pka/ras2/ * ucp1 * respiratory state Subject RIV: EE - Microbiology, Virology Impact factor: 10.456, year: 2003

  17. Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

    International Nuclear Information System (INIS)

    Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Audemard, Eric; Montermini, Laura; Meehan, Brian; Rak, Janusz

    2014-01-01

    Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellular vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells

  18. Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Hoon; Chennakrishnaiah, Shilpa [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Audemard, Eric [McGill University and Genome Quebec Innovation Centre, Montreal, Quebec (Canada); Montermini, Laura; Meehan, Brian [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Rak, Janusz, E-mail: janusz.rak@mcgill.ca [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada)

    2014-08-22

    Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellular vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.

  19. Role of Bone Marrow Derived Mesenchymal Stem Cells and the Protective Effect of Silymarin in Cisplatin-Induced Acute Renal Failure in Rats.

    Science.gov (United States)

    Ibrahim, Mohamed El-Tantawy; Bana, Eman El; El-Kerdasy, Hanan I

    2018-01-01

    Cisplatin is a highly effective antitumor agent whose clinical application is limited by its nephrotoxicity, which is associated with high mortality and morbidity rates. We aimed to study the protective role of silymarin and mesenchymal stem cells as a therapeutic tool of cisplatin nephrotoxicity. We injected rats with cisplatin in a dose of 5mg/kg body weight for 5 days to induce acute renal failure (ARF). Silymarin was administrated 6 hours before cisplatin injection and mesenchymal stem cells were injected 24 hours after cisplatin-induced ARF. We assessed the ARF biochemically by elevation of kidney function tests and histopathologically by an alteration of the histological architecture of the renal cortex in the form of shrinkage of glomeruli, lobulated tufts and glomerular hypertrophy with narrowing capsular space. The tubules showed extensive tubular degeneration with cellular hyaline materials and debris in the lumen of the renal tubules. The renal blood vessels appeared sclerotic with marked thickened walls. When silymarin was given in different doses before cisplatin, it decreased the toxic effect of cisplatin in the kidney but sclerotic blood vessels remained. Injection of mesenchymal stem cells in rats with cisplatin-induced ARF improved the histopathological effects of cisplatin in renal tissues and kidney function tests were significantly improved. There was a significant improvement in kidney function tests and renal histopathology by using silymarin as protective mechanism in cisplatin-induced ARF. Administration of mesenchymal stem cells denoted a more remarkable therapeutic effect in ARF. Copyright © 2018 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

  20. Ras mutations are rare in solitary cold and toxic thyroid nodules.

    Science.gov (United States)

    Krohn, K; Reske, A; Ackermann, F; Müller, A; Paschke, R

    2001-08-01

    Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

  1. ArF short-pulse extraction studies. Final technical report, 18 September 1981-18 February 1983

    International Nuclear Information System (INIS)

    Mandl, A.

    1983-02-01

    The experimental apparatus including e-beam pulse diagnostics is discussed. The relative fluorescence and laser output for various gas mixtures is presented and the significant improvement in laser performance for Ne buffered mixtures, allowing scaling to high pressures and high Joules per liter, is discussed. The energy deposition measurements for Ar and Ne buffered mixtures are presented. Accurate deposition measurements are necessary for a meaningful measure of the laser output efficiency. Background absorption measurements important for accessing the scalability of the ArF system are presented. The sidelight suppression experiments are discussed and the laser efficiency measurements are shown. Finally, a discussion of the measurements with their implications for the ArF system is presented in the concluding section

  2. Absence of mutations in the coding sequence of the potential tumor suppressor 3pK in metastatic melanoma

    Directory of Open Access Journals (Sweden)

    Houben Roland

    2005-12-01

    Full Text Available Abstract Background Activation of Ras or Raf contributes to tumorigenesis of melanoma. However, constitutive Raf activation is also a characteristic of the majority of benign melanocytic nevi and high intensity signaling of either Ras or Raf was found to induce growth inhibition and senescence rather than transformation. Since the chromosome 3p kinase (3pK is a target of the Ras/Raf/Mek/Erk signaling pathway which antagonizes the function of the oncogene and anti-differentiation factor Bmi-1, 3pK may function as a tumor suppressor in tumors with constitutive Ras/Raf activation. Consequently, we tested whether inactivating 3pK mutations are present in melanoma. Methods 30 metastatic melanoma samples, which were positive for activating mutations of either BRaf or NRas, were analyzed for possible mutations in the 3pk gene. The 10 coding exons and their flanking intron sequences were amplified by PCR and direct sequencing of the PCR products was performed. Results This analysis revealed that besides the presence of some single nucleotide polymorphisms in the 3pk gene, we could not detect any possible loss of function mutation in any of these 30 metastatic melanoma samples selected for the presence of activating mutations within the Ras/Raf/Mek/Erk signaling pathway. Conclusion Hence, in melanoma with constitutively active Ras/Raf inactivating mutations within the 3pk gene do not contribute to the oncogenic phenotype of this highly malignant tumor.

  3. The Ras antagonist, farnesylthiosalicylic acid (FTS, decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Yoram Nevo

    Full Text Available The Ras superfamily of guanosine-triphosphate (GTP-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J/dy(2J mouse model of merosin deficient congenital muscular dystrophy. The dy(2J/dy(2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J/dy(2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J/dy(2J mouse model of congenital muscular dystrophy.

  4. Identification and preliminary characterization of protein-cysteine farnesyltransferase

    International Nuclear Information System (INIS)

    Manne, V.; Roberts, D.; Tobin, A.; O'Rourke, E.; Barbacid, M.; De Virgilio, M.; Meyers, C.; Ahmed, N.; Kurz, B.; Resh, M.; Kung, Hsiang-Fu

    1990-01-01

    Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2 Valendash19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an onocogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, the authors describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway

  5. The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

    Directory of Open Access Journals (Sweden)

    Geldner Niko

    2005-02-01

    Full Text Available Abstract Background Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7. Conclusion Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

  6. Struktūras elementi Dž.K. Roulingas darbā "Harijs Poters"

    OpenAIRE

    Goškina, Elīna

    2009-01-01

    Nosaukums: Struktūras elementi Dž. K. Roulingas darbā “Harijs Poters”. Pamattēma: Struktūras elementi, intertekstualitāte, tās vēsturiskās attīstības posmi un klasifikācija. Intertekstualitātes nozīme tekstā, lietojot simbolu un kodu sistēmu. Atsauces uz citiem darbiem, lietojot simbolu un kodu sistēmu ar intertekstualitātes palīdzību. Intertekstualitātes veidošanās process. Mērķis: Darba mērķis ir pētīt intertekstualitātes attīstības posmus izmantojot simbolu un kodu sistēmu, sekot...

  7. Acute Alcohol Intoxication Exacerbates Rhabdomyolysis-Induced Acute Renal Failure in Rats.

    Science.gov (United States)

    Tsai, Jen-Pi; Lee, Chung-Jen; Subeq, Yi-Maun; Lee, Ru-Ping; Hsu, Bang-Gee

    2017-01-01

    Traumatic and nontraumatic rhabdomyolysis can lead to acute renal failure (ARF), and acute alcohol intoxication can lead to multiple abnormalities of the renal tubules. We examined the effect of acute alcohol intoxication in a rat model of rhabdomyolysis and ARF. Intravenous injections of 5 g/kg ethanol were given to rats over 3 h, followed by glycerol-induced rhabdomyolysis. Biochemical parameters, including blood urea nitrogen (BUN), creatinine (Cre), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and creatine phosphokinase (CPK), were measured before and after induction of rhabdomyolysis. Renal tissue injury score, renal tubular cell expression of E-cadherin, nuclear factor-κB (NF-κB), and inducible nitric oxide synthase (iNOS) were determined. Relative to rats in the vehicle group, rats in the glycerol-induced rhabdomyolysis group had significantly increased serum levels of BUN, Cre, GOT, GPT, and CPK, elevated renal tissue injury scores, increased expression of NF-κB and iNOS, and decreased expression of E-cadherin. Ethanol exacerbated all of these pathological responses. Our results suggest that acute alcohol intoxication exacerbates rhabdomyolysis-induced ARF through its pro-oxidant and inflammatory effects.

  8. v-Ha-ras oncogene insertion: A model for tumor progression of human small cell lung cancer

    International Nuclear Information System (INIS)

    Mabry, M.; Nakagawa, Toshitaro; Nelkin, B.D.; McDowell, E.; Gesell, M.; Eggleston, J.C.; Casero, R.A. Jr.; Baylin, S.B.

    1988-01-01

    Small cell lung cancer (SCLC) manifests a range of phenotypes in culture that may be important in understanding its relationship to non-SCLCs and to tumor progression events in patients. Most SCLC-derived cell lines, termed classic SCLC lines, have properties similar to SCLC tumors in patients. To delineate further the relationships between these phenotypes and the molecular events involved, the authors inserted the v-Ha-ras gene in SCLC cell lines with (biochemical variant) and without (classic) an amplified c-myc gene. These two SCLC subtypes had markedly different phenotypic responses to similar levels of expression of v-Ha-ras RNA. No biochemical or morphologic changes were observed in classic SCLC cells. In contrast, in biochemical variant SCLC cells, v-Ha-ras expression induced features typical of large cell undifferentiated lung carcinoma. Expression of v-Ha-ras in biochemical variant SCLC cells directly demonstrates that important transitions can occur between phenotypes of human lung cancer cells and that these may play a critical role in tumor progression events in patients. The finding provide a model system to study molecular events involved in tumor progression steps within a series of related tumor types

  9. Flavopiridol induces apoptosis in glioma cell lines independent of retinoblastoma and p53 tumor suppressor pathway alterations by a caspase-independent pathway.

    Science.gov (United States)

    Alonso, Michelle; Tamasdan, Cristina; Miller, Douglas C; Newcomb, Elizabeth W

    2003-02-01

    Flavopiridol is a synthetic flavone, which inhibits growth in vitro and in vivo of several solid malignancies such as renal, prostate, and colon cancers. It is a potent cyclin-dependent kinase inhibitor presently in clinical trials. In this study, we examined the effect of flavopiridol on a panel of glioma cell lines having different genetic profiles: five of six have codeletion of p16(INK4a) and p14(ARF); three of six have p53 mutations; and one of six shows overexpression of mouse double minute-2 (MDM2) protein. Independent of retinoblastoma and p53 tumor suppressor pathway alterations, flavopiridol induced apoptosis in all cell lines but through a caspase-independent mechanism. No cleavage products for caspase 3 or its substrate poly(ADP-ribose) polymerase or caspase 8 were detected. The pan-caspase inhibitor Z-VAD-fmk did not inhibit flavopiridol-induced apoptosis. Mitochondrial damage measured by cytochrome c release and transmission electron microscopy was not observed in drug-treated glioma cells. In contrast, flavopiridol treatment induced translocation of apoptosis-inducing factor from the mitochondria to the nucleus. The proteins cyclin D(1) and MDM2 involved in the regulation of retinoblastoma and p53 activity, respectively, were down-regulated early after flavopiridol treatment. Given that MDM2 protein can confer oncogenic properties under certain circumstances, loss of MDM2 expression in tumor cells could promote increased chemosensitivity. After drug treatment, a low Bcl-2/Bax ratio was observed, a condition that may favor apoptosis. Taken together, the data indicate that flavopiridol has activity against glioma cell lines in vitro and should be considered for clinical development in the treatment of glioblastoma multiforme.

  10. Key roles of Arf small G proteins and biosynthetic trafficking for animal development.

    Science.gov (United States)

    Rodrigues, Francisco F; Harris, Tony J C

    2017-04-14

    Although biosynthetic trafficking can function constitutively, it also functions specifically for certain developmental processes. These processes require either a large increase to biosynthesis or the biosynthesis and targeted trafficking of specific players. We review the conserved molecular mechanisms that direct biosynthetic trafficking, and discuss how their genetic disruption affects animal development. Specifically, we consider Arf small G proteins, such as Arf1 and Sar1, and their coat effectors, COPI and COPII, and how these proteins promote biosynthetic trafficking for cleavage of the Drosophila embryo, the growth of neuronal dendrites and synapses, extracellular matrix secretion for bone development, lumen development in epithelial tubes, notochord and neural tube development, and ciliogenesis. Specific need for the biosynthetic trafficking system is also evident from conserved CrebA/Creb3-like transcription factors increasing the expression of secretory machinery during several of these developmental processes. Moreover, dysfunctional trafficking leads to a range of developmental syndromes.

