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Sample records for activating ras mutations

  1. Activating FGFR2-RAS-BRAF mutations in ameloblastoma.

    Science.gov (United States)

    Brown, Noah A; Rolland, Delphine; McHugh, Jonathan B; Weigelin, Helmut C; Zhao, Lili; Lim, Megan S; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

    2014-11-01

    Ameloblastoma is an odontogenic neoplasm whose overall mutational landscape has not been well characterized. We sought to characterize pathogenic mutations in ameloblastoma and their clinical and functional significance with an emphasis on the mitogen-activated protein kinase (MAPK) pathway. A total of 84 ameloblastomas and 40 non-ameloblastoma odontogenic tumors were evaluated with a combination of BRAF V600E allele-specific PCR, VE1 immunohistochemistry, the Ion AmpliSeq Cancer Hotspot Panel, and Sanger sequencing. Efficacy of a BRAF inhibitor was evaluated in an ameloblastoma-derived cell line. Somatic, activating, and mutually exclusive RAS-BRAF and FGFR2 mutations were identified in 88% of cases. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 were also identified. BRAF V600E was the most common mutation, found in 62% of ameloblastomas and in ameloblastic fibromas/fibrodentinomas but not in other odontogenic tumors. This mutation was associated with a younger age of onset, whereas BRAF wild-type cases arose more frequently in the maxilla and showed earlier recurrences. One hundred percent concordance was observed between VE1 immunohistochemistry and molecular detection of BRAF V600E mutations. Ameloblastoma cells demonstrated constitutive MAPK pathway activation in vitro. Proliferation and MAPK activation were potently inhibited by the BRAF inhibitor vemurafenib. Our findings suggest that activating FGFR2-RAS-BRAF mutations play a critical role in the pathogenesis of most cases of ameloblastoma. Somatic mutations in SMO, CTNNB1, PIK3CA, and SMARCB1 may function as secondary mutations. BRAF V600E mutations have both diagnostic and prognostic implications. In vitro response of ameloblastoma to a BRAF inhibitor suggests a potential role for targeted therapy. ©2014 American Association for Cancer Research.

  2. Activating Ras mutations fail to ensure efficient replication of adenovirus mutants lacking VA-RNA

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses...... to grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA...... mutational status, upon infection with VA-less adenoviruses in the presence of interferon, but also upon addition of the PKR activator polyIC to cells. When comparing two isogenic cell lines that differ solely with regard to the presence or absence of mutant Ras, no difference was observed concerning...

  3. Absence of activating mutations in ras and gsp oncogenes in a cohort of nine patients with sporadic pediatric thyroid tumors.

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    Pauws, E; Tummers, R F; Ris-Stalpers, C; de Vijlder, J J; Voûte, T

    2001-06-01

    Characterization of the genetic background of pediatric thyroid carcinomas could aid in distinguishing between differently staged tumors with respect to treatment and prognosis. Two known genetic factors associated with thyroid carcinoma, the proto-oncogenes gsp and ras were investigated. DNA was extracted from paraffin sections from both tumor and normal thyroid tissue of nine patients (ages 9-16 years). Of these patients, eight were diagnosed with papillary carcinoma and one with follicular adenoma. The coding exons of gsp and the three known ras genes (H, K, and N-ras) were screened for mutations using SSCP-analysis. There were no mutations present in the ras and gsp proto-oncogenes hot spots, however, LOH of H-ras (chromosome location 11p15.5) was found in tumor tissue from one patient and a homozygous mutation in exon 12 of gsp causing a Pro-->Ser conversion was present in the thyroid tumor tissue from another patient. Two silent polymorphisms were detected, H-ras exon1, 86T-->C and gsp exon 5, 81T-->C. Our results indicate that the ras/gsp mutations found are probably late events in the tumorigenesis representing general oncogenic stress. In conclusion, it seems that ras/gsp activation is not a factor in the mechanism causing sporadic thyroid carcinoma in children. Copyright 2001 Wiley-Liss, Inc.

  4. A screen for mutations that prevent lethality caused by expression of activated sevenless and Ras1 in the Drosophila embryo.

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    Maixner, A; Hecker, T P; Phan, Q N; Wassarman, D A

    1998-01-01

    Ras1 plays a critical role in receptor tyrosine kinase (RTK) signal transduction pathways that function during Drosophila development. We demonstrate that mis-expression of constitutively active forms of Ras1 (Ras1V12) and the Sevenless (Sev) RTK (SevS11) during embryogenesis causes lethality due to inappropriate activation of RTK/Ras1 signaling pathways. Genetic and molecular data indicate that the rate of SevS11/sev-Ras1V12 lethality is sensitive to the expression level of both transgenes. To identify genes that encode components of RTK/Ras1 signaling pathways or modulators of RNA polymerase II transcription, we took advantage of the dose-sensitivity of the system and screened for second site mutations that would dominantly suppress the lethality. The collection of identified suppressors includes the PR55 subunit of Protein Phosphatase 2A indicating that downstream of Sev and Ras1 this subunit acts as a negative regulator of phosphatase activity. The isolation of mutations in the histone deacetylase RPD3 suggests that it functions as positive regulator of sev enhancer-driven transcription. Finally, the isolation of mutations in the Trithorax group gene devenir and the characterized allelism with the Breathless RTK encoding gene provides evidence for Ras1-mediated regulation of homeotic genes.

  5. Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms.

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    Mackenzie, Robertson; Kommoss, Stefan; Winterhoff, Boris J; Kipp, Benjamin R; Garcia, Joaquin J; Voss, Jesse; Halling, Kevin; Karnezis, Anthony; Senz, Janine; Yang, Winnie; Prigge, Elena-Sophie; Reuschenbach, Miriam; Doeberitz, Magnus Von Knebel; Gilks, Blake C; Huntsman, David G; Bakkum-Gamez, Jamie; McAlpine, Jessica N; Anglesio, Michael S

    2015-05-19

    Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be "RAS-pathway alteration negative", using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell

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

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    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.

  7. Ras activation by SOS

    DEFF Research Database (Denmark)

    Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen

    2014-01-01

    SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS...... by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather......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...

  8. Association of H-ras mutations with adenocarcinomas of the parotid gland

    NARCIS (Netherlands)

    van Halteren, H. K.; Top, B.; Mooi, W. J.; Balm, A. J.; Rodenhuis, S.

    1994-01-01

    We have examined 17 adenocarcinomas and 2 mixed tumors of the salivary glands for mutational activation of the oncogenes H-ras, K-ras and N-ras. The presence of mutations was determined by in vitro amplification of gene fragments spanning codons 12, 13 and 61 and the use of mutation-specific

  9. Oncogenic Effects of High MAPK Activity in Colorectal Cancer Mark Progenitor Cells and Persist Irrespective of RAS Mutations.

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    Blaj, Cristina; Schmidt, Eva Marina; Lamprecht, Sebastian; Hermeking, Heiko; Jung, Andreas; Kirchner, Thomas; Horst, David

    2017-04-01

    About 40% of colorectal cancers have mutations in KRAS accompanied by downstream activation of MAPK signaling, which promotes tumor invasion and progression. Here, we report that MAPK signaling shows strong intratumoral heterogeneity and unexpectedly remains regulated in colorectal cancer irrespective of KRAS mutation status. Using primary colorectal cancer tissues, xenograft models, and MAPK reporter constructs, we showed that tumor cells with high MAPK activity resided specifically at the leading tumor edge, ceased to proliferate, underwent epithelial-mesenchymal transition (EMT), and expressed markers related to colon cancer stem cells. In KRAS-mutant colon cancer, regulation of MAPK signaling was preserved through remaining wild-type RAS isoforms. Moreover, using a lineage tracing strategy, we provide evidence that high MAPK activity marked a progenitor cell compartment of growth-fueling colon cancer cells in vivo Our results imply that differential MAPK signaling balances EMT, cancer stem cell potential, and tumor growth in colorectal cancer. Cancer Res; 77(7); 1763-74. ©2017 AACR . ©2017 American Association for Cancer Research.

  10. Silencing of SPRY1 Triggers Complete Regression of Rhabdomyosarcoma Tumors Carrying a Mutated RAS Gene

    NARCIS (Netherlands)

    Schaaf, Gerben; Hamdi, Mohamed; Zwijnenburg, Danny; Lakeman, Arjan; Geerts, Dirk; Versteeg, Rogier; Kool, Marcel

    2010-01-01

    RAS oncogenes are among the most frequently mutated genes in human cancer, but effective strategies for therapeutic inhibition of the RAS pathway have been elusive. Sprouty1 (SPRY1) is an upstream antagonist of RAS that is activated by extracellular signal-related kinase (ERK), providing a negative

  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. Ras pathway activation in malignant mesothelioma.

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    Patel, Manish R; Jacobson, Blake A; De, Arpita; Frizelle, Sandra P; Janne, Pasi; Thumma, Saritha C; Whitson, Brian A; Farassati, Faris; Kratzke, Robert A

    2007-09-01

    Mutations in Ras family genes are rare in malignant mesothelioma. The role of activation of the Ras signaling pathway in the pathogenesis of mesothelioma is not clear. We studied the activation status of the Ras pathway and the status of other Ras-associated kinases in a panel of human mesothelioma cell lines. In addition, we tested the effect of inhibition of several kinase pathways on mesothelioma cell proliferation. The potential role of kinase signaling on the regulation of cap-dependent translation was also studied. In general, Ras-guanosine triphosphate (GTP) was higher in mesothelioma cell lines when compared with a nontransformed mesothelial cell line (LP9). Furthermore, known Ras effectors such as extracellular-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase were found to be active in most of the mesothelioma cell lines tested. Exposure to specific inhibitors of extracellular-regulated kinase 1/2 (U0126) and c-Jun N-terminal kinase (SP600125) significantly decreased the proliferation of H2596 and H2373 cells compared with mock-treated cells. SP600125-mediated c-Jun N-terminal kinase inhibition, but not extracellular-regulated kinase 1/2 inhibition, resulted in a decrease in phosphorylation of 4E-BP1, consequently decreasing cap-dependent activation. These experiments provide a rationale for targeting Ras and associated signaling pathways in mesothelioma and also suggest cap-dependent translation as one mechanism by which Ras induces proliferation in this disease.

  13. Coexistence of K-ras mutations and HPV infection in colon cancer

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    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.

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

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    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.

  15. 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

  16. MEK inhibition is a promising therapeutic strategy for MLL-rearranged infant acute lymphoblastic leukemia patients carrying RAS mutations.

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    Kerstjens, Mark; Driessen, Emma M C; Willekes, Merel; Pinhanços, Sandra S; Schneider, Pauline; Pieters, Rob; Stam, Ronald W

    2017-02-28

    Acute lymphoblastic leukemia (ALL) in infants is an aggressive malignancy with a poor clinical outcome, and is characterized by translocations of the Mixed Lineage Leukemia (MLL) gene. Previously, we identified RAS mutations in 14-24% of infant ALL patients, and showed that the presence of a RAS mutation decreased the survival chances even further. We hypothesized that targeting the RAS signaling pathway could be a therapeutic strategy for RAS-mutant infant ALL patients. Here we show that the MEK inhibitors Trametinib, Selumetinib and MEK162 severely impair primary RAS-mutant MLL-rearranged infant ALL cells in vitro. While all RAS-mutant samples were sensitive to MEK inhibitors, we found both sensitive and resistant samples among RAS-wildtype cases. We confirmed enhanced RAS pathway signaling in RAS-mutant samples, but found no apparent downstream over-activation in the wildtype samples. However, we did confirm that MEK inhibitors reduced p-ERK levels, and induced apoptosis in the RAS-mutant MLL-rearranged ALL cells. Finally, we show that MEK inhibition synergistically enhances prednisolone sensitivity, both in RAS-mutant and RAS-wildtype cells. In conclusion, MEK inhibition represents a promising therapeutic strategy for MLL-rearranged ALL patients harboring RAS mutations, while patients without RAS mutations may benefit through prednisolone sensitization.

  17. Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

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    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

  18. Absence of activating mutations in ras and gsp oncogenes in a cohort of nine patients with sporadic pediatric thyroid tumors

    NARCIS (Netherlands)

    Pauws, E.; Tummers, R. F.; Ris-Stalpers, C.; de Vijlder, J. J.; Voûte, T.

    2001-01-01

    BACKGROUND: Characterization of the genetic background of pediatric thyroid carcinomas could aid in distinguishing between differently staged tumors with respect to treatment and prognosis. Two known genetic factors associated with thyroid carcinoma, the proto-oncogenes gsp and ras were

  19. 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

  20. Diet, lifestyle and risk of K-ras mutation-positive and -negative colorectal adenomas.

    NARCIS (Netherlands)

    Wark, P.A.; Kuil, W. van der; Ploemacher, J.; Muijen, G.N.P. van; Mulder, C.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

  1. c-Ha-ras BamHI RFLP in human urothelial tumors and point mutations in hot codons

    International Nuclear Information System (INIS)

    Weismanova, E; Skovraga, M.; Kaluz, S.

    1993-01-01

    High-molecular weights DNAs from 30 bladder and renal cell carcinomas (RCC) were isolated and the c-Ha-ras the c-Ha-ras gene BamHI RFLP was examined. Amplification of c-Ha-ras with normal localization with regard to the size of alleles was found only in the case. One of the normally localized c-Ha-ras allele termed RCC c-H-ras of a length of about 6.6 kbp was cloned and an oncogene-activating point mutation was identified using two restriction enzymes. After comparison of CfrI and Cfr10I cleavage maps of RCC c-Ha-ras to complete nucleotide sequences of EJ/T24 c-Ha-ras oncogene and its normal counterpart, a point mutation was identified within codon 11 or 12. The use of CfrI and Cfr10I is of value for clinical practice in identification of point mutations in c-Ha-ras PCR product in neoplasia accompanied by somatic mutation of c-Ha-ras. The correlation among c-Ha-ras allele, amplification/loss, presence of point mutation and progression of neoplasia is discussed. (author)

  2. Calcium activation of Ras mediated by neuronal exchange factor Ras-GRF.

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    Farnsworth, C L; Freshney, N W; Rosen, L B; Ghosh, A; Greenberg, M E; Feig, L A

    1995-08-10

    Tyrosine kinase receptors stimulate the Ras signalling pathway by enhancing the activity of the SOS nucleotide-exchange factor. This occurs, at least in part, by the recruitment of an SOS-GRB2 complex to Ras in the plasma membrane. Here we describe a different signalling pathway to Ras that involves activation of the Ras-GRF exchange factor in response to Ca2+ influx. In particular, we show that the ability of Ras-GRF to activate Ras in vivo is markedly enhanced by raised Ca2+ concentrations. Activation is mediated by calmodulin binding to an IQ motif in Ras-GRF, because substitutions in conserved amino acids in this motif prevent both calmodulin binding to Ras-GRF and Ras-GRF activation in vivo. So far, full-length Ras-GRF has been detected only in brain neurons. Our findings implicate Ras-GRF in the regulation of neuronal functions that are influenced by Ca2+ signals.

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

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    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

  4. Somatic Activation of rasK Gene in a Human Ovarian Carcinoma

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    Feig, L. A.; Bast, R. C.; Knapp, R. C.; Cooper, G. M.

    1984-02-01

    A tumor isolate from a patient with serous cystadenocarcinoma of the ovary contained an activated rasK gene detected by transfection of NIH/3T3 cells. In contrast, DNA from normal cells of the same patient lacked transforming activity, indicating that activation of this transforming gene was the consequence of somatic mutation in the neoplastic cells. The transforming gene product displayed an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels that differed from the mobilities of rasK transforming proteins in other tumors, indicating that a previously undescribed mutation was responsible for activation of rasK in this ovarian carcinoma.

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

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    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

  6. Activation of the exchange factor Ras-GRF by calcium requires an intact Dbl homology domain.

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    Freshney, N W; Goonesekera, S D; Feig, L A

    1997-04-21

    Ras-GRF is a guanine nucleotide exchange factor that activates Ras proteins. Its activity on Ras in cells is enhanced upon calcium influx. Activation follows calcium-induced binding of calmodulin to an IQ motif near the N-terminus of Ras-GRF. Ras-GRF also contains a Dbl homology (DH) domain C-terminal to the IQ motif. In many proteins, DH domains act as exchange factors for Rho-GTPase family members. However, we failed to detect exchange activity of this domain on well characterized Rho family members. Instead, we found that mutations analogous to those that block exchange activity of Dbl prevented Ras-GRF activation by calcium/ calmodulin in vivo. All DH domains are followed immediately by a pleckstrin homology (PH) domain. We found that a mutation at a conserved site within the PH domain following the DH domain also prevented Ras-GRF activation by calcium in vivo. These results suggest that in addition to playing a role as activators of Rho proteins, DH domains can also contribute to the coupling of cellular signals to Ras activation.

  7. Skin tumorigenesis and Ki-ras and Ha-ras mutations in tumors from adult mice exposed in utero to 3'-azido-2',3'-dideoxythymidine.