  11. Impairment of cocaine-mediated behaviours in mice by clinically relevant Ras-ERK inhibitors

    Science.gov (United States)

    Papale, Alessandro; Morella, Ilaria Maria; Indrigo, Marzia Tina; Bernardi, Rick Eugene; Marrone, Livia; Marchisella, Francesca; Brancale, Andrea; Spanagel, Rainer; Brambilla, Riccardo; Fasano, Stefania

    2016-01-01

    Ras-ERK signalling in the brain plays a central role in drug addiction. However, to date, no clinically relevant inhibitor of this cascade has been tested in experimental models of addiction, a necessary step toward clinical trials. We designed two new cell-penetrating peptides - RB1 and RB3 - that penetrate the brain and, in the micromolar range, inhibit phosphorylation of ERK, histone H3 and S6 ribosomal protein in striatal slices. Furthermore, a screening of small therapeutics currently in clinical trials for cancer therapy revealed PD325901 as a brain-penetrating drug that blocks ERK signalling in the nanomolar range. All three compounds have an inhibitory effect on cocaine-induced ERK activation and reward in mice. In particular, PD325901 persistently blocks cocaine-induced place preference and accelerates extinction following cocaine self-administration. Thus, clinically relevant, systemically administered drugs that attenuate Ras-ERK signalling in the brain may be valuable tools for the treatment of cocaine addiction. DOI: http://dx.doi.org/10.7554/eLife.17111.001 PMID:27557444

  12. Characteristics and Efficacy of a Sterile Hyphomycete (ARF18), a New Biocontrol Agent for Heterodera glycines and Other Nematodes

    OpenAIRE

    Kim, D. G.; Riggs, R. D.

    1991-01-01

    A filamentous, nonsporulating fungus, designated Arkansas Fungus 18 (ARF18), was isolated from 9 of 95 populations of Heterodera glycines, the soybean cyst nematode, in Arkansas. In petri dishes, ARF18 parasitized 89% of H. glycines eggs in cysts. The fungus also infected eggs of Meloidogyne incognita and eggs in cysts of Cactodera betulae, H. graminophila, H. lespedezae, H. leuceilyma, H. schachtii, and H. trifolii. In pot tests, reproduction of SCN was 70% less in untreated field soil that ...

  13. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

    Science.gov (United States)

    Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

    1993-05-06

    Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

  14. Long term protection after immunization with P. berghei sporozoites correlates with sustained IFNγ responses of hepatic CD8+ memory T cells.

    Science.gov (United States)

    Nganou-Makamdop, Krystelle; van Gemert, Geert-Jan; Arens, Theo; Hermsen, Cornelus C; Sauerwein, Robert W

    2012-01-01

    Protection against P. berghei malaria can successfully be induced in mice by immunization with both radiation attenuated sporozoites (RAS) arresting early during liver stage development, or sporozoites combined with chloroquine chemoprophylaxis (CPS), resulting in complete intra-hepatic parasite development before killing of blood-stages by chloroquine takes place. We assessed the longevity of protective cellular immune responses by RAS and CPS P. berghei immunization of C57BL/6j mice. Strong effector and memory (T(EM)) CD8+ T cell responses were induced predominantly in the liver of both RAS and CPS immunized mice while CD4+ T cells with memory phenotype remained at base line levels. Compared to unprotected naïve mice, we found high sporozoite-specific IFNγ ex vivo responses that associated with induced levels of in vivo CD8+ T(EM) cells in the liver but not spleen. Long term evaluation over a period of 9 months showed a decline of malaria-specific IFNγ responses in RAS and CPS mice that significantly correlated with loss of protection (r(2) = 0.60, pmemory CD8+ T cells could be boosted by re-exposure to wild-type sporozoites. Our data show that sustainable protection against malaria associates with distinct intra-hepatic immune responses characterized by strong IFNγ producing CD8+ memory T cells.

  15. Upregulation of c-mesenchymal epithelial transition expression and RAS mutations are associated with late lung metastasis and poor prognosis in colorectal carcinoma.

    Science.gov (United States)

    Liu, Jianhua; Zeng, Weiqiang; Huang, Chengzhi; Wang, Junjiang; Xu, Lishu; Ma, Dong

    2018-05-01

    The present study aimed to investigate whether c-mesenchymal epithelial transition factor (C-MET) overexpression combined with RAS (including KRAS, NRAS and HRAS ) or BRAF mutations were associated with late distant metastases and the prognosis of patients with colorectal cancer (CRC). A total of 374 patients with stage III CRC were classified into 4 groups based on RAS/BRAF and C-MET status for comprehensive analysis. Mutations in RAS / BRAF were determined using Sanger sequencing and C-MET expression was examined using immunohistochemistry. The associations between RAS/BRAF mutations in combination with C-MET overexpression and clinicopathological variables including survival were evaluated. In addition, their predictive value for late distant metastases were statistically analyzed via logistic regression and receiver operating characteristic analysis. Among 374 patients, mutations in KRAS, NRAS, HRAS, BRAF and C-MET overexpression were observed in 43.9, 2.4, 0.3, 5.9 and 71.9% of cases, respectively. Considering RAS/BRAF mutations and C-MET overexpression, vascular invasion (P=0.001), high carcino-embryonic antigen level (P=0.031) and late distant metastases (PC-MET overexpression, although they were both predictive factors for adverse prognosis. Further logistic regression suggested that RAS/BRAF mutations and C-MET overexpression may predict late distant metastases. In conclusion, RAS/BRAF mutations and C-MET overexpression may serve as predictive indicators for metastatic behavior and poor prognosis of CRC.

  16. Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells

    International Nuclear Information System (INIS)

    Ho, P.-Y.; Hsu, S.-P.; Liang, Y.-C.; Kuo, M.-L.; Ho, Y.-S.; Lee, W.-S.

    2008-01-01

    Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstrated that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest

  17. Stressing fish in Recirculating Aquaculture Systems (RAS): Does stress induced in one group of fish affect the feeding motivation of other fish sharing the same RAS?

    NARCIS (Netherlands)

    Martins, C.I.; Eding, E.H.; Verreth, J.A.J.

    2011-01-01

    As a consequence of water re-use and high stocking densities, Recirculating Aquaculture Systems (RAS) may lead to an accumulation of substances released by the fish into the water, e.g. cortisol and alarm pheromones. This study investigated the effect of stressing fish on the feeding motivation of

  18. Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Fiorella Guadagni

    2012-01-01

    Full Text Available The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine at codon 57. In addition, we found in the same patient’s sample a silent polymorphism at codon 11 (Ala11Ala of exon 1. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 729–733

  19. Ras oncogenes in oral cancer: the past 20 years.

    Science.gov (United States)

    Murugan, Avaniyapuram Kannan; Munirajan, Arasambattu Kannan; Tsuchida, Nobuo

    2012-05-01

    Oral squamous cell carcinoma (OSCC) of head and neck is associated with high morbidity and mortality in both Western and Asian countries. Several risk factors for the development of oral cancer are very well established, including tobacco chewing, betel quid, smoking, alcohol drinking and human papilloma virus (HPV) infection. Apart from these risk factors, many genetic factors such as oncogenes, tumor suppressor genes and regulatory genes are identified to involve in oral carcinogenesis with these risk factors dependent and independent manner. Ras is one of the most frequently genetically deregulated oncogene in oral cancer. In this review, we analyze the past 22years of literature on genetic alterations such as mutations and amplifications of the isoforms of the ras oncogene in oral cancer. Further, we addressed the isoform-specific role of the ras in oral carcinogenesis. We also discussed how targeting the Akt and MEK, downstream effectors of the PI3K/Akt and MAPK pathways, respectively, would probably pave the possible molecular therapeutic target for the ras driven tumorigenesis in oral cancer. Analysis of these ras isoforms may critically enlighten specific role of a particular ras isoform in oral carcinogenesis, enhance prognosis and pave the way for isoform-specific molecular targeted therapy in OSCC. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. K-RasV14I recapitulates Noonan syndrome in mice

    Science.gov (United States)

    Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J.; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R.; Guerra, Carmen; Barbacid, Mariano

    2014-01-01

    Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-RasV14I, a recurrent KRAS mutation in NS patients. K-RasV14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-RasV14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome. PMID:25359213

  1. Involvement of ZFPIP/Zfp462 in chromatin integrity and survival of P19 pluripotent cells

    International Nuclear Information System (INIS)

    Masse, Julie; Laurent, Audrey; Nicol, Barbara; Guerrier, Daniel; Pellerin, Isabelle; Deschamps, Stephane

    2010-01-01

    Toti- or pluripotent cells proliferation and/or differentiation have been shown to be strongly related to nuclear chromatin organization and structure over the last past years. We have recently identified ZFPIP/Zfp462 as a zinc finger nuclear factor necessary for correct cell division during early embryonic developmental steps of vertebrates. We thus questioned whether this factor was playing a general role during cell division or if it was somehow involved in embryonic cell fate or differentiation. To achieve this goal, we performed a knock-down experiment in the pluripotent P19 and differentiated 3T3 cell lines, both expressing endogenous ZFPIP/Zfp462. Using specific shRNA directed against ZFPIP/Zfp462 transcripts, we demonstrated that depletion of this protein induced cell death in P19 but had no effect in 3T3 cells. In addition, in the absence of the protein, the P19 cells exhibited a complete destructuration of pericentromeric domains associated with a redistribution of the HP1α proteins and an increase in DNA satellites transcribed RNAs level. These data suggested an instrumental role of ZFPIP/Zfp462 in maintaining the chromatin structure of pluripotent cells.

  2. High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27Kip Cdk-complexes

    DEFF Research Database (Denmark)

    Groth, Anja; Willumsen, Berthe Marie

    2005-01-01

    The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells...

  3. Trabecular meshwork ECM remodeling in glaucoma: could RAS be a target?

    Science.gov (United States)

    Agarwal, Puneet; Agarwal, Renu

    2018-06-14

    Disturbances of extracellular matrix (ECM) homeostasis in trabecular meshwork (TM) cause increased aqueous outflow resistance leading to elevated intraocular pressure (IOP) in glaucomatous eyes. Therefore, restoration of ECM homeostasis is a rational approach to prevent disease progression. Since renin-angiotensin system (RAS) inhibition positively alters ECM homeostasis in cardiovascular pathologies involving pressure and volume overload, it is likely that RAS inhibitors reduce IOP primarily by restoring ECM homeostasis. Areas covered: Current evidence showing the presence of RAS components in ocular tissue and its role in regulating aqueous humor dynamics is briefly summarized. The role of RAS in ECM remodeling is discussed both in terms of its effects on ECM synthesis and its breakdown. The mechanisms of ECM remodeling involving interactions of RAS with transforming growth factor-β, Wnt/β-catenin signaling, bone morphogenic proteins, connective tissue growth factor, and matrix metalloproteinases in ocular tissue are discussed. Expert opinion: Current literature strongly indicates a significant role of RAS in ECM remodeling in TM of hypertensive eyes. Hence, IOP-lowering effect of RAS inhibitors may primarily be attributed to restoration of ECM homeostasis in aqueous outflow pathways rather than its vascular effects. However, the mechanistic targets for RAS inhibitors have much wider distribution and consequences, which remain relatively unexplored in TM.

  4. Prenatal Exposure to LPS Alters The Intrarenal RAS in Offspring, Which Is Ameliorated by Adipose Tissue-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Ding, Xian-Fei; Sun, Mou; Guan, Fang-Xia; Guo, Li-Na; Zhang, Yan-Yan; Wan, You-Dong; Zhang, Xiao-Juan; Yu, Yan-Wu; Ma, Shan-Shan; Yao, Hai-Mu; Yao, Rui; Zhang, Rui-Fang; Sun, Tong-Wen; Kan, Quan-Cheng

    2017-11-06

    Prenatal lipopolysaccharide (LPS) exposure causes hypertension in rat offspring through an unknown mechanism. Here, we investigated the role of the intrarenal renin-angiotensin system (RAS) in hypertension induced by prenatal LPS exposure and also explored whether adipose tissue-derived mesenchymal stem cells (ADSCs) can ameliorate the effects of prenatal LPS exposure in rat offspring. Sixty-four pregnant rats were randomly divided into 4 groups (n = 16 in each), namely, a control group and an LPS group, which were intraperitoneally injected with vehicle and 0.79 mg/kg LPS, respectively, on the 8th, 10th, and 12th days of gestation; an ADSCs group, which was intravenously injected with 1.8 × 107 ADSCs on the 8th, 10th, and 12th days of gestation; and an LPS + ADSCs group, which received a combination of the treatments administered to the LPS and ADSCs groups. Prenatal LPS exposure increased blood pressure, Ang II expression, Ang II-positive, monocyte and lymphocyte, apoptotic cells in the kidney, and induced renal histological changes in offspring; however, the LPS and control groups did not differ significantly with respect to plasma renin activity levels, Ang II levels, or renal function. ADSCs treatment attenuated the blood pressure and also ameliorated the other effects of LPS-treated adult offspring. Prenatal exposure to LPS activates the intrarenal RAS but not the circulating RAS and thus induces increases in blood pressure in adult offspring; however, ADSCs treatment attenuates the blood pressure increases resulting from LPS exposure and also ameliorates the other phenotypic changes induced by LPS treatment by inhibiting intrarenal RAS activation. © American Journal of Hypertension, Ltd 2017. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  5. Morus alba Leaf Lectin (MLL) Sensitizes MCF-7 Cells to Anoikis by Inhibiting Fibronectin Mediated Integrin-FAK Signaling through Ras and Activation of P38 MAPK