    Science.gov (United States)

    Zhang, Z; Diwan, B A; Anderson, L M; Logsdon, D; Olivero, O A; Haines, D C; Rice, J M; Yuspa, S H; Poirier, M C

    1998-09-01

    This study was designed to evaluate the potential initiating effects of transplacental 3'-azido-2',3'-dideoxythymine (AZT) and the role of ras mutational activation in skin tumors induced in a two-stage mouse skin model. In addition, mouse liver and lung tumors from a transplacental AZT tumorigenicity study reported elsewhere (Olivero et al., J Natl Cancer Inst 89:1602-1608, 1997) were examined for evidence of ras activation. For both tumor studies, pregnant CD-1 mice were given either vehicle or 25 mg of AZT daily on days 12-18 of gestation. In the 1997 study, the offspring were given no further exposure and were killed at 1 yr of age. For the skin tumor study, all mice received twice-weekly topical 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment from weeks 5-35; half of the mice had been exposed to AZT in utero. At weeks 16-18, 30, 31, and 34-41, the skin tumor incidences in mice given AZT and TPA were significantly higher than in mice given TPA alone (P A transition in the second base, and the major mutation in codon 13 (six in seven tumors examined) was a G-->T transversion in the second base. In skin tumors, AZT exposure did not increase the number of Ha-ras codon 61 mutations, and no Ki-ras mutations were observed. Analysis of ras mutations in liver and lung tumors from mice exposed to AZT in utero (Olivero et al., J Natl Cancer Inst 89:16021608, 1997) with no TPA promotion showed no significant AZT-related increases.

  8. 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

  9. Noninvasive detection through REMS-PCR technique of K-ras mutations in stool DNA of patients with colorectal cancer.

    Science.gov (United States)

    Mixich, Francisc; Ioana, Mihai; Voinea, Florea; Săftoiu, Adrian; Ciurea, Tudorel

    2007-03-01

    Tumor exfoliated cells that shed into stool are attractive targets for molecular screening and early detection of colon malignancies. Many studies have suggested that the detection of activated ras may have diagnostic or prognostic importance. The aim of this study was to establish the suitability for use in diagnostic laboratories of the noninvasive screening test of K-ras mutation determination in the stool and its routine prognostic value in colorectal cancer. Paired stool and tissue specimens obtained after polypectomy and colorectal biopsy from 28 patients diagnosed solely by histopathological findings with primary colorectal carcinoma, were prospectively studied for K-ras codon 12 mutations by restriction endonuclease-mediated selective (REMS)-PCR. DNA was obtained in 28 of tissue samples (100%) and 26 of stool samples (92.8%). K-ras codon 12 mutation was seen in 14 (50.0%) paired stool and tissue samples. Mutation detection was possible in 1000-fold excess of wild-type sequence. These results may be important in the design of genetic screening programs, determination of prognosis, early detection and treatment for patients with colon malignancy. The sensitivity and specificity of K-ras determination on stool-derived DNA in patients with colorectal carcinoma, support the opportunity of a large-scale trial to validate its use as a screening test. REMS- PCR is not labor intensive, but a sensitive, rapid, and robust assay for the detection of point mutations, and was introduced by us in a routine diagnostic laboratory.

  10. Langerhans cell histiocytosis: A neoplastic disorder driven by Ras-ERK pathway mutations.

    Science.gov (United States)

    Tran, Gary; Huynh, Thy N; Paller, Amy S

    2018-03-01

    Langerhans cell histiocytosis (LCH) is a disorder of myeloid neoplasia of dendritic cells that affects 1 in 200,000 children <15 years of age and even fewer adults. LCH presents with a spectrum of clinical manifestations. High-risk stratification is reserved for infiltration of blood, spleen, liver, and lungs. After decades of debate on the disease pathogenesis, a neoplastic mechanism is now favored on the basis of LCH cell clonality, rare cases of familial clustering, and recent evidence of mutations involving the Ras/Raf/MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase) pathway in lesional biopsy specimens. Somatic mutations are most often found in BRAF (BRAF V600E in 47.1% of reported patients) and MAP2K1 (21.7%) and uncommonly found in MAP3K1 or ARAF. Increased levels of phospho-ERK in lesional tissue, activation of Ras/Raf/MEK/ERK signaling with these mutations in vitro, and the mutual exclusivity of these mutations in a given patient suggest a central role for activation of the Ras/Raf/MEK/ERK oncogenic pathway in LCH. Immunohistochemical assessment of lesional tissue using the VE1 BRAF V600E mutation-specific antibody can serve as a screening tool for BRAF V600E -positive LCH. Case reports suggest that BRAF V600E -positive LCH unresponsive to standard therapy might respond to B-Raf-MEK pathway inhibition, but rigorous randomized clinical trials have yet to be performed. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  11. 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...

  12. Detecting N-RAS Q61R Mutated Thyroid Neoplasias by Immunohistochemistry.

    Science.gov (United States)

    Crescenzi, A; Fulciniti, F; Bongiovanni, M; Giovanella, L; Trimboli, Pierpaolo

    2017-03-01

    Recently, the immunohistochemistry (IHC) for N-RAS Q61R has been developed and commercialized for clinical practice. Here, we investigated the reliability of IHC to identify N-RAS Q61R mutated thyroid neoplasia. A series of 24 consecutive thyroid lesions undergone surgery following indeterminate cytology were enrolled. Paraffin sections were stained for IHC using the rabbit monoclonal anti-human N-RAS Q61R, clone SP174. N-RAS mutations in codon 61 were also investigated by automated sequencing. At histology, 12 cases of follicular carcinoma, cytologically defined as follicular lesions, 1 papillary cancer, 7 follicular adenomas, and 4 hyperplastic nodules were found. Of these, 4 showed a positive IHC for anti N-RAS antibody where N-RAS expression was detected mainly at cytoplasmic level with similar intensity of reaction. The remaining cases had negative IHC. A 100% concordance between IHC and molecular analysis for N-RAS Q61R was observed. In conclusion, this study shows high reliability of IHC to identify N-RAS Q61R mutated thyroid lesions with high cost-effectiveness. These data indicate the reliability of IHC to identify N-RAS Q61R mutated thyroid neoplasia and suggest to adopt this approach for a more accurate management of patients, when indicated.

  13. RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma.

    Science.gov (United States)

    Chen, Xu; Wu, Qiuxia; Depeille, Philippe; Chen, Peirong; Thornton, Sophie; Kalirai, Helen; Coupland, Sarah E; Roose, Jeroen P; Bastian, Boris C

    2017-05-08

    Constitutive activation of Gαq signaling by mutations in GNAQ or GNA11 occurs in over 80% of uveal melanomas (UMs) and activates MAPK. Protein kinase C (PKC) has been implicated as a link, but the mechanistic details remained unclear. We identified PKC δ and ɛ as required and sufficient to activate MAPK in GNAQ mutant melanomas. MAPK activation depends on Ras and is caused by RasGRP3, which is significantly and selectively overexpressed in response to GNAQ/11 mutation in UM. RasGRP3 activation occurs via PKC δ- and ɛ-dependent phosphorylation and PKC-independent, DAG-mediated membrane recruitment, possibly explaining the limited effect of PKC inhibitors to durably suppress MAPK in UM. The findings nominate RasGRP3 as a therapeutic target for cancers driven by oncogenic GNAQ/11. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Decrease in specific micronutrient intake in colorectal cancer patients with tumors presenting Ki-ras mutation

    OpenAIRE

    JORDI SALAS; NURIA LASO; SERGI MAS; M. JOSE LAFUENTE; XAVIER CASTERAD; MANUEL TRIAS; ANTONIO BALLESTA; RAFAEL MOLINA; CARLOS ASCASO; SHICHUN ZHENG; JOHN K. WIENCKE; AMALIA LAFUENTE

    2004-01-01

    Decrease in specific micronutrient intake in colorectal cancer patients with tumors presenting Ki-ras mutation BACKGROUND: The diversity of the Mediterranean diet and the heterogeneity of acquired genetic alterations in colorectal cancer (CRC) led us to examine the possible association between dietary factors and mutations, such as Ki-ras mutations, in genes implicated in the pathogenesis of these neoplasms. PATIENTS AND METHODS: The study was based on 246 cases and 296 controls. For th...

  15. Genetic and Molecular Analysis of Suppressors of Ras Mutations

    National Research Council Canada - National Science Library

    Sieburth, Derek

    1998-01-01

    .... elegans vulval development. We describe the identification and characterization of a novel gene, sur-8, that functions to regulate a receptor tyrosine kinase-Ras-MAP kinase-mediated signal transduction pathway during C...

  16. Genetic and Molecular Analysis of Suppressors of Ras Mutations

    National Research Council Canada - National Science Library

    Sieburth, Derek

    1999-01-01

    .... elegans vulvaZ development. We describe the identification and characterization of a novel gene, sur-8, that functions to regulate a receptor tyrosine kinase-Ras-MAp kinase- mediated signal transduction pathway during C...

  17. Ras activation and symmetry breaking during Dictyostelium chemotaxis.

    Science.gov (United States)

    Kortholt, Arjan; Keizer-Gunnink, Ineke; Kataria, Rama; Van Haastert, Peter J M

    2013-10-01

    Central to chemotaxis is the molecular mechanism by which a shallow spatial gradient of chemoattractant induces symmetry breaking of activated signaling molecules. Previously, we have used Dictyostelium mutants to investigate the minimal requirements for chemotaxis, and identified a basal signaling module providing activation of Ras and F-actin at the leading edge. Here, we show that Ras activation after application of a pipette releasing the chemoattractant cAMP has three phases, each depending on specific guanine-nucleotide-exchange factors (GEFs). Initially a transient activation of Ras occurs at the entire cell boundary, which is proportional to the local cAMP concentrations and therefore slightly stronger at the front than in the rear of the cell. This transient Ras activation is present in gα2 (gpbB)-null cells but not in gβ (gpbA)-null cells, suggesting that Gβγ mediates the initial activation of Ras. The second phase is symmetry breaking: Ras is activated only at the side of the cell closest to the pipette. Symmetry breaking absolutely requires Gα2 and Gβγ, but not the cytoskeleton or four cAMP-induced signaling pathways, those dependent on phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)P3], cGMP, TorC2 and PLA2. As cells move in the gradient, the crescent of activated Ras in the front half of the cell becomes confined to a small area at the utmost front of the cell. Confinement of Ras activation leads to cell polarization, and depends on cGMP formation, myosin and F-actin. The experiments show that activation, symmetry breaking and confinement of Ras during Dictyostelium chemotaxis uses different G-protein subunits and a multitude of Ras GEFs and GTPase-activating proteins (GAPs).

  18. RAS2/PKA pathway activity is involved in the nitrogen regulation of L-leucine uptake in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sáenz, D A; Chianelli, M S; Stella, C A; Mattoon, J R; Ramos, E H

    1997-03-01

    The aim of the present work is to study the participation of RAS2/PKA signal pathway in the nitrogen regulation of L-leucine transport in yeast cells. The study was performed on Saccharomyces cerevisiae isogenic strains with the normal RAS2 gene, the RAS2val19 mutant and the disrupted ras2::LEU2. These strains bring about different activities of the RAS2/PKA signal pathway, L-(14C)-Amino acid uptake measurements were determined in cells grown in a rich YPD medium with a mixed nitrogen source or in minimal media containing NH4+ or L-proline as the sole nitrogen source. We report herein that in all strains used, even in those grown in a minimal proline medium, the activity of the general amino acid permease (GAP1) was not detected. L-Leucine uptake in these strains is mediated by two kinetically characterized transport systems. Their KT values are of the same order as those of S1 and S2 L-leucine permeases. Mutation in the RAS2 gene alters initial velocities and Jmax values in both high and low affinity L-leucine transport systems. Activation of the RAS2/PKA signalling pathway by the RAS2val19 mutation, blocks the response to a poor nitrogen source whereas inactivation of RAS2 by gene disruption, results in an increase of the same response.

  19. Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras

    NARCIS (Netherlands)

    Medema, R.H.; Vries-Smits, A.M. de; Zon, G.C.M. van der; Maassen, J.A.; Bos, J.L.

    1993-01-01

    A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate

  20. In Silico Screening of Mutated K-Ras Inhibitors from Malaysian Typhonium flagelliforme for Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ayesha Fatima

    2014-01-01

    Full Text Available K-ras is an oncogenic GTPase responsible for at least 15–25% of all non-small cell lung cancer cases worldwide. Lung cancer of both types is increasing with an alarming rate due to smoking habits in Malaysia among men and women. Natural products always offer alternate treatment therapies that are safe and effective. Typhonium flagelliforme or Keladi Tikus is a local plant known to possess anticancer properties. The whole extract is considered more potent than individual constituents. Since K-ras is the key protein in lung cancer, our aim was to identify the constituents of the plant that could target the mutated K-ras. Using docking strategies, reported potentially active compounds of Typhonium flagelliforme were docked into the allosteric surface pockets and switch regions of the K-ras protein to identify possible inhibitors. The selected ligands were found to have a high binding affinity for the switch II and the interphase region of the ras-SOS binding surface.

  1. MEK-1 Activates C-Raf Through a Ras-Independent Mechanism

    Science.gov (United States)

    Leicht, Deborah T.; Balan, Vitaly; Zhu, Jun; Kaplun, Alexander; Bronisz, Agnieszka; Rana, Ajay; Tzivion, Guri

    2013-01-01

    C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using 32P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the downregulation of RKIP and MST2. PMID:23360980

  2. The prognostic value of simultaneous tumor and serum RAS/RAF mutations in localized colon cancer

    DEFF Research Database (Denmark)

    Brenner Thomsen, Caroline Emilie; Appelt, Ane Lindegaard; Andersen, Rikke Fredslund

    2017-01-01

    The impact of RAS/RAF mutations in localized colon cancer needs clarification. Based on analysis of tumor-specific DNA, this study aimed at elucidating the prognostic influence of mutational status in tumor and serum using an extended panel of mutations. The study retrospectively included 294...... patients with curatively resected stage I-III adenocarcinoma of the colon. Mutations in tumor and serum were determined at time of surgery. Analyses were performed with droplet digital PCR technology. Hazard ratio (HR) for the association between mutational status and survival was estimated in multivariate...

  3. How do K-RAS-activated cells evade cellular defense mechanisms?

    Science.gov (United States)

    Lee, Y-S; Bae, S-C

    2016-02-18

    Lung adenocarcinomas, like other cancers, develop through the accumulation of epigenetic and genetic alterations. Numerous studies have shown that K-RAS mutation is among the most important early events in carcinogenesis of the lung. However, it is also well established that growth-stimulating signals feed back into growth-suppressing pathways, and any imbalance in these signaling networks will cause the cell to exit the cell cycle, thereby preventing uncontrolled cell growth. How, then, do K-RAS-activated cells evade cellular defense mechanisms? To answer this question, it is necessary to identify the molecular event(s) responsible for the development of early dysplastic lesions that are unable to defend against aberrant oncogene activation. Lineage-determining transcriptional regulators govern differentiation status during normal lung development, as well as in lung adenocarcinoma. Among the genes involved in K-RAS-induced lung tumorigenesis, RUNX3 is unique: inactivation of Runx3 in mouse lung induces lung adenoma and abrogates the ARF-p53 pathway. This observation raises the possibility of intimate cross-talk between the differentiation program and oncogene surveillance. In this review, we summarized evidences suggesting that K-RAS-activated cells do not evade cellular defense mechanisms per se; instead, cells with K-RAS mutations are selected only if they occur in cells in which defense mechanism is abrogated.

  4. Gamma band activity in the reticular activating system (RAS

    Directory of Open Access Journals (Sweden)

    Francisco J Urbano

    2012-01-01

    Full Text Available This review considers recent evidence showing that cells in three regions of the reticular activating system (RAS exhibit gamma band activity, and describes the mechanisms behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN, intralaminar parafascicular nucleus (Pf, and pontine Subcoeruleus nucleus dorsalis (SubCD all fire in the beta/gamma band range when maximally activated, but no higher. The mechanisms behind this ceiling effect have been recently elucidated. We describe recent findings showing that every cell in the PPN have high threshold, voltage-dependent P/Q-type calcium channels that are essential, while N-type calcium channels are permissive, to gamma band activity. Every cell in the Pf also showed that P/Q-type and N-type calcium channels are responsible for this activity. On the other hand, every SubCD cell exhibited sodium-dependent subthreshold oscillations. A novel mechanism for sleep-wake control based on well-known transmitter interactions, electrical coupling, and gamma band activity is described. The data presented here on inherent gamma band activity demonstrates the global nature of sleep-wake oscillation that is orchestrated by brainstem-thalamic mechanism, and questions the undue importance given to the hypothalamus for regulation of sleep-wakefulness. The discovery of gamma band activity in the RAS follows recent reports of such activity in other subcortical regions like the hippocampus and cerebellum. We hypothesize that, rather than participating in the temporal binding of sensory events as seen in the cortex, gamma band activity manifested in the RAS may help stabilize coherence related to arousal, providing a stable activation state during waking and paradoxical sleep. Most of our thoughts and actions are driven by preconscious processes. We speculate that continuous sensory input will induce gamma band activity in the RAS that could participate in the

  5. The Anticancer Peptide TAT-RasGAP317−326 Exerts Broad Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Mathieu Heulot

    2017-06-01

    Full Text Available Antibiotic resistance has become a major health issue. Nosocomial infections and the prevalence of resistant pathogenic bacterial strains are rising steadily. Therefore, there is an urgent need to develop new classes of antibiotics effective on multi-resistant nosocomial pathogenic bacteria. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP, called TAT-RasGAP317−326, induces cancer cell death, inhibits metastatic progression, and sensitizes tumor cells to various anti-cancer treatments in vitro and in vivo. We here report that TAT-RasGAP317−326 also possesses antimicrobial activity. In vitro, TAT-RasGAP317−326, but not mutated or truncated forms of the peptide, efficiently killed a series of bacteria including Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa. In vivo experiments revealed that TAT-RasGAP317−326 protects mice from lethal E. coli-induced peritonitis if administrated locally at the onset of infection. However, the protective effect was lost when treatment was delayed, likely due to rapid clearance and inadequate biodistribution of the peptide. Peptide modifications might overcome these shortcomings to increase the in vivo efficacy of the compound in the context of the currently limited antimicrobial options.