    Science.gov (United States)

    Saranya, Jayaram; Shilpa, Ganesan; Raghu, Kozhiparambil G.; Priya, Sulochana

    2017-01-01

    Lectins are a unique class of carbohydrate binding proteins/glycoproteins, and many of them possess anticancer properties. They can induce cell cycle arrest and apoptosis, inhibit protein synthesis, telomerase activity and angiogenesis in cancer cells. In the present study, we have demonstrated the effect of Morus alba leaf lectin (MLL) on anoikis induction in MCF-7 cells. Anoikis induction in cancer cells has a significant role in preventing early stage metastasis. MLL treatment in monolayers of MCF-7 cells caused significant detachment of cells in a time and concentration dependent manner. The detached cells failed to re-adhere and grew even to culture plates coated with different matrix proteins. DNA fragmentation, membrane integrity studies, annexin V staining, caspase 9 activation and upregulation of Bax/Bad confirmed that the detached cells underwent apoptosis. Upregulation of matrix metalloproteinase 9 (MMP-9) caused a decrease in fibronectin (FN) production which facilitated the cells to detach by blocking the FN mediated downstream signaling. On treatment with MLL, we have observed downregulation of integrin expression, decreased phosphorylation of focal adhesion kinase (FAK), loss in FAK-integrin interaction and active Ras. MLL treatment downregulated the levels of phosphorylated Akt and PI3K. Also, we have studied the effect of MLL on two stress activated protein kinases p38 MAPK and JNK. p38 MAPK activation was found to be elevated, but there was no change in the level of JNK. Thus our study substantiated the possible antimetastatic effect of MLL by inducing anoikis in MCF-7 cells by activation of caspase 9 and proapoptotic Bax/Bad by blockage of FN mediated integrin/FAK signaling and partly by activation of p38 MAPK. PMID:28223935

  6. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

    Science.gov (United States)

    Su, L N; Little, J B

    1992-08-01

    Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

  7. Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

    Science.gov (United States)

    Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

    2014-03-04

    Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

  8. Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice.

    Directory of Open Access Journals (Sweden)

    Alexander E Urban

    Full Text Available Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis.

  9. Illuga saga Gríðarfóstra in Sweden

    DEFF Research Database (Denmark)

    Lavender, Philip Thomas

    2014-01-01

    This article looks at the intellectual history behind and scholarly preparation towards the first edition of Illuga saga Gríðarfóstra in Sweden in 1695. One of the main questions which the article tries to answer is why an edition of a saga about Danish kings and heroes would end up being produce...

  10. Spectrum of K ras mutations in Pakistani colorectal cancer patients

    International Nuclear Information System (INIS)

    Murtaza, B.N.; Bibi, A.; Rashid, M.U.; Khan, Y.I.; Chaudri, M.S.; Shakoori, A.R.

    2013-01-01

    The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients

  11. Spectrum of K ras mutations in Pakistani colorectal cancer patients

    Energy Technology Data Exchange (ETDEWEB)

    Murtaza, B.N.; Bibi, A. [School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore (Pakistan); Rashid, M.U.; Khan, Y.I. [Shaukat Khanum Memorial Cancer Hospital and Research Centre, Johar Town, Lahore (Pakistan); Chaudri, M.S. [Services Institute of Medical Sciences, Lahore (Pakistan); Shakoori, A.R. [School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore (Pakistan)

    2013-11-29

    The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

  12. Japāņu mode un subkultūras 20.-21.gadsimtā

    OpenAIRE

    Murašova, Kristīne

    2013-01-01

    Bakalaura darba nosaukums ir „Japāņu mode un subkultūras 20.-21.gadsimtā”. Šis darbs sevī iekļauj pētījumu par Japānas populārākajām subkultūrām un modes kustībām pēdējo divu gadsimtu laikā. Pētījums tiek veikts no psiholoģiskā skatu punkta. Darba mērķis ir sniegt lasītājam izpratni par tradicionālas japāņu kultūras konfliktu ar mūsdienu kultūru,analizēt un izprast subkultūru patieso būtību, kā arī uztvert vēstījumu, ko subkultūru pārstāvji cenšas nodot sabiedrībai.Bakalaura darbs sastāv n...

  13. Genetic and Molecular Analysis of Suppressors of Ras Mutations

    National Research Council Canada - National Science Library

    Eastburn, Dennis

    2000-01-01

    .... The study of Caenorhabditis elegans and other model systems has demonstrated that Ras is part of a conserved Ras/MAPK signaling pathway involved in many aspects of development and cell regulation. The C...

  14. Small kinetochore associated protein (SKAP promotes UV-induced cell apoptosis through negatively regulating pre-mRNA processing factor 19 (Prp19.

    Directory of Open Access Journals (Sweden)

    Shan Lu

    Full Text Available Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19. We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.

  15. Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

    Science.gov (United States)

    The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

  16. An Actomyosin-Arf-GEF Negative Feedback Loop for Tissue Elongation under Stress.

    Science.gov (United States)

    West, Junior J; Zulueta-Coarasa, Teresa; Maier, Janna A; Lee, Donghoon M; Bruce, Ashley E E; Fernandez-Gonzalez, Rodrigo; Harris, Tony J C

    2017-08-07

    In response to a pulling force, a material can elongate, hold fast, or fracture. During animal development, multi-cellular contraction of one region often stretches neighboring tissue. Such local contraction occurs by induced actomyosin activity, but molecular mechanisms are unknown for regulating the physical properties of connected tissue for elongation under stress. We show that cytohesins, and their Arf small G protein guanine nucleotide exchange activity, are required for tissues to elongate under stress during both Drosophila dorsal closure (DC) and zebrafish epiboly. In Drosophila, protein localization, laser ablation, and genetic interaction studies indicate that the cytohesin Steppke reduces tissue tension by inhibiting actomyosin activity at adherens junctions. Without Steppke, embryogenesis fails, with epidermal distortions and tears resulting from myosin misregulation. Remarkably, actomyosin network assembly is necessary and sufficient for local Steppke accumulation, where live imaging shows Steppke recruitment within minutes. This rapid negative feedback loop provides a molecular mechanism for attenuating the main tension generator of animal tissues. Such attenuation relaxes tissues and allows orderly elongation under stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. File list: Unc.PSC.50.AllAg.P19 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.PSC.50.AllAg.P19 mm9 Unclassified Pluripotent stem cell P19 SRX1098121,SRX10981...20,SRX1098122,SRX1098124,SRX1098123,SRX1098125 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.PSC.50.AllAg.P19.bed ...

  18. File list: Unc.PSC.05.AllAg.P19 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.PSC.05.AllAg.P19 mm9 Unclassified Pluripotent stem cell P19 SRX1098120,SRX10981...23,SRX1098121,SRX1098122,SRX1098125,SRX1098124 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.PSC.05.AllAg.P19.bed ...

  19. File list: Unc.PSC.20.AllAg.P19 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.PSC.20.AllAg.P19 mm9 Unclassified Pluripotent stem cell P19 SRX1098121,SRX10981...20,SRX1098123,SRX1098122,SRX1098124,SRX1098125 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.PSC.20.AllAg.P19.bed ...

  20. File list: Unc.PSC.10.AllAg.P19 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.PSC.10.AllAg.P19 mm9 Unclassified Pluripotent stem cell P19 SRX1098120,SRX10981...21,SRX1098122,SRX1098124,SRX1098123,SRX1098125 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.PSC.10.AllAg.P19.bed ...

  1. Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest

    Directory of Open Access Journals (Sweden)

    Dina Dikovskaya

    2015-09-01

    Full Text Available Oncogene-induced senescence (OIS is a tumor suppression mechanism that blocks cell proliferation in response to oncogenic signaling. OIS is frequently accompanied by multinucleation; however, the origin of this is unknown. Here, we show that multinucleate OIS cells originate mostly from failed mitosis. Prior to senescence, mutant H-RasV12 activation in primary human fibroblasts compromised mitosis, concordant with abnormal expression of mitotic genes functionally linked to the observed mitotic spindle and chromatin defects. Simultaneously, H-RasV12 activation enhanced survival of cells with damaged mitoses, culminating in extended mitotic arrest and aberrant exit from mitosis via mitotic slippage. ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects. Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16. In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

  2. CDK2 and PKA mediated-sequential phosphorylation is critical for p19INK4d function in the DNA damage response.

    Directory of Open Access Journals (Sweden)

    Mariela C Marazita

    Full Text Available DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with (32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage.

  3. Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

    Science.gov (United States)

    DeSmet, Marsha L; Fleet, James C

    2017-10-01

    High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH) 2 D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH) 2 D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH) 2 D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH) 2 D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Diet, Lifestyle and risk of K-ras mutation-positive and -negative colorectal adenomas

    NARCIS (Netherlands)

    Wark, P.A.; Kuil, van der W.; Ploemacher, J.; Muijen, van G.N.P.; Mulder, Ch.J.J.; Weijenberg, M.P.; Kok, F.J.; Kampman, E.

    2006-01-01

    K-ras mutation-positive (K-ras+) and -negative (K-ras-) colorectal adenomas may differ clinically and pathologically. As environmental compounds may cause mutations in the growth-related K-ras oncogene or affect clonal selection depending on mutational status, we evaluated whether the aetiology of

  5. Msn2p/Msn4p act as a key transcriptional activator of yeast cytoplasmic thiol peroxidase II.

    Science.gov (United States)

    Hong, Seung-Keun; Cha, Mee-Kyung; Choi, Yong-Soo; Kim, Won-Cheol; Kim, Il-Han

    2002-04-05

    We observed that the transcription of Saccharomyces cerevisiae cytoplasmic thiol peroxidase type II (cTPx II) (YDR453C) is regulated in response to various stresses (e.g. oxidative stress, carbon starvation, and heat-shock). It has been suggested that both transcription-activating proteins, Yap1p and Skn7p, regulate the transcription of cTPx II upon exposure to oxidative stress. However, a dramatic loss of transcriptional response to various stresses in yeast mutant strains lacking both Msn2p and Msn4p suggests that the transcription factors act as a principal transcriptional activator. In addition to two Yap1p response elements (YREs), TTACTAA and TTAGTAA, the presence of two stress response elements (STREs) (CCCCT) in the upstream sequence of cTPx II also suggests that Msn2p/Msn4p could control stress-induced expression of cTPx II. Analysis of the transcriptional activity of site-directed mutagenesis of the putative STREs (STRE1 and STRE2) and YREs (TRE1 and YRE2) in terms of the activity of a lacZ reporter gene under control of the cTPx II promoter indicates that STRE2 acts as a principal binding element essential for transactivation of the cTPx II promoter. The transcriptional activity of the cTPx II promoter was exponentially increased after postdiauxic growth. The transcriptional activity of the cTPx II promoter is greatly increased by rapamycin. Deletion of Tor1, Tor2, Ras1, and Ras2 resulted in a considerable induction when compared with their parent strains, suggesting that the transcription of cTPx II is under negative control of the Ras/cAMP and target of rapamycin signaling pathways. Taken together, these results suggest that cTPx II is a target of Msn2p/Msn4p transcription factors under negative control of the Ras-protein kinase A and target of rapamycin signaling pathways. Furthermore, the accumulation of cTPx II upon exposure to oxidative stress and during the postdiauxic shift suggests an important antioxidant role in stationary phase yeast cells.

  6. Influence of K-ras status and anti-tumour treatments on complications due to colorectal self-expandable metallic stents: a retrospective multicentre study.

    Science.gov (United States)

    Fuccio, Lorenzo; Correale, Loredana; Arezzo, Alberto; Repici, Alessandro; Manes, Gianpiero; Trovato, Cristina; Mangiavillano, Benedetto; Manno, Mauro; Cortelezzi, Claudio Camillo; Dinelli, Marco; Cennamo, Vincenzo; de Bellis, Mario

    2014-06-01

    This study aimed to explore the relationship between K-ras status, anti-tumour treatments, and the complications of colorectal self-expandable metallic stenting in colorectal cancer. This is a retrospective, multicentre study of 91 patients with obstructive advanced colorectal cancer palliated with enteral stents between 2007 and 2011. K-ras wild-type tumours were diagnosed in 44 patients (48.4%); 82 (90.1%) received chemotherapy and 45 (49.4%) had additional biological therapy (34 bevacizumab, 11 cetuximab). Twenty-one (23.1%) experienced stent-related complications: 11 (52.4%) occurred in the K-ras mutant group (P=0.9). K-ras wild-type patients were not less likely to develop adverse events than K-ras mutant patients (OR, 0.99; 95% CI: 0.4-2.7). Overall mean time to complication was 167.6 days (range 4-720 days), with no difference between the two groups (141 vs. 197 days; P=0.5). Chemotherapy did not influence the risk of complications (OR, 0.56; 95% CI: 0.14-2.9), and there was no evidence that patients treated with chemotherapy and cetuximab were more likely to experience stent-related complications than patients treated with chemotherapy alone, or untreated (OR, 1.2; 95% CI: 0.2-5.9). Although perforation rates were higher with bevacizumab-based treatment (11.8% vs. 7%), this result was not statistically significant (P=0.69). K-ras mutation status, chemotherapy, and biological treatments should not influence colorectal stent-related complication rates. Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  7. RAS signalling in energy metabolism and rare human diseases.