  6. Activated Ras alters lens and corneal development through induction of distinct downstream targets

    Directory of Open Access Journals (Sweden)

    Reneker Lixing

    2010-01-01

    Full Text Available Abstract Background Mammalian Ras genes regulate diverse cellular processes including proliferation and differentiation and are frequently mutated in human cancers. Tumor development in response to Ras activation varies between different tissues and the molecular basis for these variations are poorly understood. The murine lens and cornea have a common embryonic origin and arise from adjacent regions of the surface ectoderm. Activation of the fibroblast growth factor (FGF signaling pathway induces the corneal epithelial cells to proliferate and the lens epithelial cells to exit the cell cycle. The molecular mechanisms that regulate the differential responses of these two related tissues have not been defined. We have generated transgenic mice that express a constitutively active version of human H-Ras in their lenses and corneas. Results Ras transgenic lenses and corneal epithelial cells showed increased proliferation with concomitant increases in cyclin D1 and D2 expression. This initial increase in proliferation is sustained in the cornea but not in the lens epithelial cells. Coincidentally, cdk inhibitors p27Kip1 and p57Kip2 were upregulated in the Ras transgenic lenses but not in the corneas. Phospho-Erk1 and Erk2 levels were elevated in the lens but not in the cornea and Spry 1 and Spry 2, negative regulators of Ras-Raf-Erk signaling, were upregulated more in the corneal than in the lens epithelial cells. Both lens and corneal differentiation programs were sensitive to Ras activation. Ras transgenic embryos showed a distinctive alteration in the architecture of the lens pit. Ras activation, though sufficient for upregulation of Prox1, a transcription factor critical for cell cycle exit and initiation of fiber differentiation, is not sufficient for induction of terminal fiber differentiation. Expression of Keratin 12, a marker of corneal epithelial differentiation, was reduced in the Ras transgenic corneas. Conclusions Collectively, these

  7. Mutations of p53 and ras genes in radon-associated lung cancer from uranium miners

    Energy Technology Data Exchange (ETDEWEB)

    Vaehaekangas, K.H.; Metcalf, R.A.; Welsh, J.A.; Bennett, W.P.; Harris, C.C. (National Cancer Inst., Bethesda, MD (United States)); Samet, J.M. (New Mexico Univ., Albuquerque, NM (United States). UNM Medical Center); Lane, D.P. (Dundee Univ. (United Kingdom). Dept. of Biochemistry)

    1992-03-07

    Radon increases the risk of lung cancer in smoking and non-smoking underground miners. To investigate the mutational spectrum associated with exposure to high levels of radon, the authors sequenced exons 5-9 of the p53 tumour suppressor gene and codons 12-13 of the Ki-ras protooncogene in 19 lung cancers from uranium miners exposed to radon and tobacco smoke. Mutations were not found in Ki-ras, but 9 p53 mutations, including 2 deletions, were found in 7 patients by direct DNA sequencing after polymerase chain reaction amplification of DNA from formalin-fixed, paraffin-embedded tissue. In tumours from 5 patients, the mutation produced an aminoacid change and an increased nuclear content of p53 protein. (author).

  8. 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 ...

  9. GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

    Science.gov (United States)

    Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2015-01-01

    K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

  10. GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site.

    Science.gov (United States)

    Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2015-11-27

    K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4B(WT)-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4B(WT)-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Ki-ras gene mutations are invariably present in low-grade mucinous tumors of the vermiform appendix.

    Science.gov (United States)

    Zauber, Peter; Berman, Errol; Marotta, Stephen; Sabbath-Solitare, Marlene; Bishop, Timothy

    2011-07-01

    Low-grade mucinous tumors of the appendix appear to have a simple histological structure. Paradoxically, reports have suggested a greater frequency of Ki-ras gene mutation in these lesions than in more complex lesions such as benign colonic adenomas and carcinomas. We assessed several molecular genetic changes, including Ki-ras gene mutations, in a large series of low-grade mucinous tumors of the appendix. We retrospectively ascertained low-grade mucinous tumors of the appendix from computerized pathology records. Extracted DNA was analyzed for APC and DCC gene loss of heterozygosity, microsatellite instability and for the presence of Ki-ras gene mutation using standard molecular techniques. Controls consisted of normal appendices, other appendiceal neoplasms, and ovarian mucinous cystadenomas. A total of 31 low-grade appendiceal mucinous tumors were identified. All were microsatellite stable and none demonstrated loss of heterozygosity for the APC or DCC genes. By contrast, all 31 lesions contained a Ki-ras gene mutation. The presence of a Ki-ras gene mutation in all lesions, with no other molecular changes identified, strongly suggests a possible etiological role of the Ki-ras mutation in the development of this particular lesion of the appendix. Based on other work regarding intestinal bacteria, we hypothesize a relationship between chronic inflammation of the appendix from bacterial overgrowth and Ki-ras gene mutation.

  12. Machine Learning Detects Pan-cancer Ras Pathway Activation in The Cancer Genome Atlas.

    Science.gov (United States)

    Way, Gregory P; Sanchez-Vega, Francisco; La, Konnor; Armenia, Joshua; Chatila, Walid K; Luna, Augustin; Sander, Chris; Cherniack, Andrew D; Mina, Marco; Ciriello, Giovanni; Schultz, Nikolaus; Sanchez, Yolanda; Greene, Casey S

    2018-04-03

    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. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

    Directory of Open Access Journals (Sweden)

    de Bruïne Adriaan P

    2005-12-01

    Full Text Available Abstract Background The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1 and Ras (K-ras pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. Methods In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Results Mutations at the phosphorylation sites (codons 31, 33, 37, and 45 in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656 and 36% (235/656, respectively. Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656. Nine percent of all tumours (58/656 lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. Conclusion CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.

  14. Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

    International Nuclear Information System (INIS)

    Lüchtenborg, Margreet; Weijenberg, Matty P; Wark, Petra A; Saritas, A Merdan; Roemen, Guido MJM; Muijen, Goos NP van; Bruïne, Adriaan P de; Brandt, Piet A van den; Goeij, Anton FPM de

    2005-01-01

    The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency

  15. Growth factor-dependent activation of the Ras-Raf-MEK-MAPK pathway in the human pancreatic carcinoma cell line PANC-1 carrying activated K-ras: implications for cell proliferation and cell migration.

    Science.gov (United States)

    Giehl, K; Skripczynski, B; Mansard, A; Menke, A; Gierschik, P

    2000-06-08

    Human ductal adenocarcinoma of the pancreas frequently carry activating point mutations in the K-ras protooncogene. We have analysed the activity of the Ras-Raf-MEK-MAPK cascade in the human pancreatic carcinoma cell line PANC-1 carrying an activating K-ras mutation. Serum-starved cells and cells grown in medium with serum did not show constitutively activated c-Raf, MEK-1, or p42 MAPK. Stimulation of cells with epidermal growth factor (EGF) or fetal calf serum (FCS) resulted in activation of N-Ras, but not K-Ras, as well as activation of c-Raf, MEK-1, and p42 MAPK. Preincubation of serum-starved cells with MEK-1 inhibitor PD98059 abolished EGF- and FCS-induced MAPK activation, identifying MEK as the upstream activator of MAPK. PANC-1 cells exhibited marked serum-dependence of anchorage-dependent and -independent cell growth as well as cell migration. EGF, alone or in combination with insulin and transferrin, did not induce cell proliferation of serum-starved PANC-1 cells, indicating that activation of MAPK alone was not sufficient to induce cell proliferation. FCS-induced DNA synthesis was inhibited by 40% by the MEK-1 inhibitor. On the other hand, treatment with either FCS or EGF alone resulted in marked, MEK-dependent increase of directed cell migration. Collectively, our results show that the activating K-ras mutation in PANC-1 cells does not result in constitutively increased Raf-MEK-MAPK signaling. Signal transduction via the Ras-Raf-MEK-MAPK cascade is maintained in these cells and is required for growth factor-induced cell proliferation and directed cell migration. Oncogene (2000).

  16. Rational combination of MEK inhibitor and the STAT3 pathway modulator for the therapy in K-Ras mutated pancreatic and colon cancer cells.

    Science.gov (United States)

    Zhao, Chengguang; Xiao, Hui; Wu, Xiaojuan; Li, Chenglong; Liang, Guang; Yang, Shulin; Lin, Jiayuh

    2015-06-10

    K-Ras mutations are frequently detected in pancreatic and colon cancers, which are associated with the resistance to MEK inhibitors targeting the Ras pathway. Identifying the underlying mechanisms for the acquired resistance is essential for the future clinical development of MEK inhibitors. Here, we identified that Signal Transducer and Activator of Transcription 3 (STAT3) was significantly activated following the MEK inhibition using AZD6244, PD98059 and Trametinib in K-Ras mutant pancreatic and colon cancer cells. The STAT3 activation may be important for the MEK inhibitor resistance in these K-Ras mutant cancer cells. We have shown that dual inhibition of STAT3 and MEK using the STAT3 inhibitor LY5 and MEK inhibitor Trametinib exerts significant anti-tumor cell efficacy in K-Ras mutant pancreatic and colon cancer cells in vitro. In addition, Trametinib showed increased suppression on tumor growth in vivo in STAT3 knockdown pancreatic cancer cells compared with tumor growth of control cells without STAT3 knockdown. Taken together, our results suggest the induced STAT3 activation as a possible mechanism for the resistance to MEK inhibitor and demonstrate the potentials of a combination therapy using MEK and STAT3 inhibitors in pancreatic and colon cancers harboring K-Ras mutant proteins.

  17. Detection of K-ras gene mutations in feces by magnetic nanoprobe in patients with pancreatic cancer: A preliminary study.

    Science.gov (United States)

    Wang, Xiaoguang; Wang, Jingshuai; Chen, Fei; Zhong, Zhengxiang; Qi, Lifeng

    2018-01-01

    The present study aimed to investigate the feasibility and effectiveness of detecting K-ras mutation by using magnetic nanoparticles in fecal samples of patients with pancreatic cancer at different stages. The novel methodology of K-ras mutation detection was compared to the existing methodology of cancer antigen (CA)19-9 examination. Patients with pancreatic cancer (n=88), pancreatic benign diseases who displayed chronic pancreatitis (n=35), pancreatic mucinous cyst neoplasms (n=10) and pancreatic serous cyst (n=9) admitted to the Department of Surgery, Jiaxing Second Hospital were enrolled in the present study. Fecal samples were collected from all patients, DNA was extracted and magnetic nanoprobe was then used to detect K-ras mutation. The results obtained using the novel magnetic nanoprobe detection technique showed a K-ras mutation rate of 81.8% (72/88) in the patients with pancreatic cancer and 18.5% (10/54) in patients with pancreatic benign diseases. In patients with pancreatic cancer, the K-ras mutation rate was comparable in stages I + IIA and IIB + III + IV (78.9 vs. 84.0%; P>0.05). The sensitivity and specificity of K-ras mutation for detection of pancreatic cancer was 81.8 and 81.5%, respectively. Sixty-eight pancreatic cancer patients had >37 U/ml CA99 with a sensitivity and specificity for pancreatic cancer detection of 77.3 and 77.8%, which was not significantly lower than detection by the fecal K-ras mutations (P>0.05). Combinational detection of fecal K-ras mutations and serum CA19-9 significantly increased the sensitivity regarding pancreatic cancer detection to 97.7% (P0.05) compared with fecal K-ras mutations or CA19-9 alone. The findings showed that the magnetic nanoprobe is able to detect fecal K-ras mutations in different stages of pancreatic cancer, with comparable sensitivity and specificity to CA19-9 examination for differentiating pancreatic cancer. Furthermore, combined detection of CA19-9 and K-ras mutations has enhanced sensitivity

  18. The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium.

    Science.gov (United States)

    Buchsbaum, R; Telliez, J B; Goonesekera, S; Feig, L A

    1996-09-01

    We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location. This requires the PH domain and the adjacent coiled-coil and IQ sequences. Remarkably, this form of Ras-GRF is constitutively activated. The PH and coiled-coil domains must also perform an additional function, since targeting to the particulate fraction of cells is not sufficient to allow Ras-GRF activation by calcium. A Ras-GRF mutant containing the PH domain from Ras-GTPase-activating protein in place of its own N-terminal PH domain localizes to the particulate fraction of cells but does not respond to calcium. Similar phenotypes are seen with mutant Ras-GRFs containing point mutations in either the PH or coiled-coil domain. These findings argue that the N-terminal PH, coiled-coil, and IQ domains of Ras-GRF function together to connect Ras-GRF to multiple components in the particulate fractions of cells that are required for responsiveness of the protein to calcium signaling.

  19. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

    Directory of Open Access Journals (Sweden)

    Jianhua Gong

    Full Text Available Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency, hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

  20. 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

  1. The dual RAF/MEK inhibitor CH5126766/RO5126766 may be a potential therapy for RAS-mutated tumor cells.

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    Makoto Wada

    Full Text Available Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model. The present study indicates that CH5126766/RO5126766 is an attractive RAF/MEK inhibitor in RAS-mutated malignant tumor cells including melanoma.

  2. The dual RAF/MEK inhibitor CH5126766/RO5126766 may be a potential therapy for RAS-mutated tumor cells.

    Science.gov (United States)

    Wada, Makoto; Horinaka, Mano; Yamazaki, Toshikazu; Katoh, Norito; Sakai, Toshiyuki

    2014-01-01

    Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model. The present study indicates that CH5126766/RO5126766 is an attractive RAF/MEK inhibitor in RAS-mutated malignant tumor cells including melanoma.

  3. Nonrandom duplication of the chromosome bearing a mutated Ha-ras-1 allele in mouse skin tumors

    International Nuclear Information System (INIS)

    Bianchi, A.B.; Aldaz, C.M.; Conti, C.J.

    1990-01-01

    The authors analyzed the normal/mutated allelic ratio of the Ha-ras-1 gene in mouse skin squamous cell carcinomas induced by initation with dimethylbenz[a]anthracene and promotion with phorbol 12-myristate 13-acetate. DNA for these studies was obtained from short-term tumor cultures (24-72 hr) to eliminate the contribution of stromal and inflammatory cells to the sample. The alelotypic analysis was performed in 25 squamous cell carcinomas by quantitative radio-analysis of the Xba I restriction fragment length polymorphism as detected by BS9, a v-Ha-ras probe, and rehybridization of the Southern blots with probes for chromosomes 7 and 9. Approximately 85% of the tumors presented overrepresentation of the mutated allele in the form of 1 normal/2 mutated (12 tumors), 0 normal/3 mutated (4 tumors), 0 normal/2 mutated (3 tumors), and gene amplification (3 tumors). No tumor was found with a 2 normal/1 mutated allelic ratio. These results support their previous cytogenetic studies, indicating that trisomy of chromosome 7 is present in themajority of these tumors show that nonrandom duplication of the chromosome carrying the mutated Ha-ras-1 allel appears to be a major mechanism by which the mutated gene is overrepresented

  4. A new mutation-independent approach to cancer therapy: Inhibiting oncogenic RAS and MYC, by targeting mitochondrial biogenesis.

    Science.gov (United States)

    Ozsvari, Bela; Sotgia, Federica; Lisanti, Michael P

    2017-10-27

    Here, we used MCF7 cells as a model system to interrogate how MYC/RAS co-operativity contributes to metabolic flux and stemness in breast cancer cells. We compared the behavior of isogenic MCF7 cell lines transduced with c-Myc or H-Ras (G12V), either individually or in combination. Cancer stem cell (CSC) activity was measured using the mammosphere assay. c-Myc augmented both mammosphere formation and mitochondrial respiration, without any effects on glycolytic flux. In contrast, H-Ras (G12V) synergistically augmented both mammosphere formation and glycolysis, but only in combination with c-Myc, directly demonstrating MYC/RAS co-operativity. As c-Myc is known to exert its effects, in part, by stimulating mitochondrial biogenesis, we next examined the effects of another stimulus known to affect mitochondrial biogenesis, i.e. ROS production. To pharmacologically induce oxidative stress, we used Rotenone (a mitochondrial inhibitor) to target mitochondrial complex I. Treatment with Rotenone showed bi-phasic effects; low-dose Rotenone (1 to 2.5 nM) elevated mammosphere formation, while higher doses (10 to 100 nM) were inhibitory. Importantly, the stimulatory effects of Rotenone on CSC propagation were blocked using a mitochondrial-specific anti-oxidant, namely Mito-tempo. Thus, "mild" mitochondrial oxidative stress, originating at Complex I, was sufficient to pheno-copy the effects of c-Myc, effectively promoting CSC propagation. To validate the idea that mitochondrial biogenesis is required to stimulate CSC propagation, we employed Doxycycline, a well-established inhibitor of mitochondrial protein translation. Treatment with Doxycycline was indeed sufficient to block the stimulatory effects of H-Ras (G12V), c-Myc, and Rotenone on CSC propagation. As such, Doxycycline provides a strong rationale for designing new therapeutics to target mitochondrial biogenesis, suggesting a new "mutation-independent" approach to cancer therapy. In support of this notion, most currently

  5. Correlations of IGF-1R and COX-2 Expressions with Ras and BRAF Genetic Mutations, Clinicopathological Features and Prognosis of Colorectal Cancer Patients.