    Science.gov (United States)

    Dard, L; Bellance, N; Lacombe, D; Rossignol, R

    2018-05-08

    The RAS pathway is a highly conserved cascade of protein-protein interactions and phosphorylation that is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Recent findings indicate that the RAS pathway plays a role in the regulation of energy metabolism via the control of mitochondrial form and function but little is known on the participation of this effect in RAS-related rare human genetic diseases. Germline mutations that hyperactivate the RAS pathway have been discovered and linked to human developmental disorders that are known as RASopathies. Individuals with RASopathies, which are estimated to affect approximately 1/1000 human birth, share many overlapping characteristics, including cardiac malformations, short stature, neurocognitive impairment, craniofacial dysmorphy, cutaneous, musculoskeletal, and ocular abnormalities, hypotonia and a predisposition to developing cancer. Since the identification of the first RASopathy, type 1 neurofibromatosis (NF1), which is caused by the inactivation of neurofibromin 1, several other syndromes have been associated with mutations in the core components of the RAS-MAPK pathway. These syndromes include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), which was formerly called LEOPARD syndrome, Costello syndrome (CS), cardio-facio-cutaneous syndrome (CFC), Legius syndrome (LS) and capillary malformation-arteriovenous malformation syndrome (CM-AVM). Here, we review current knowledge about the bioenergetics of the RASopathies and discuss the molecular control of energy homeostasis and mitochondrial physiology by the RAS pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Coexistence of K-ras mutations and HPV infection in colon cancer

    Directory of Open Access Journals (Sweden)

    Tezol Ayda

    2006-05-01

    Full Text Available Abstract Background Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer. Methods K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact tests Results HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis. Conclusion Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype.

  9. Acute interstitial nephritis induced by intermittent use of Rifampicin in patient with Brucellosis

    International Nuclear Information System (INIS)

    Salih, S. Bin; Kharal, M.; Qahtani, M.; Dahneem, L.; Nohair, S.

    2008-01-01

    Acute oliguric renal failure (ARF) developed in a patient 2 days after she was started on intermittent anti-Brucella therapy including rifampicin. The clinical picture was compatible with acute allergic interstitial nephritis. Renal histology revealed mainly acute tubular necrosis with mild tubulo-intertitial mononuclear cellular infiltrate. Intermittent therapy, as in our patient, has been the major factor in the development of rifampicin induced ARF in cases reviewed in literature. (author)

  10. Gastroprotective effects of several H2RAs on ibuprofen-induced gastric ulcer in rats.

    Science.gov (United States)

    Liu, Jing; Sun, Dan; He, Jinfeng; Yang, Chengli; Hu, Tingting; Zhang, Lijing; Cao, Hua; Tong, Ai-Ping; Song, Xiangrong; Xie, Yongmei; He, Gu; Guo, Gang; Luo, Youfu; Cheng, Ping; Zheng, Yu

    2016-03-15

    Ibuprofen is the first line of treatment for osteoarthritis and arthritis. The main side effects of ibuprofen especially in long-term treatment include gastric ulcer, duodenal ulcer and indigestion etc. Therefore, screening drugs with effective gastric protective effects and low toxicity for combination therapy with ibuprofen is necessary. The mechanism of gastric damage induced by ibuprofen is still unclear, however, cell damage caused by reactive oxygen species (ROS) is considered as the main reason. Preliminary screening of literature with the criteria of low toxicity led to four histamine-2 receptor antagonists (H2RAs): nizatidine, famotidine, lafutidine, and roxatidine acetate, which were selected for further investigation. These drugs were evaluated systemically by examining the gastric ulcer index, lipid peroxidation (LPO), membrane permeability, toxicity to main organs, and the influence on the activity of antioxidant enzymes, and myeloperoxidase (MPO). Nizatidine was found to be the best gastric protective agent. It exhibited excellent protective effect by increasing antioxidant enzyme activity, decreasing MPO activity, reducing LPO, and membrane permeability. Combination treatment with nizatidine and ibuprofen did not show any significant toxicity. Nizatidine was considered as a good option for combination therapy with ibuprofen especially for diseases that require long-term treatment such as arthritis and osteoarthritis. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

    Energy Technology Data Exchange (ETDEWEB)

    Su, L.-N.; Little, J.B. (Harvard School of Public Health, Boston, MA (United States))

    1992-08-01

    Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author).

  12. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

    International Nuclear Information System (INIS)

    Su, L.-N.; Little, J.B.

    1992-01-01

    Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author)

  13. Ras activation by SOS

    DEFF Research Database (Denmark)

    Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen

    2014-01-01

    Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual ...

  14. The ArfGAP2/3 Glo3 and ergosterol collaborate in transport of a subset of cargoes

    Directory of Open Access Journals (Sweden)

    Alejandro F. Estrada

    2015-07-01

    Full Text Available Proteins reach the plasma membrane through the secretory pathway in which the trans Golgi network (TGN acts as a sorting station. Transport from the TGN to the plasma membrane is maintained by a number of different pathways that act either directly or via the endosomal system. Here we show that a subset of cargoes depends on the ArfGAP2/3 Glo3 and ergosterol to maintain their proper localization at the plasma membrane. While interfering with neither ArfGAP2/3 activity nor ergosterol biosynthesis individually significantly altered plasma membrane localization of the tryptophan transporter Tat2, the general amino acid permease Gap1 and the v-SNARE Snc1, in a Δglo3 Δerg3 strain those proteins accumulated in internal endosomal structures. Export from the TGN to the plasma membrane and recycling from early endosomes appeared unaffected as the chitin synthase Chs3 that travels along these routes was localized normally. Our data indicate that a subset of proteins can reach the plasma membrane efficiently but after endocytosis becomes trapped in endosomal structures. Our study supports a role for ArfGAP2/3 in recycling from endosomes and in transport to the vacuole/lysosome.

  15. Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

    Science.gov (United States)

    Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

    2001-10-01

    Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

  16. Utilizing ras signaling pathway to direct selective replication of herpes simplex virus-1.

    Directory of Open Access Journals (Sweden)

    Weihong Pan

    Full Text Available Re-engineering the tropism of viruses is an attractive translational strategy for targeting cancer cells. The Ras signal transduction pathway is a central hub for a variety of pro-oncogenic events with a fundamental role in normal and neoplastic physiology. In this work we were interested in linking Ras activation to HSV-1 replication in a direct manner in order to generate a novel oncolytic herpes virus which can target cancer cells. To establish such link, we developed a mutant HSV-1 in which the expression of ICP4 (infected cell protein-4, a viral protein necessary for replication is controlled by activation of ELK, a transcription factor down-stream of the Ras pathway and mainly activated by ERK (extracellular signal-regulated kinase, an important Ras effector pathway. This mutant HSV-1 was named as Signal-Smart 1 (SS1. A series of prostate cells were infected with the SS1 virus. Cells with elevated levels of ELK activation were preferentially infected by the SS1 virus, as demonstrated by increased levels of viral progeny, herpetic glycoprotein C and overall SS1 viral protein production. Upon exposure to SS1, the proliferation, invasiveness and colony formation capabilities of prostate cancer cells with increased ELK activation were significantly decreased (p<0.05, while the rate of apoptosis/necrosis in these cells was increased. Additionally, high Ras signaling cells infected with SS1 showed a prominent arrest in the G1 phase of the cell cycle as compared to cells exposed to parental HSV-1. The results of this study reveal the potential for re-modeling the host-herpes interaction to specifically interfere with the life of cancer cells with increased Ras signaling. SS1 also serves as a "prototype" for development of a family of signal-smart viruses which can target cancer cells on the basis of their signaling portfolio.

  17. Aplasia Ras homologue member Ⅰ overexpression inhibits tumor growth and induces apoptosis through inhibition of PI3K/Akt survival pathways in human osteosarcoma MG-63 cells in culture.

    Science.gov (United States)

    Ye, Kaishan; Wang, Shuanke; Yang, Yong; Kang, Xuewen; Wang, Jing; Han, Hua

    2015-09-01

    Aplasia Ras homologue member Ⅰ (ARHI), an imprinted tumor-suppressor gene, is downregulated in various types of cancer. However, the expression, function and specific mechanisms of ARHI in human osteosarcoma (OS) cells remain unclear. The aim of the present study was to assess the effect of ARHI on OS cell proliferation and apoptosis and its associated mechanism. In the study, ARHI mRNA and protein levels were markedly downregulated in OS cells compared with the human osteoblast precursor cell line hFOB1.19. By generating stable transfectants, ARHI was overexpressed in OS cells that had low levels of ARHI. Overexpression of ARHI inhibited cell viability and proliferation and induced apoptosis. However, caspase‑3 activity was not changed by ARHI overexpression. In addition, phosphorylated Akt protein expression decreased in the ARHI overexpression group compared to that in the control vector group. The knockdown of ARHI also resulted in the promotion of cell proliferation and the attenuation of apoptosis in MG‑63 cells. Additionally, ARHI silencing increased the level of p‑Akt. The present results indicate that ARHI inhibits OS cell proliferation and may have a key role in the development of OS.

  18. Evaluation of Control and Adjustment Structures of the Shibab Main Canal Using HEC-RAS

    Directory of Open Access Journals (Sweden)

    maryam mohammadi

    2017-02-01

    Full Text Available Introduction: Poor performance of irrigation and drainage networks causes to reduce the transfer and distribution throughputs and in result comes useless water and makes too much consumption in forming. A significant portion of water losses in irrigation and drainage networksis related to transmission and distribution. Therefor more consideration in thrirrigation network management, improve irrigation efficiency and also the exploitation of water resources, especially in the agricultural sector is necessary. Controlling and adjusting structures of water level in the direction of drainage and irrigation canals can influence on increasing of throughput and decrease the use of water. So, right choosing and recognition of the deficiencies of these structures helps carry up the throughput of the networks and prevents to waste water. It is hard to solve equations of water flow in canals and related institutions by using analytic methods. For this reason, this research was done with HEC-RAS hydraulical model in the main channelof irrigation and drainage network of Sistan plain. Materials and Methods: Sistan plain is located in southeastern of Iran with good potential for agricultural production because of the alluvial sediments from Hirmand River. 23820 ha of the Sistan plain is covered by 5 blocksof the Shibab irrigation and drainage network. While Sistan’sShibab irrigation network efficiency is low, HEC-RAS Hydraulics model in unsteady condition was performed to control and adjust this network’s main canal in approximately 19 Km length. In this research, the evaluation model in the canal was performed for more suitable intakingwater in the quadruple order 2 canals . So, the existing structure’s operation was analyzed on controlling structures in management and lack of management situations.This research was assessed during a 15-day impounding period using hydraulic model of HEC-RAS with the aim of performance and operation evaluation of

  19. k-RAS mutations in non-small cell lung cancer patients treated with TKIs among smokers and non-smokers: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Ai-Gui Jiang

    2016-06-01

    Full Text Available Aim of the study : Recent studies have suggested that k-RAS mutations are related to the response to epidermal growth factor receptor (EGFR tyrosine-kinase inhibitions (TKIs in advanced non-small cell lung cancer (NSCLC treatment. The aim of this meta-analysis was to assess the relationship between smoking history and k-RAS mutations in NSCLC treated with TKIs. Material and methods : We searched MEDLINE and Web of Science up to 15 March 2014. The pooled relative risk (RR was estimated by using fixed effect model or random effect model, according to heterogeneity between studies. We also carried out power analyses. Results : We identified 12 studies with 1193 patients, including 196 patients (16.4% with k-RAS mutations. The pooled k-RAS mutations incidence was 22.8% (174/764 in patients with smoke expose vs. 5.4% (23/429 in those with no smoke exposure. The pooled RR was 2.991 (95% CI: 1.884–4.746; Z = 4.65, p = 0.000. No publication bias was found (Begg’s test: z = 1.09, p = 0.274 and Egger’s test: t = 1.38, p = 0.201. In subgroup analyses, the pooled RR was 3.336 (95% CI: 1.925–5.779; Z = 4.30, p = 0.000 in the Caucasian subgroup, while in the Asian subgroup the pooled RR was 2.093 (95% CI: 0.909–4.822; Z = 1.73, p = 0.083, but the sample size was underpowered (0.465. Conclusions : The current meta-analysis found that smoking was related to increased incidence of k-RAS mutations in non-small cell lung cancer treated with TKIs. This may be further evidence that smoking will lead to a worse prognosis in NSCLC patients treated with TKIs.