    Science.gov (United States)

    Jin, Mei; Long, Zi-Wen; Yang, Jing; Lin, Xiang

    2018-01-01

    This case-control study aims to investigate the correlations of insulin-like growth factor receptor 1 (IGF-1R) and cyclooxygenase 2 (COX-2) expressions with Ras and BRAF genetic mutations, clinicopathological features and prognosis of colorectal cancer (CRC) patients. A total of 213 CRC patients (case group) and 200 healthy individuals (control group) were selected from our hospital. Ras (K-Ras/N-Ras) and BRAF genetic mutations were detected by direct sequencing. The positive expression rates of IGF-IR and COX-2 in CRC and normal tissues were detected using immunohistochemistry. RT-qPCR and Western blotting were applied to detect the mRNA and protein expressions of IGF-IR and COX-2 in CRC tissues and normal tissues. Total mutation rate of N-Ras, BRAF and K-Ras in case group were 5.2%, 12.2% and 47.4%, respectively. The expressions of IGF-IR and COX-2 were higher in CRC tissues with Ras and BRAF mutations than in those without. CRC tissues with Ras mutation showed higher COX-2 expression than those with BRAF mutation. IGF-IR and COX-2 expressions were correlated to infiltration degree, lymphatic metastasis (in CRC tissues with and without Ras and BRAF mutations), and Dukes stages (only in CRC tissues with Ras and BRAF mutations). CRC patients with negative expressions of IGF-IR and COX-2 had significantly higher accumulative survival rate and longer mean survival duration than those with positive expressions of IGF-IR and COX-2. These findings indicate that IGF-1R and COX-2 expressions may be associated with Ras and BRAF genetic mutations, clinicopathological feature and prognosis of CRC patients.

  6. Real-time PCR-based method for the rapid detection of extended RAS mutations using bridged nucleic acids in colorectal cancer.

    Science.gov (United States)

    Iida, Takao; Mizuno, Yukie; Kaizaki, Yasuharu

    2017-10-27

    Mutations in RAS and BRAF are predictors of the efficacy of anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Therefore, simple, rapid, cost-effective methods to detect these mutations in the clinical setting are greatly needed. In the present study, we evaluated BNA Real-time PCR Mutation Detection Kit Extended RAS (BNA Real-time PCR), a real-time PCR method that uses bridged nucleic acid clamping technology to rapidly detect mutations in RAS exons 2-4 and BRAF exon 15. Genomic DNA was extracted from 54 formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from mCRC patients. Among the 54 FFPE samples, BNA Real-time PCR detected 21 RAS mutations (38.9%) and 5 BRAF mutations (9.3%), and the reference assay (KRAS Mutation Detection Kit and MEBGEN™ RASKET KIT) detected 22 RAS mutations (40.7%). The concordance rate of detected RAS mutations between the BNA Real-time PCR assay and the reference assays was 98.2% (53/54). The BNA Real-time PCR assay proved to be a more simple, rapid, and cost-effective method for detecting KRAS and RAS mutations compared with existing assays. These findings suggest that BNA Real-time PCR is a valuable tool for predicting the efficacy of early anti-EGFR therapy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance.

    Science.gov (United States)

    McCubrey, James A; Steelman, Linda S; Chappell, William H; Abrams, Stephen L; Franklin, Richard A; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M

    2012-10-01

    The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance.

  8. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance

    Science.gov (United States)

    McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Abrams, Stephen L.; Franklin, Richard A.; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M.

    2012-01-01

    The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance. PMID:23085539

  9. Noninvasive image texture analysis differentiates K-ras mutation from pan-wildtype NSCLC and is prognostic.

    Directory of Open Access Journals (Sweden)

    Glen J Weiss

    Full Text Available Non-invasive characterization of a tumor's molecular features could enhance treatment management. Quantitative computed tomography (CT based texture analysis (QTA has been used to derive tumor heterogeneity information, and the appearance of the tumors has been shown to relate to patient outcome in non-small cell lung cancer (NSCLC and other cancers. In this study, we examined the potential of tumoral QTA to differentiate K-ras mutant from pan-wildtype tumors and its prognostic potential using baseline pre-treatment non-contrast CT imaging in NSCLC.Tumor DNA from patients with early-stage NSCLC was analyzed on the LungCarta Panel. Cases with a K-ras mutation or pan-wildtype for 26 oncogenes and tumor suppressor genes were selected for QTA. QTA was applied to regions of interest in the primary tumor. Non-parametric Mann Whitney test assessed the ability of the QTA, clinical and patient characteristics to differentiate between K-ras mutation from pan-wildtype. A recursive decision tree was developed to determine whether the differentiation of K-ras mutant from pan-wildtype tumors could be improved by sequential application of QTA parameters. Kaplan-Meier survival analysis assessed the ability of these markers to predict survival.QTA was applied to 48 cases identified, 27 had a K-ras mutation and 21 cases were pan-wildtype. Positive skewness and lower kurtosis were significantly associated with the presence of a K-ras mutation. A five node decision tree had sensitivity, specificity, and accuracy values (95% CI of 96.3% (78.1-100, 81.0% (50.5-97.4, and 89.6% (72.9-97.0; respectively. Kurtosis was a significant predictor of OS and DFS, with a lower kurtosis value linked with poorer survival.Lower kurtosis and positive skewness are significantly associated with K-ras mutations. A QTA feature such as kurtosis is prognostic for OS and DFS. Non-invasive QTA can differentiate the presence of K-ras mutation from pan-wildtype NSCLC and is associated with

  10. [Correlation analysis between abundance of K-ras mutation in plasma free DNA and its correlation with clinical outcome and prognosis in patients with metastatic colorectal cancer].

    Science.gov (United States)

    Bai, Yan-qing; Liu, Xiao-jing; Wang, Yan; Ge, Fei-jiao; Zhao, Chuan-hua; Fu, Ya-li; Lin, Li; Xu, Jian-ming

    2013-09-01

    To detect K-ras gene mutations in plasma free DNA by peptide nucleic acid clamp PCR assay (PNA-PCR) and nested primer PCR, and to analyze the correlation between K-ras mutations and prognosis in patients with metastatic colorectal cancer (mCRC). Peripheral blood was collected and free DNA was extracted from plasma in 106 patients with mCRC. Nested primer PCR and PNA-PCR were used to detect K-ras gene mutation in the plasma free DNA. The patients were divided into three groups by K-ras status: wild-type group (wild-type determined by both methods), low mutation group (mutation by PNA-PCR method, wild-type by nested primer PCR method) and high mutation group (mutation by two methods). The correlation between K-ras mutations and prognosis was analyzed. The mutation rate of K-ras in tumor tissues of the 106 patients was 40.6%. The Mutation rate of K-ras in plasma free DNA detected by PNA-PCR was 31.1%, significantly higher than that of 15.1% detected by nested primer PCR (P = 0.006). The consistent rate of the K-ras status in plasma free DNA detected by PNA-PCR and that in tumor tissue detected by traditional method was up to 83.0%. The median overall survival (OS) of patients of the wild type, low mutation and high mutation groups was 23.5 months, 17.3 months and 13.9 months, respectively (P = 0.002). The median progression-free survival (PFS) of the K-ras wild-type, low mutation and high mutation groups with first-line chemotherapy was 6.8 months, 6.1 months and 3.2 months, respectively (P = 0.002), and the median OS of them were 23.0 months, 15.5 months and 13.9 months, respectively (P = 0.036). The overall response rate (ORR) was improved in the K-ras wide-type patients who received cetuximab combined with chemotherapy as first-line therapy (75.0% vs. 23.4%, P = 0.058). Cetuximab combined with in second-line therapy chemotherapy led to a significant improvement in disease control rate (DCR) ( 100% vs. 35.7%, P mutation in plasma free DNA can be used to substitute

  11. Meat consumption and K-ras mutations in sporadic colon and rectal cancer in The Netherlands Cohort Study

    NARCIS (Netherlands)

    Brink, M.; Weijenberg, M.P.; Goeij, A.F.P.M. de; Roemen, G.M.J.M.; Lentjes, M.H.F.M.; Bruïne, A.P. de; Goldbohm, R.A.; Brandt, P.A. van den

    2005-01-01

    Case-cohort analyses were performed on meat and fish consumption in relation to K-ras mutations in 448 colon and 160 rectal cancers that occurred during 7.3 years of follow-up, excluding the first 2.3 years, and 2948 subcohort members of The Netherlands Cohort Study on diet and cancer. Adjusted

  12. MEK inhibition is a promising therapeutic strategy for MLL-rearranged infant acute lymphoblastic leukemia patients carrying RAS mutations

    NARCIS (Netherlands)

    Kerstjens, M. (Mark); E.M.C. Driessen; M. Willekes (Merel); Pinhanços, S.S. (Sandra S.); P. Schneider (Pauline); R. Pieters (Rob); R.W. Stam (Ronald)

    2017-01-01

    textabstractAcute lymphoblastic leukemia (ALL) in infants is an aggressive malignancy with a poor clinical outcome, and is characterized by translocations of the Mixed Lineage Leukemia (MLL) gene. Previously, we identified RAS mutations in 14-24% of infant ALL patients, and showed that the presence

  13. Associations between primary tumor RAS, BRAF and PIK3CA mutation status and metastatic site in patients with chemo-resistant metastatic colorectal cancer

    DEFF Research Database (Denmark)

    Christensen, Troels Dreier; Palshof, Jesper Andreas; Larsen, Finn Ole

    2018-01-01

    , after fluoropyrimidine, oxaliplatin and irinotecan treatment failure. The database contained information regarding tumor mutation status of KRAS, NRAS, BRAF and PIK3CA genes. RESULTS: Totally, 448 patients were included. On multivariate analyses, RAS mutations were significantly associated...... investigated the association between RAS (KRAS or NRAS), BRAF, PIK3CA mutations and metastatic pattern in patients with metastatic (m) CRC. MATERIAL AND METHODS: This study reviewed Danish biobank and database of patients with mCRC who received cetuximab and irinotecan, independent of RAS mutation status...... = 1.86-25.02) and PIK3CA mutations with decreased hazard of peritoneal metastases (HR = 0.31; 95%CI = 0.11-0.86). CONCLUSIONS: This study indicated that in patients with mCRC, RAS mutations are associated with increased risk of lung and ovary metastases. BRAF V600E is associated with increased risk...