  20. Clinical comparison of overlapping deletions of 19p13.3.

    Science.gov (United States)

    Risheg, Hiba; Pasion, Romela; Sacharow, Stephanie; Proud, Virginia; Immken, LaDonna; Schwartz, Stuart; Tepperberg, Jim H; Papenhausen, Peter; Tan, Tiong Y; Andrieux, Joris; Plessis, Ghislaine; Amor, David J; Keitges, Elisabeth A

    2013-05-01

    We present three patients with overlapping interstitial deletions of 19p13.3 identified by high resolution SNP microarray analysis. All three had a similar phenotype characterized by intellectual disability or developmental delay, structural heart abnormalities, large head relative to height and weight or macrocephaly, and minor facial anomalies. Deletion sizes ranged from 792 Kb to 1.0 Mb and included a common region arr [hg19] 19p13.3 (3,814,392-4,136,989), containing eight genes: ZFR2, ATCAY, NMRK2, DAPK3, EEF2, PIAS4, ZBTB7A, MAP2K2, and two non-coding RNA's MIR637 and SNORDU37. The patient phenotypes were compared with three previous single patient reports with similar interstitial 19p13.3 deletions and six additional patients from the DECIPHER and ISCA databases to determine if a common haploinsufficient phenotype for the region can be established. Copyright © 2013 Wiley Periodicals, Inc.

  1. Impact of The Protective Renin-Angiotensin System (RAS) on The Vasoreparative Function of CD34+ CACs in Diabetic Retinopathy

    Science.gov (United States)

    Duan, Yaqian; Moldovan, Leni; Miller, Rehae C.; Beli, Eleni; Salazar, Tatiana; Hazra, Sugata; Al-Sabah, Jude; Chalam, KV; Raghunandan, Sneha; Vyas, Ruchi; hide

    2016-01-01

    Purpose: In diabetes, the impaired vasoreparative function of Circulating Angiogenic Cells (CACs) is believed to contribute to the progression of diabetic retinopathy (DR). Accumulating evidence suggests that the protective arm of renin-angiotensin system (RAS) ACE2 Angiotensin-(1-7) Mas plays an important role in restoring the function of diabetic CACs. We examined the protective RAS in CACs in diabetic individuals with different stages of retinopathy. Methods: Study subjects (n43) were recruited as controls or diabetics with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fundus photography and fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ CACs were isolated from peripheral blood of diabetics and control subjects. RAS gene expressions in CACs were measured by qPCR. The vasoreparative function of CACs was assessed by migration ability toward CXCL12 using the QCM 5M 96-well chemotaxis cell migration assay. Results: ACE2 gene is a key enzyme converting the deleterious Angiotensin II to the beneficial Angiotensin-(1-7). ACE2 expression in CACs from diabetic subjects without DR was increased compared to controls, suggestive of compensation (p0.0437). The expression of Mas (Angiotensin-(1-7) receptor) in CACs was also increased in diabetics without DR, while was reduced in NPDR compared to controls (p0.0002), indicating a possible loss of compensation of the protective RAS at this stage of DR. The presence of even mild NPDR was associated with CD34+ CAC migratory dysfunction. When pretreating CACs of DR subjects with Angiotensin-(1-7), migratory ability to a chemoattractant CXCL12 was restored (p0.0008). By VESGEN analysis, an increase in small vessel density was observed in NPDR subjects when compared with the controls. Conclusions: These data suggest the protective RAS axis within diabetic CACs may help maintain their vasoreparative potential

  2. Changes in microbial water quality in RAS following altered feed loading

    DEFF Research Database (Denmark)

    Rojas-Tirado, Paula Andrea; Pedersen, Per Bovbjerg; Vadstein, Olav

    2018-01-01

    and inorganic nutrients available for microbial growth in RAS. How these nutrient inputs affect and regulate bacteria in RAS water is, however, unclear. To investigate this relationship and the associated water quality dynamics, the effects of altered feed loading on microbial water quality in RAS was studied....... The study included six independent, identical pilot-scale RAS, each with a total volume of 1.7 m3 (make-up water: 80 L/day) stocked with juvenile rainbow trout (Oncorhynchus mykiss). All systems had been operating with constant and identical feed loading of 3.13 kg feed/m3 make-up water for a period......Intensive recirculating aquaculture systems (RAS) with its hyper-eutrophic water offer ideal conditions for bacterial growth, abundance and activity, potentially affecting fish and system performance. Feed composition and feed loading in particular will have significant impact on organic...

  3. Prostaglandin E2 blocks menadione-induced apoptosis through the Ras/Raf/Erk signaling pathway in promonocytic leukemia cell lines.

    Science.gov (United States)

    Yeo, Hyun-Seok; Shehzad, Adeeb; Lee, Young Sup

    2012-04-01

    Altered oxidative stress has long been observed in cancer cells, and this biochemical property of cancer cells represents a specific vulnerability that can be exploited for therapeutic benefit. The major role of an elevated oxidative stress for the efficacy of molecular targeted drugs is under investigation. Menadione is considered an attractive model for the study of oxidative stress, which can induce apoptosis in human leukemia HL-60 cell lines. Prostaglandin E(2) (PGE(2)) via its receptors not only promotes cell survival but also reverses apoptosis and promotes cancer progression. Here, we present evidence for the biological role of PGE(2) as a protective agent of oxidative stress-induced apoptosis in monocytic cells. Pretreatment of HL-60 cells with PGE(2) markedly ameliorated the menadione-induced apoptosis and inhibited the degradation of PARP and lamin B. The EP(2) receptor antagonist AH6809 abrogated the inhibitory effect of PGE(2), suggesting the role of the EP(2)/cAMP system. The PKA inhibitor H89 also reversed apoptosis and decreased the PKA activity that was elevated 10-fold by PGE(2). The treatment of HL-60 cells with NAC or zinc chloride showed a similar protective effect as with PGE(2) on menadione-treated cells. Furthermore, PGE(2) activated the Ras/Raf/MEK pathway, which in turn initiated ERK activation, and ultimately protected menadione-induced apoptosis. These results imply that PGE(2) via cell survival pathways may protect oxidative stress-induced apoptosis in monocytic cells. This study warrants further pre-clinical investigation as well as application towards leukemia clinics.

  4. Mevalonates, Ras and Breast Cancer

    National Research Council Canada - National Science Library

    White, Michael

    2001-01-01

    .... This selective inhibition appears to be a consequence of expression of oncogenic Ras. Here we are evaluating the ability of Fmev to selectively interfere with proliferation of breast cancer cells...

  5. Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

    Science.gov (United States)

    Lechuga, Carmen G; Simón-Carrasco, Lucía; Jacob, Harrys K C; Drosten, Matthias

    2017-01-01

    Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Ras lox (H-Ras -/- , N-Ras -/- , K-Ras lox/lox , RERT ert/ert ) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

  6. Measurement of the fluorine content of three NBS standard reference materials by use of the /sup 19/F(p, p'. gamma. )/sup 19/F reaction

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, A L; Kraner, H W; Shroy, R E; Jones, K W [Brookhaven National Lab., Upton, NY (USA)

    1984-08-01

    The fluorine contents of National Bureau of Standards (NBS) Standard Reference Materials (SRM) 91, opal glass; 120b, phosphate rock; and 2671a, freeze-dried urine; have been measured using the /sup 19/F(p,p'..gamma..)/sup 19/F reaction at a proton energy of 3.1 MeV. The results are in good agreement with the values certified by the NBS.

  7. Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

    Science.gov (United States)

    Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

    2012-01-01

    Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the

  8. Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

    International Nuclear Information System (INIS)

    Chang, Eun-Ju; Kim, Hong-Hee; Huh, Jung-Eun; Kim, In-Ae; Seung Ko, Jea; Chung, Chong-Pyoung; Kim, Hyun-Man

    2005-01-01

    The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growth factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue

  9. Structuring of poly ether ether ketone by ArF excimer laser radiation in different atmospheres

    International Nuclear Information System (INIS)

    Feng, Y.; Gottmann, J.; Kreutz, E.W.

    2003-01-01

    Structuring of poly ether ether ketone (PEEK) by 193 nm ArF excimer laser radiation has been investigated. Experiments were carried out in different atmospheres (air, vacuum, Ar, O 2 ) in order to study its influence on the quality of the structures and the formation of the debris. Repetition rate makes little effect on the ablation rate and roughness of the structure in presence of any kind of atmosphere, indicating for the structuring of PEEK by ArF laser radiation a large window of processing. The roughness at the bottom of the structures and the morphology of the side walls are strongly affected by the properties of the atmosphere. The smallest roughness is achieved at 0.6 J/cm 2 for all kinds of processing gases. Debris around the structures can be diminished by structuring in vacuum. Plasma expansion speed has been measured by using high speed photography

  10. Gasochromic performance of WO3-nanorod thin films fabricated with an ArF excimer laser

    International Nuclear Information System (INIS)

    Yaacob, M. H.; Ou, J. Z.; Wlodarski, W.; Kim, C. S.; Lee, J. Y.; Kim, Y. H.; Oh, C. M.; Dhakal, K. P.; Kim, J. Y.; Kang, J. H.

    2012-01-01

    Thin films with tungsten trioxide (WO 3 ) nanorods were fabricated by using an ArF pulsed laser deposition system. Because the ArF excimer laser operates at a very short wavelength of 193 nm, short enough to expect strong absorption of the photons in the semiconductor oxide targets, and because the clusters incoming to the substrates have high momentum, we could build thin films with good surface morphology. Highly homogeneous arrays of nanorods with sizes mostly in the range of 30 - 40 nm were observed. The absorbance response towards hydrogen (H 2 ) gas was investigated for a WO 3 film coated with 25-A-thick palladium (Pd). The Pd/WO 3 -nanorod thin films exhibited excellent gasochromic response when measured in the visible-NIR range (400 - 1000 nm). As low as 0.06% H 2 concentration was clearly sensed. A significant reversible absorbance change and fast recovery ( 2 at different concentrations.

  11. LncRNA H19 and Target Gene-mediated Cleft Palate Induced by TCDD.

    Science.gov (United States)

    Gao, Li Yun; Zhang, Feng Quan; Zhao, Wei Hui; Han, Guang Liang; Wang, Xiao; Li, Qiang; Gao, Shan Shan; Wu, Wei Dong

    2017-09-01

    This study investigated the role of long non-coding RNAs (lncRNAs) in the development of the palatal tissues. Cleft palates in mice were induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression levels of long non-coding RNA H19 (lncRNA H19) and insulin-like growth factor 2 (IGF2) gene were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The rate of occurrence of cleft palate was found to be 100% by TCDD exposure, and TCDD could cause short upper limb, cerebral fissure, webbed neck, and short neck. The expression levels of lncRNA H19 and IGF2 gene specifically showed embryo age-related differences on E13, E14, and E15 in the palatal tissues. The expression levels of lncRNA H19 and IGF2 gene showed an inverse relationship on E13, E14, and E15. These findings demonstrated that lncRNA H19 and IGF2 can mediate the development of mouse cleft palate. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  12. Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

    Science.gov (United States)

    Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

    1993-06-25

    Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

  13. Sequence Ready Characterization of the Pericentromeric Region of 19p12

    Energy Technology Data Exchange (ETDEWEB)

    Evan E. Eichler

    2006-08-31

    Current mapping and sequencing strategies have been inadequate within the proximal portion of 19p12 due, in part, to the presence of a recently expanded ZNF (zinc-finger) gene family and the presence of large (25-50 kb) inverted beta-satellite repeat structures which bracket this tandemly duplicated gene family. The virtual of absence of classically defined “unique” sequence within the region has hampered efforts to identify and characterize a suitable minimal tiling path of clones which can be used as templates required for finished sequencing of the region. The goal of this proposal is to develop and implement a novel sequence-anchor strategy to generate a contiguous BAC map of the most proximal portion of chromosome 19p12 for the purpose of complete sequence characterization. The target region will be an estimated 4.5 Mb of DNA extending from STS marker D19S450 (the beginning of the ZNF gene cluster) to the centromeric (alpha-satellite) junction of 19p11. The approach will entail 1) pre-selection of 19p12 BAC and cosmid clones (NIH approved library) utilizing both 19p12 -unique and 19p12-SPECIFIC repeat probes (Eichler et al., 1998); 2) the generation of a BAC/cosmid end-sequence map across the region with a density of one marker every 8kb; 3) the development of a second-generation of STS (sequence tagged sites) which will be used to identify and verify clonal overlap at the level of the sequence; 4) incorporation of these sequence-anchored overlapping clones into existing cosmid/BAC restriction maps developed at Livermore National Laboratory; and 5) validation of the organization of this region utilizing high-resolution FISH techniques (extended chromatin analysis) on monochromosomal 19 somatic cell hybrids and parental cell lines of source material. The data generated will be used in the selection of the most parsimonious tiling path of BAC clones to be sequenced as part of the JGI effort on chromosome 19 and should serve as a model for the sequence

  14. Hotair mediates hepatocarcinogenesis through suppressing miRNA-218 expression and activating P14 and P16 signaling.

    Science.gov (United States)

    Fu, Wei-Ming; Zhu, Xiao; Wang, Wei-Mao; Lu, Ying-Fei; Hu, Bao-Guang; Wang, Hua; Liang, Wei-Cheng; Wang, Shan-Shan; Ko, Chun-Hay; Waye, Mary Miu-Yee; Kung, Hsiang-Fu; Li, Gang; Zhang, Jin-Fang

    2015-10-01

    Long non-coding RNA Hotair has been considered as a pro-oncogene in multiple cancers. Although there is emerging evidence that reveals its biological function and the association with clinical prognosis, the precise mechanism remains largely elusive. We investigated the function and mechanism of Hotair in hepatocellular carcinoma (HCC) cell models and a xenograft mouse model. The regulatory network between miR-218 and Hotair was elucidated by RNA immunoprecipitation and luciferase reporter assays. Finally, the correlation between Hotair, miR-218 and the target gene Bmi-1 were evaluated in 52 paired HCC specimens. In this study, we reported that Hotair negatively regulated miR-218 expression in HCC, which might be mediated through an EZH2-targeting-miR-218-2 promoter regulatory axis. Further investigation revealed that Hotair knockdown dramatically inhibited cell viability and induced G1-phase arrest in vitro and suppressed tumorigenicity in vivo by promoting miR-218 expression. Oncogene Bmi-1 was shown to be a functional target of miR-218, and the main downstream targets signaling, P16(Ink4a) and P14(ARF), were activated in Hotair-suppressed tumorigenesis. In primary human HCC specimens, Hotair and Bmi-1 were concordantly upregulated whereas miR-218 was downregulated in these tissues. Furthermore, Hotair was inversely associated with miR-218 expression and positively correlated with Bmi-1 expression in these clinical tissues. Hotair silence activates P16(Ink4a) and P14(ARF) signaling by enhancing miR-218 expression and suppressing Bmi-1 expression, resulting in the suppression of tumorigenesis in HCC. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  15. R-Ras regulates migration through an interaction with filamin A in melanoma cells.

    Directory of Open Access Journals (Sweden)

    Joanna E Gawecka

    2010-06-01

    Full Text Available Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins.We identified Filamin A (FLNa as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin beta1, beta2 and beta7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaDelta3 abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaDelta3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly.These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration.

  16. Human hypervariable sequences in risk assessment: rare Ha-ras alleles in cancer patients

    International Nuclear Information System (INIS)

    Krontiris, T.G.; DiMartino, N.A.; Mitcheson, H.D.; Lonergan, J.A.; Begg, C.; Parkinson, D.R.

    1987-01-01

    A variable tandem repeat (VTR) is responsible for the hyperallelism one kilobase 3' to the human c-Ha-ras-1 (Ha-ras) gene. Thirty-two distinct restriction fragments, comprising 3 allelic classes by frequency of occurrence, have thus far been detected in a sample size of approximately 800 caucasians. Rare Ha-ras alleles, 21 in all, are almost exclusively confined to the genomes of cancer patients. From their data the authors have computed the relative cancer risk associated with possession of a rare Ha-ras allele to be 27. To understand the molecular basis for this phenomenon, they have begun to clone Ha-ras fragments from nontumor DNA of cancer patients. They report here the weak activation, as detected by transfection and transformation of NIH 3T3 mouse cells, of two Ha-ras genes which were obtained from lymphocyte DNA of a melanoma patient. They have mapped the regions that confer this transforming activity to the fragment containing the VTR in one Ha-ras clone and the fragment containing gene coding sequences in the other

  17. Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

    Science.gov (United States)

    Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

    1994-11-04

    Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

  18. Renin-angiotensin system inhibition ameliorates CCl4-induced liver fibrosis in mice through the inactivation of nuclear transcription factor kappa B.

    Science.gov (United States)

    Saber, Sameh; Mahmoud, Amr A A; Helal, Noha S; El-Ahwany, Eman; Abdelghany, Rasha H

    2018-06-01

    Therapeutic interventions for liver fibrosis are still limited due to the complicated molecular pathogenesis. Renin-angiotensin system (RAS) seems to contribute to the development of hepatic fibrosis. Therefore, we aimed to examine the effect of RAS inhibition on CCl 4 -induced liver fibrosis. Mice were treated with silymarin (30 mg·kg -1 ), perindopril (1 mg·kg -1 ), fosinopril (2 mg·kg -1 ), or losartan (10 mg·kg -1 ). The administration of RAS inhibitors improved liver histology and decreased protein expression of alpha smooth muscle actin (α-SMA) and hepatic content of hydroxyproline. These effects found to be mediated via inactivation of nuclear transcription factor kappa B (NFκB) pathway by the inhibition of NFκB p65 phosphorylation at the Ser536 residue and phosphorylation-induced degradation of nuclear factor kappa-B inhibitor alpha (NFκBia) subsequently inhibited NFκB-induced TNF-α and TGF-β1, leading to lower levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF). We concluded that the tissue affinity of the angiotensin converting enzyme inhibitors (ACEIs) has no impact on its antifibrotic activity and that interfering the RAS either through the inhibition of ACE or the blockade of AT1R has the same therapeutic benefit. These results suggest RAS inhibitors as promising candidates for further clinical trials in the management of hepatic fibrosis.

  19. Literature review : performance of RAP/RAS mixes and new direction.

    Science.gov (United States)

    2014-04-01

    In the last several years reclaimed asphalt pavement (RAP) and recycled asphalt shingles (RAS) have been : widely used in asphalt mixes in Texas. The use of RAP/RAS can significantly reduce the initial cost of : asphalt mixtures, conserve energy, and...

  20. Nf1 Loss and Ras Hyperactivation in Oligodendrocytes Induce NOS-Driven Defects in Myelin and Vasculature

    Directory of Open Access Journals (Sweden)

    Debra A. Mayes

    2013-09-01

    Full Text Available Patients with neurofibromatosis type 1 (NF1 and Costello syndrome Rasopathy have behavioral deficits. In NF1 patients, these may correlate with white matter enlargement and aberrant myelin. To model these features, we induced Nf1 loss or HRas hyperactivation in mouse oligodendrocytes. Enlarged brain white matter tracts correlated with myelin decompaction, downregulation of claudin-11, and mislocalization of connexin-32. Surprisingly, non-cell-autonomous defects in perivascular astrocytes and the blood-brain barrier (BBB developed, implicating a soluble mediator. Nitric oxide (NO can disrupt tight junctions and gap junctions, and NO and NO synthases (NOS1–NOS3 were upregulated in mutant white matter. Treating mice with the NOS inhibitor NG-nitro-L-arginine methyl ester or the antioxidant N-acetyl cysteine corrected cellular phenotypes. CNP-HRasG12V mice also displayed locomotor hyperactivity, which could be rescued by antioxidant treatment. We conclude that Nf1/Ras regulates oligodendrocyte NOS and that dysregulated NO signaling in oligodendrocytes can alter the surrounding vasculature. The data suggest that antioxidants may improve some behavioral deficits in Rasopathy patients.

  1. Direct mapping of 19F in 19FDG-6P in brain tissue at subcellular resolution using soft X-ray fluorescence

    Science.gov (United States)

    Poitry-Yamate, C.; Gianoncelli, A.; Kourousias, G.; Kaulich, B.; Lepore, M.; Gruetter, R.; Kiskinova, M.

    2013-10-01

    Low energy x-ray fluorescence (LEXRF) detection was optimized for imaging cerebral glucose metabolism by mapping the fluorine LEXRF signal of 19F in 19FDG, trapped as intracellular 19F-deoxyglucose-6-phosphate (19FDG-6P) at 1μm spatial resolution from 3μm thick brain slices. 19FDG metabolism was evaluated in brain structures closely resembling the general cerebral cytoarchitecture following formalin fixation of brain slices and their inclusion in an epon matrix. 2-dimensional distribution maps of 19FDG-6P were placed in a cytoarchitectural and morphological context by simultaneous LEXRF mapping of N and O, and scanning transmission x-ray (STXM) imaging. A disproportionately high uptake and metabolism of glucose was found in neuropil relative to intracellular domains of the cell body of hypothalamic neurons, showing directly that neurons, like glial cells, also metabolize glucose. As 19F-deoxyglucose-6P is structurally identical to 18F-deoxyglucose-6P, LEXRF of subcellular 19F provides a link to in vivo 18FDG PET, forming a novel basis for understanding the physiological mechanisms underlying the 18FDG PET image, and the contribution of neurons and glia to the PET signal.

  2. Direct mapping of 19F in 19FDG-6P in brain tissue at subcellular resolution using soft X-ray fluorescence

    International Nuclear Information System (INIS)

    Poitry-Yamate, C; Lepore, M; Gruetter, R; Gianoncelli, A; Kourousias, G; Kiskinova, M; Kaulich, B

    2013-01-01

    Low energy x-ray fluorescence (LEXRF) detection was optimized for imaging cerebral glucose metabolism by mapping the fluorine LEXRF signal of 19 F in 19 FDG, trapped as intracellular 19 F-deoxyglucose-6-phosphate ( 19 FDG-6P) at 1μm spatial resolution from 3μm thick brain slices. 19 FDG metabolism was evaluated in brain structures closely resembling the general cerebral cytoarchitecture following formalin fixation of brain slices and their inclusion in an epon matrix. 2-dimensional distribution maps of 19 FDG-6P were placed in a cytoarchitectural and morphological context by simultaneous LEXRF mapping of N and O, and scanning transmission x-ray (STXM) imaging. A disproportionately high uptake and metabolism of glucose was found in neuropil relative to intracellular domains of the cell body of hypothalamic neurons, showing directly that neurons, like glial cells, also metabolize glucose. As 19 F-deoxyglucose-6P is structurally identical to 18 F-deoxyglucose-6P, LEXRF of subcellular 19 F provides a link to in vivo 18 FDG PET, forming a novel basis for understanding the physiological mechanisms underlying the 18 FDG PET image, and the contribution of neurons and glia to the PET signal

  3. RAS in Pregnancy and Preeclampsia and Eclampsia

    Directory of Open Access Journals (Sweden)

    M. Rodriguez

    2012-01-01

    Full Text Available Preeclampsia is a common disease of pregnancy characterized by the presence of hypertension and commitment of many organs, including the brain, secondary to generalized endothelial dysfunction. Its etiology is not known precisely, but it involved several factors, highlighting the renin angiotensin system (RAS, which would have an important role in the origin of multisystem involvement. This paper reviews the evidence supporting the involvement of RAS in triggering the disease, in addition to the components of this system that would be involved and how it eventually produces brain engagement.

  4. Zonation of heme synthesis enzymes in mouse liver and their regulation by β-catenin and Ha-ras.

    Science.gov (United States)

    Braeuning, Albert; Schwarz, Michael

    2010-11-01

    Cytochrome P450 (CYP) hemoproteins play an important role in hepatic biotransformation. Recently, β-catenin and Ha-ras signaling have been identified as players controlling transcription of various CYP genes in mouse liver. The aim of the present study was to analyze the role of β-catenin and Ha-ras in the regulation of heme synthesis. Heme synthesis-related gene expression was analyzed in normal liver, in transgenic mice expressing activated β-catenin or Ha-ras, and in hepatomas. Regulation of the aminolevulinate dehydratase promoter was studied in vitro. Elevated expression of mRNAs and proteins involved in heme biosynthesis was linked to β-catenin activation in perivenous hepatocytes, in transgenic hepatocytes, and in hepatocellular tumors. Stimulation of the aminolevulinate dehydratase promoter by β-catenin was independent of the β-catenin/T-cell-specific transcription factor dimer. By contrast, activation of Ha-ras repressed heme synthesis-related gene expression. The present data suggest that β-catenin enhances the expression of both CYPs and heme synthesis-related genes, thus coordinating the availability of CYP apoprotein and its prosthetic group heme. The reciprocal regulation of heme synthesis by β-catenin and Ha-ras-dependent signaling supports our previous hypothesis that antagonistic action of these pathways plays a major role in the control of zonal gene expression in healthy mouse liver and aberrant expression patterns in hepatocellular tumors.

  5. Interaction between Wnt/β-catenin and RAS-ERK pathways and an anti-cancer strategy via degradations of β-catenin and RAS by targeting the Wnt/β-catenin pathway.

    Science.gov (United States)

    Jeong, Woo-Jeong; Ro, Eun Ji; Choi, Kang-Yell

    2018-01-01

    Aberrant activation of the Wnt/β-catenin and RAS-extracellular signal-regulated kinase (ERK) pathways play important roles in the tumorigenesis of many different types of cancer, most notably colorectal cancer (CRC). Genes for these two pathways, such as adenomatous polyposis coli ( APC ) and KRAS are frequently mutated in human CRC, and involved in the initiation and progression of the tumorigenesis, respectively. Moreover, recent studies revealed interaction of APC and KRAS mutations in the various stages of colorectal tumorigenesis and even in metastasis accompanying activation of the cancer stem cells (CSCs). A key event in the synergistic cooperation between Wnt/β-catenin and RAS-ERK pathways is a stabilization of both β-catenin and RAS especially mutant KRAS by APC loss, and pathological significance of this was indicated by correlation of increased β-catenin and RAS levels in human CRC where APC mutations occur as high as 90% of CRC patients. Together with the notion of the protein activity reduction by lowering its level, inhibition of both β-catenin and RAS especially by degradation could be a new ideal strategy for development of anti-cancer drugs for CRC. In this review, we will discuss interaction between the Wnt/β-catenin and RAS-ERK pathways in the colorectal tumorigenesis by providing the mechanism of RAS stabilization by aberrant activation of Wnt/β-catenin. We will also discuss our small molecular anti-cancer approach controlling CRC by induction of specific degradations of both β-catenin and RAS via targeting Wnt/β-catenin pathway especially for the KYA1797K, a small molecule specifically binding at the regulator of G-protein signaling (RGS)-domain of Axin.

  6. Interaction between the triglyceride lipase ATGL and the arf1 activator GBF1

    KAUST Repository

    Ellong, Emy Njoh

    2011-07-18

    The Arf1 exchange factor GBF1 (Golgi Brefeldin A resistance factor 1) and its effector COPI are required for delivery of ATGL (adipose triglyceride lipase) to lipid droplets (LDs). Using yeast two hybrid, co-immunoprecipitation in mammalian cells and direct protein binding approaches, we report here that GBF1 and ATGL interact directly and in cells, through multiple contact sites on each protein. The C-terminal region of ATGL interacts with N-terminal domains of GBF1, including the catalytic Sec7 domain, but not with full-length GBF1 or its entire N-terminus. The N-terminal lipase domain of ATGL (called the patatin domain) interacts with two C-terminal domains of GBF1, HDS (Homology downstream of Sec7) 1 and HDS2. These two domains of GBF1 localize to lipid droplets when expressed alone in cells, but not to the Golgi, unlike the full-length GBF1 protein, which localizes to both. We suggest that interaction of GBF1 with ATGL may be involved in the membrane trafficking pathway mediated by GBF1, Arf1 and COPI that contributes to the localization of ATGL to lipid droplets.

  7. Interaction between the triglyceride lipase ATGL and the arf1 activator GBF1

    KAUST Repository

    Ellong, Emy Njoh; Soni, Krishnakant G.; Bui, Quynh-Trang; Sougrat, Rachid; Golinelli-Cohen, Marie-Pierre; Jackson, Catherine L.

    2011-01-01

    The Arf1 exchange factor GBF1 (Golgi Brefeldin A resistance factor 1) and its effector COPI are required for delivery of ATGL (adipose triglyceride lipase) to lipid droplets (LDs). Using yeast two hybrid, co-immunoprecipitation in mammalian cells and direct protein binding approaches, we report here that GBF1 and ATGL interact directly and in cells, through multiple contact sites on each protein. The C-terminal region of ATGL interacts with N-terminal domains of GBF1, including the catalytic Sec7 domain, but not with full-length GBF1 or its entire N-terminus. The N-terminal lipase domain of ATGL (called the patatin domain) interacts with two C-terminal domains of GBF1, HDS (Homology downstream of Sec7) 1 and HDS2. These two domains of GBF1 localize to lipid droplets when expressed alone in cells, but not to the Golgi, unlike the full-length GBF1 protein, which localizes to both. We suggest that interaction of GBF1 with ATGL may be involved in the membrane trafficking pathway mediated by GBF1, Arf1 and COPI that contributes to the localization of ATGL to lipid droplets.

  8. Ablation and cone formation mechanism on CR-39 by ArF laser irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Shakeri Jooybari, B., E-mail: baninshakery@gmail.com, E-mail: hafarideh@aut.ac.ir [Department of Energy Engineering and Physics, Amirkabir University of Technology, P.O. Box 15875-4413, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of); Nuclear Science and Technology Research Institute NSRT, Tehran (Iran, Islamic Republic of); Afarideh, H., E-mail: baninshakery@gmail.com, E-mail: hafarideh@aut.ac.ir [Department of Energy Engineering and Physics, Amirkabir University of Technology, P.O. Box 15875-4413, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of); Lamehi-Rachti, M. [Nuclear Science and Technology Research Institute NSRT, Tehran (Iran, Islamic Republic of); Ghergherehchi, M. [Department of Energy Engineering and Physics, Amirkabir University of Technology, P.O. Box 15875-4413, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of); College of Information and Communication Engineering, Sungkyunkwan University, Suwon (Korea, Republic of)

    2015-03-07

    In this work, chemical properties, surface modification, and micro structures formation on ablated polyallyl di-glycol carbonate (CR-39) polymer by ArF laser irradiation (λ = 193 nm) at various fluences and pulse number were investigated. CR-39 samples have been irradiated with an ArF laser (193 nm) at a repetition rate of 1 Hz. Threshold fluence of ablation and effective absorption coefficient of CR-39 were determined. Conical microstructures (Taylor cone) formed on laser-ablated CR-39 exhibit: smooth, Taylor cone shape walls and sharp tips together with interference and well defined fringe-structure with a period of 230 nm, around cone base. Mechanism of cone formation and cone evolution of CR-39 ablated surface were investigated by change of fluences (at a given pulse number) and pulse number (at a given fluence). Cone height, cone base, and region of interface were increased in micrometer steps by increasing the total fluence. Depression on the base of the cone and the circular fringe were simulated. FTIR spectra were measured and energy dispersive x-ray analysis of irradiated and un-irradiated samples was performed.

  9. A design of energy detector for ArF excimer lasers

    Science.gov (United States)

    Feng, Zebin; Han, Xiaoquan; Zhou, Yi; Bai, Lujun

    2017-08-01

    ArF excimer lasers with short wavelength and high photon energy are widely applied in the field of integrated circuit lithography, material processing, laser medicine, and so on. Excimer laser single pulse energy is a very important parameter in the application. In order to detect the single pulse energy on-line, one energy detector based on photodiode was designed. The signal processing circuit connected to the photodiode was designed so that the signal obtained by the photodiode was amplified and the pulse width was broadened. The amplified signal was acquired by a data acquisition card and stored in the computer for subsequent data processing. The peak of the pulse signal is used to characterize the single pulse energy of ArF excimer laser. In every condition of deferent pulse energy value levels, a series of data about laser pulses energy were acquired synchronously using the Ophir energy meter and the energy detector. A data set about the relationship between laser pulse energy and the peak of the pulse signal was acquired. Then, by using the data acquired, a model characterizing the functional relationship between the energy value and the peak value of the pulse was trained based on an algorithm of machine learning, Support Vector Regression (SVR). By using the model, the energy value can be obtained directly from the energy detector designed in this project. The result shows that the relative error between the energy obtained by the energy detector and by the Ophir energy meter is less than 2%.

  10. Curcumin homing to the nucleolus: mechanism for initiation of an apoptotic program.

    Science.gov (United States)

    Ghosh, Mistuni; Ryan, Robert O

    2014-11-01

    Curcumin is a plant-derived polyphenol that displays antitumor properties. Incubation of cultured SF-767 glioma cells with curcumin gave rise to intense intranuclear foci of curcumin fluorescence. In vitro studies revealed that nuclear homing by curcumin is not a result of DNA/chromatin binding. On the other hand, curcumin fluorescence colocalized with nucleophosmin, a nucleolus marker protein. To determine the temporal relationship between curcumin-induced apoptosis and nucleolar homing, confocal live cell imaging was performed. The data show that curcumin localization to the nucleolus occurs prior to cell surface exposure of phosphatidylserine. In studies of the mechanism of curcumin-induced apoptosis in SF-767 cells, its effect on the subcellular location of p14(ARF) was determined. Whereas p14(ARF) was confined to the nucleolus in untreated cells, 2 h following incubation with curcumin, it displayed a diffuse nuclear distribution. Given the role of nuclear p14(ARF) in binding the E3 ubiquitin ligase, mouse double minute 2 homolog (MDM2), the effect of curcumin treatment on cellular levels of the tumor suppressor protein, p53, was examined. Between 2 and 4 h following curcumin treatment, p53 levels increased with maximum levels reached by 8 h. Thus, curcumin homing to the nucleolus induces redistribution of p14(ARF) to the nucleoplasm where interaction with MDM2 leads to stabilization of p53, with subsequent initiation of apoptosis. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.

    Science.gov (United States)

    Severyn, Bryan; Nguyen, Thi; Altman, Michael D; Li, Lixia; Nagashima, Kumiko; Naumov, George N; Sathyanarayanan, Sriram; Cook, Erica; Morris, Erick; Ferrer, Marc; Arthur, Bill; Benita, Yair; Watters, Jim; Loboda, Andrey; Hermes, Jeff; Gilliland, D Gary; Cleary, Michelle A; Carroll, Pamela M; Strack, Peter; Tudor, Matt; Andersen, Jannik N

    2016-10-01

    The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway. © 2016 Society for Laboratory Automation and Screening.

  12. Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway.

    OpenAIRE

    Puil, L; Liu, J; Gish, G; Mbamalu, G; Bowtell, D; Pelicci, P G; Arlinghaus, R; Pawson, T

    1994-01-01

    The cytosolic 185 and 210 kDa Bcr-Abl protein tyrosine kinases play important roles in the development of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (Ph+ ALL). p185 and p210 Bcr-Abl contain identical abl-encoded sequences juxtaposed to a variable number of bcr-derived amino acids. As the mitogenic and transforming activities of tyrosine kinases involve stimulation of the Ras pathway, we analyzed Bcr-Abl oncoproteins for interacti...

  13. Machine Learning Detects Pan-cancer Ras Pathway Activation in The Cancer Genome Atlas

    Directory of Open Access Journals (Sweden)

    Gregory P. Way

    2018-04-01

    Full Text Available Summary: Precision oncology uses genomic evidence to match patients with treatment but often fails to identify all patients who may respond. The transcriptome of these “hidden responders” may reveal responsive molecular states. We describe and evaluate a machine-learning approach to classify aberrant pathway activity in tumors, which may aid in hidden responder identification. The algorithm integrates RNA-seq, copy number, and mutations from 33 different cancer types across The Cancer Genome Atlas (TCGA PanCanAtlas project to predict aberrant molecular states in tumors. Applied to the Ras pathway, the method detects Ras activation across cancer types and identifies phenocopying variants. The model, trained on human tumors, can predict response to MEK inhibitors in wild-type Ras cell lines. We also present data that suggest that multiple hits in the Ras pathway confer increased Ras activity. The transcriptome is underused in precision oncology and, combined with machine learning, can aid in the identification of hidden responders. : Way et al. develop a machine-learning approach using PanCanAtlas data to detect Ras activation in cancer. Integrating mutation, copy number, and expression data, the authors show that their method detects Ras-activating variants in tumors and sensitivity to MEK inhibitors in cell lines. Keywords: Gene expression, machine learning, Ras, NF1, KRAS, NRAS, HRAS, pan-cancer, TCGA, drug sensitivity

  14. The activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 to the distal CCAAT box of the RhoB promoter

    International Nuclear Information System (INIS)

    Ahn, Jiwon; Choi, Jeong-Hae; Won, Misun; Kang, Chang-Mo; Gyun, Mi-Rang; Park, Hee-Moon; Kim, Chun-Ho; Chung, Kyung-Sook

    2011-01-01

    Highlights: → Regulation of transcriptional activation of RhoB is still unclear. → We examine the effect of p38 MAPK inhibition, and c-Jun and RhoB depletion on UV-induced RhoB expression and apoptosis. → We identify the regions of RhoB promoter necessary to confer UV responsiveness using pRhoB-luciferase reporter assays. → c-Jun, ATF2 and p300 are dominantly associated with NF-Y on the distal CCAAT box. → The activation of p38 MAPK primarily contribute to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins on distal CCAAT box of RhoB promoter. -- Abstract: The Ras-related small GTP-binding protein RhoB is rapidly induced in response to genotoxic stresses caused by ionizing radiation. It is known that UV-induced RhoB expression results from the binding of activating transcription factor 2 (ATF2) via NF-Y to the inverted CCAAT box (-23) of the RhoB promoter. Here, we show that the association of c-Jun with the distal CCAAT box (-72) is primarily involved in UV-induced RhoB expression and p38 MAPK regulated RhoB induction through the distal CCAAT box. UV-induced RhoB expression and apoptosis were markedly attenuated by pretreatment with the p38 MAPK inhibitor. siRNA knockdown of RhoB, ATF2 and c-Jun resulted in decreased RhoB expression and eventually restored the growth of UV-irradiated Jurkat cells. In the reporter assay using luciferase under the RhoB promoter, inhibition of RhoB promoter activity by the p38 inhibitor and knockdown of c-Jun using siRNA occurred through the distal CCAAT box. Immunoprecipitation and DNA affinity protein binding assays revealed the association of c-Jun and p300 via NF-YA and the dissociation of histone deacetylase 1 (HDAC1) via c-Jun recruitment to the CCAAT boxes of the RhoB promoter. These results suggest that the activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins to the distal CCAAT box of the RhoB promoter in Jurkat cells.

  15. Peripheral blood cells from children with RASopathies show enhanced spontaneous colonies growth in vitro and hyperactive RAS signaling

    International Nuclear Information System (INIS)

    Gaipa, G; Bugarin, C; Cianci, P; Sarno, J; Bonaccorso, P; Biondi, A; Selicorni, A

    2015-01-01

    Germline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients

  16. Long term protection after immunization with P. berghei sporozoites correlates with sustained IFNgamma responses of hepatic CD8+ memory T cells.

    NARCIS (Netherlands)

    Nganou Makamdop, C.K.; Gemert, G.J.A. van; Arens, T.; Hermsen, C.C.; Sauerwein, R.W.

    2012-01-01

    Protection against P. berghei malaria can successfully be induced in mice by immunization with both radiation attenuated sporozoites (RAS) arresting early during liver stage development, or sporozoites combined with chloroquine chemoprophylaxis (CPS), resulting in complete intra-hepatic parasite

  17. Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

    Science.gov (United States)

    Ogara, María F; Sirkin, Pablo F; Carcagno, Abel L; Marazita, Mariela C; Sonzogni, Silvina V; Ceruti, Julieta M; Cánepa, Eduardo T

    2013-01-01

    The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.

  18. Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

    Directory of Open Access Journals (Sweden)

    María F Ogara

    Full Text Available The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.

  19. Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

    Science.gov (United States)

    Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

  20. Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast–ovarian cancer susceptibility locus

    Science.gov (United States)

    Lawrenson, Kate; Kar, Siddhartha; McCue, Karen; Kuchenbaeker, Karoline; Michailidou, Kyriaki; Tyrer, Jonathan; Beesley, Jonathan; Ramus, Susan J.; Li, Qiyuan; Delgado, Melissa K.; Lee, Janet M.; Aittomäki, Kristiina; Andrulis, Irene L.; Anton-Culver, Hoda; Arndt, Volker; Arun, Banu K.; Arver, Brita; Bandera, Elisa V.; Barile, Monica; Barkardottir, Rosa B.; Barrowdale, Daniel; Beckmann, Matthias W.; Benitez, Javier; Berchuck, Andrew; Bisogna, Maria; Bjorge, Line; Blomqvist, Carl; Blot, William; Bogdanova, Natalia; Bojesen, Anders; Bojesen, Stig E.; Bolla, Manjeet K.; Bonanni, Bernardo; Børresen-Dale, Anne-Lise; Brauch, Hiltrud; Brennan, Paul; Brenner, Hermann; Bruinsma, Fiona; Brunet, Joan; Buhari, Shaik Ahmad; Burwinkel, Barbara; Butzow, Ralf; Buys, Saundra S.; Cai, Qiuyin; Caldes, Trinidad; Campbell, Ian; Canniotto, Rikki; Chang-Claude, Jenny; Chiquette, Jocelyne; Choi, Ji-Yeob; Claes, Kathleen B. M.; Collonge-Rame, Marie- Agnès; Damette, Alexandre; Barouk-Simonet, Emmanuelle; Bonnet, Françoise; Bubien, Virginie; Sevenet, Nicolas; Longy, Michel; Berthet, Pascaline; Vaur, Dominique; Castera, Laurent; Ferrer, Sandra Fert; Bignon, Yves-Jean; Uhrhammer, Nancy; Coron, Fanny; Faivre, Laurence; Baurand, Amandine; Jacquot, Caroline; Bertolone, Geoffrey; Lizard, Sarab; Leroux, Dominique; Dreyfus, Hélène; Rebischung, Christine; Peysselon, Magalie; Peyrat, Jean-Philippe; Fournier, Joëlle; Révillion, Françoise; Adenis, Claude; Vénat-Bouvet, Laurence; Léone, Mélanie; Boutry-Kryza, Nadia; Calender, Alain; Giraud, Sophie; Verny-Pierre, Carole; Lasset, Christine; Bonadona, Valérie; Barjhoux, Laure; Sobol, Hagay; Bourdon, Violaine; Noguchi, Tetsuro; Remenieras, Audrey; Coupier, Isabelle; Pujol, Pascal; Sokolowska, Johanna; Bronner, Myriam; Delnatte, Capucine; Bézieau, Stéphane; Mari, Véronique; Gauthier-Villars, Marion; Buecher, Bruno; Rouleau, Etienne; Golmard, Lisa; Moncoutier, Virginie; Belotti, Muriel; de Pauw, Antoine; Elan, Camille; Fourme, Emmanuelle; Birot, Anne-Marie; Saule, Claire; Laurent, Maïté; Houdayer, Claude; Lesueur, Fabienne; Mebirouk, Noura; Coulet, Florence; Colas, Chrystelle; Soubrier, Florent; Warcoin, Mathilde; Prieur, Fabienne; Lebrun, Marine; Kientz, Caroline; Muller, Danièle; Fricker, Jean-Pierre; Toulas, Christine; Guimbaud, Rosine; Gladieff, Laurence; Feillel, Viviane; Mortemousque, Isabelle; Bressac-de-Paillerets, Brigitte; Caron, Olivier; Guillaud-Bataille, Marine; Cook, Linda S.; Cox, Angela; Cramer, Daniel W.; Cross, Simon S.; Cybulski, Cezary; Czene, Kamila; Daly, Mary B.; Damiola, Francesca; Dansonka-Mieszkowska, Agnieszka; Darabi, Hatef; Dennis, Joe; Devilee, Peter; Diez, Orland; Doherty, Jennifer A.; Domchek, Susan M.; Dorfling, Cecilia M.; Dörk, Thilo; Dumont, Martine; Ehrencrona, Hans; Ejlertsen, Bent; Ellis, Steve; Gregory, Helen; Miedzybrodzka, Zosia; Morrison, Patrick J.; Donaldson, Alan; Rogers, Mark T.; Kennedy, M. John; Porteous, Mary E.; Brady, Angela; Barwell, Julian; Foo, Claire; Lalloo, Fiona; Side, Lucy E.; Eason, Jacqueline; Henderson, Alex; Walker, Lisa; Cook, Jackie; Snape, Katie; Murray, Alex; McCann, Emma; Engel, Christoph; Lee, Eunjung; Evans, D. Gareth; Fasching, Peter A.; Feliubadalo, Lidia; Figueroa, Jonine; Flesch-Janys, Dieter; Fletcher, Olivia; Flyger, Henrik; Foretova, Lenka; Fostira, Florentia; Foulkes, William D.; Fridley, Brooke L.; Friedman, Eitan; Frost, Debra; Gambino, Gaetana; Ganz, Patricia A.; Garber, Judy; García-Closas, Montserrat; Gentry-Maharaj, Aleksandra; Ghoussaini, Maya; Giles, Graham G.; Glasspool, Rosalind; Godwin, Andrew K.; Goldberg, Mark S.; Goldgar, David E.; González-Neira, Anna; Goode, Ellen L.; Goodman, Marc T.; Greene, Mark H.; Gronwald, Jacek; Guénel, Pascal; Haiman, Christopher A.; Hall, Per; Hallberg, Emily; Hamann, Ute; Hansen, Thomas V. O.; Harrington, Patricia A.; Hartman, Mikael; Hassan, Norhashimah; Healey, Sue; Rookus, M. A.; van Leeuwen, F. E.; van der Kolk, L. E.; Schmidt, M. K.; Russell, N. S.; de Lange, J. L.; Wijnands, R.; Collée, J. M.; Hooning, M. J.; Seynaeve, C.; van Deurzen, C. H. M.; Obdeijn, I. M.; van Asperen, C. J.; Tollenaar, R. A. E. M.; van Cronenburg, T. C. T. E. F.; Kets, C. M.; Ausems, M. G. E. M.; van der Pol, C. C.; van Os, T. A. M.; Waisfisz, Q.; Meijers-Heijboer, H. E. J.; Gómez-Garcia, E. B.; Oosterwijk, J. C.; Mourits, M. J.; de Bock, G. H.; Vasen, H. F.; Siesling, S.; Verloop, J.; Overbeek, L. I. H.; Heitz, Florian; Herzog, Josef; Høgdall, Estrid; Høgdall, Claus K.; Hogervorst, Frans B. L.; Hollestelle, Antoinette; Hopper, John L.; Hulick, Peter J.; Huzarski, Tomasz; Imyanitov, Evgeny N.; Fox, Stephen; Kirk, Judy; Lindeman, Geoff; Price, Melanie; Bowtell, David; deFazio, Anna; Webb, Penny; Isaacs, Claudine; Ito, Hidemi; Jakubowska, Anna; Janavicius, Ramunas; Jensen, Allan; John, Esther M.; Johnson, Nichola; Kabisch, Maria; Kang, Daehee; Kapuscinski, Miroslav; Karlan, Beth Y.; Khan, Sofia; Kiemeney, Lambertus A.; Kjaer, Susanne Kruger; Knight, Julia A.; Konstantopoulou, Irene; Kosma, Veli-Matti; Kristensen, Vessela; Kupryjanczyk, Jolanta; Kwong, Ava; de la Hoya, Miguel; Laitman, Yael; Lambrechts, Diether; Le, Nhu; De Leeneer, Kim; Lester, Jenny; Levine, Douglas A.; Li, Jingmei; Lindblom, Annika; Long, Jirong; Lophatananon, Artitaya; Loud, Jennifer T.; Lu, Karen; Lubinski, Jan; Mannermaa, Arto; Manoukian, Siranoush; Le Marchand, Loic; Margolin, Sara; Marme, Frederik; Massuger, Leon F. A. G.; Matsuo, Keitaro; Mazoyer, Sylvie; McGuffog, Lesley; McLean, Catriona; McNeish, Iain; Meindl, Alfons; Menon, Usha; Mensenkamp, Arjen R.; Milne, Roger L.; Montagna, Marco; Moysich, Kirsten B.; Muir, Kenneth; Mulligan, Anna Marie; Nathanson, Katherine L.; Ness, Roberta B.; Neuhausen, Susan L.; Nevanlinna, Heli; Nord, Silje; Nussbaum, Robert L.; Odunsi, Kunle; Offit, Kenneth; Olah, Edith; Olopade, Olufunmilayo I.; Olson, Janet E.; Olswold, Curtis; O'Malley, David; Orlow, Irene; Orr, Nick; Osorio, Ana; Park, Sue Kyung; Pearce, Celeste L.; Pejovic, Tanja; Peterlongo, Paolo; Pfeiler, Georg; Phelan, Catherine M.; Poole, Elizabeth M.; Pylkäs, Katri; Radice, Paolo; Rantala, Johanna; Rashid, Muhammad Usman; Rennert, Gad; Rhenius, Valerie; Rhiem, Kerstin; Risch, Harvey A.; Rodriguez, Gus; Rossing, Mary Anne; Rudolph, Anja; Salvesen, Helga B.; Sangrajrang, Suleeporn; Sawyer, Elinor J.; Schildkraut, Joellen M.; Schmidt, Marjanka K.; Schmutzler, Rita K.; Sellers, Thomas A.; Seynaeve, Caroline; Shah, Mitul; Shen, Chen-Yang; Shu, Xiao-Ou; Sieh, Weiva; Singer, Christian F.; Sinilnikova, Olga M.; Slager, Susan; Song, Honglin; Soucy, Penny; Southey, Melissa C.; Stenmark-Askmalm, Marie; Stoppa-Lyonnet, Dominique; Sutter, Christian; Swerdlow, Anthony; Tchatchou, Sandrine; Teixeira, Manuel R.; Teo, Soo H.; Terry, Kathryn L.; Terry, Mary Beth; Thomassen, Mads; Tibiletti, Maria Grazia; Tihomirova, Laima; Tognazzo, Silvia; Toland, Amanda Ewart; Tomlinson, Ian; Torres, Diana; Truong, Thérèse; Tseng, Chiu-chen; Tung, Nadine; Tworoger, Shelley S.; Vachon, Celine; van den Ouweland, Ans M. W.; van Doorn, Helena C.; van Rensburg, Elizabeth J.; Van't Veer, Laura J.; Vanderstichele, Adriaan; Vergote, Ignace; Vijai, Joseph; Wang, Qin; Wang-Gohrke, Shan; Weitzel, Jeffrey N.; Wentzensen, Nicolas; Whittemore, Alice S.; Wildiers, Hans; Winqvist, Robert; Wu, Anna H.; Yannoukakos, Drakoulis; Yoon, Sook-Yee; Yu, Jyh-Cherng; Zheng, Wei; Zheng, Ying; Khanna, Kum Kum; Simard, Jacques; Monteiro, Alvaro N.; French, Juliet D.; Couch, Fergus J.; Freedman, Matthew L.; Easton, Douglas F.; Dunning, Alison M.; Pharoah, Paul D.; Edwards, Stacey L.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Gayther, Simon A.

    2016-01-01

    A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10−20), ER-negative BC (P=1.1 × 10−13), BRCA1-associated BC (P=7.7 × 10−16) and triple negative BC (P-diff=2 × 10−5). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10−3) and ABHD8 (P<2 × 10−3). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3′-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk. PMID:27601076