  14. Sensitive and specific detection of K-ras mutations in colon tumors by short oligonucleotide mass analysis

    Science.gov (United States)

    Lleonart, Matilde E.; Ramón y Cajal, Santiago; Groopman, John D.; Friesen, Marlin D.

    2004-01-01

    Short oligonucleotide mass analysis (SOMA) is a technique by which small sequences of mutated and wild-type DNA, produced by PCR amplification and restriction digestion, are characterized by HPLC-electrospray ionization tandem mass spectrometry. We have adapted the method to specifically detect two common point mutations at codon 12 of the c-K-ras gene. Mutations in DNA from 121 colon tumor samples were identified by SOMA and validated by comparison with sequencing. SOMA correctly identified 26 samples containing the 12GAT mutation and four samples containing the 12AGT mutation. Sequencing did not reveal mutant DNA in three samples out of the 26 samples shown by SOMA to contain the 12GAT mutation. In these three samples, the presence of mutant DNA was confirmed by SOMA analysis after selective PCR amplification in the presence of BstN1 restriction enzyme. Additional mutations in codons 12 and 13 were revealed by sequencing in 24 additional samples, and their presence did not interfere with the correct identification of G to A or G to T mutations in codon 12. These results provide the basis for a sensitive and specific method to detect c-K-ras codon 12-mutated DNA at levels below 10–12% of wild-type DNA. PMID:15037663

  15. [Screening for K-ras mutations in colorectal and lung cancers by using a novel real-time PCR with ADx-K-ras kit and Sanger DNA sequencing].

    Science.gov (United States)

    Zhang, Hai-Ping; Fu, Li; Chen, Pei-Qiong; Ye, Yun-Bin; Ji, Tian-Hai; Zheng, Li-Mou

    2010-11-01

    to map out the frequency and types of K-ras gene mutations present in colorectal and lung cancer patients; to evaluate the clinical applicability of a novel real-time double-loop probe PCR using the ADx-K-ras kit, and to compare its performance with the result by using traditional Sanger DNA sequencing in detection of somatic mutations of the tumor genes. a total of 827 formalin-fixed paraffin-embedded (FFPE) blocks including 583 from the colorectal and 244 from the lung cancer patients were assayed. Genomic DNA of the sample tissues was extracted, purified and subjected to PCR amplification of K-ras gene codon 12 and 13 and DNA sequencing was carried on using both the traditional Sanger sequencing method and the ADx's K-ras mutation detection kit, respectively. The mutation rates for K-ras gene at codon 12 and 13, and the mutation frequencies detected by using both methods were analyzed. 533 out of 583 (91.4%) colorectal cancer samples and 144 out of 244 lung cancer samples (59.0%) were detected using the traditional Sanger DNA sequencing technique, and 583 out of 583 (100.0%) colorectal plus 244 out of 244(100.0%) lung cancers were detected, respectively by using the ADx-K-ras kit. Of the 583 colorectal cancer samples, 192 (32.9%) showed mutations by using the ADx-K-ras kit in comparing with a result of 160 samples (27.4%) with K-ras gene mutation by using the traditional Sanger DNA sequencing technique. Of the 244 lung cancer samples, 26 (10.7%) showed K-ras gene mutations by using ADx-K-ras kit, while in 144 samples detected by using the traditional Sanger DNA sequencing technique, only 12 samples (8.3%) showed K-ras gene mutations. In colorectal cancer analyzed, GGT→GAT at codon 12 was the most common event with 35.1% (66/188) mutations, followed by GGC→GAC at codon 13 with 26.6% (50/188) and GGT→GTT at codon 12 with 18.6% (35/188), while GGT→GCT at codon12 was the most rare with only 1.6% (3/188) of the total mutation cases. In patients with lung

  16. Piceatannol suppresses the metastatic potential of MCF10A human breast epithelial cells harboring mutated H-ras by inhibiting MMP-2 expression.

    Science.gov (United States)

    Song, Nu Ry; Hwang, Mun Kyung; Heo, Yong-Seok; Lee, Ki Won; Lee, Hyong Joo

    2013-10-01

    Metastasis is one of the most threatening features of the oncogenic process and the main cause of cancer-related mortality. Several studies have demonstrated that matrix metalloproteinases (MMPs) are critical for tumor invasion and metastasis. Resveratrol (3,5,4'-trihydroxystilbene), a phenolic compound of red wine, has been reported to be a natural chemopreventive agent. However, the cancer preventive effects of piceatannol (3,5,3',4'-tetrahydroxystilbene), a metabolite of resveratrol and the underlying molecular mechanisms have not yet been fully elucidated. In this study, we report that piceatannol inhi-bits H-ras-induced MMP-2 activity and the invasive phenotype of MCF10A human breast epithelial cells harboring mutated H-ras (H-ras MCF10A cells) more effectively than resveratrol. Piceatannol attenuated the H-ras-induced phosphorylation of Akt in a time- and dose-dependent manner, whereas resveratrol, at the same concentrations, did not exert an inhibitory effect. In vitro kinase assays demonstrated that piceatannol significantly inhibited phosphatidylinositol 3-kinase (PI3K) activity and suppressed phospha-tidylinositol (3,4,5)-trisphosphate (PIP3) expression in the H-ras MCF10A cells. Ex vivo pull-down assays revealed that piceatannol directly bound to PI3K, inhibiting PI3K activity. Data from molecular docking suggested that piceatannol is a more tight-binding inhibitor than resveratrol due to the additional hydrogen bond between the hydroxyl group and the backbone amide group of Val882 in the ATP-binding pocket of PI3K.

  17. 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.

  18. 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.

  19. Evaluation of Intraductal Ultrasonography, Endoscopic Brush Cytology and K-ras, P53 Gene Mutation in the Early Diagnosis of Malignant Bile Duct Stricture

    Directory of Open Access Journals (Sweden)

    Ping Huang

    2015-01-01

    Conclusions: IDUS combined with brush cytology and K-ras, P53 gene mutation detection is better than the separate detection and contribute to the early diagnosis of malignant biliary stricture. Its more widespread use is recommended.

  20. Specific regulation of point-mutated K-ras-immortalized cell proliferation by a photodynamic antisense strategy.

    Science.gov (United States)

    Higuchi, Maiko; Yamayoshi, Asako; Kato, Kiyoko; Kobori, Akio; Wake, Norio; Murakami, Akira

    2010-02-01

    It has been reported that point mutations in genes are responsible for various cancers, and the selective regulation of gene expression is an important factor in developing new types of anticancer drugs. To develop effective drugs for the regulation of point-mutated genes, we focused on photoreactive antisense oligonucleotides. Previously, we reported that photoreactive oligonucleotides containing 2'-O-psoralenylmethoxyethyl adenosine (2'-Ps-eom) showed drastic photoreactivity in a strictly sequence-specific manner. Here, we demonstrated the specific gene regulatory effects of 2'-Ps-eom on [(12)Val]K-ras mutant (GGT --> GTT). Photo-cross-linking between target mRNAs and 2'-Ps-eom was sequence-specific, and the effect was UVA irradiation-dependent. Furthermore, 2'-Ps-eom was able to inhibit K-ras-immortalized cell proliferation (K12V) but not Vco cells that have the wild-type K-ras gene. These results suggest that the 2'-Ps-eom will be a powerful nucleic acid drug to inhibit the expression of disease-causing point mutation genes, and has great therapeutic potential in the treatment of cancer.

  1. Lateral flow strip for visual detection of K-ras mutations based on allele-specific PCR.

    Science.gov (United States)

    Wang, Cong; Chen, Xiaomin; Wu, Yuying; Li, Hao; Wang, Yu; Pan, Xiaofu; Tang, Tingting; Liu, Ziying; Li, Xiaokun

    2016-10-01

    To develop a convenient and sensitive point-of-care test for detecting gene mutations based on allele-specific PCR. To develop a lateral flow strip for visual detection of K-ras mutations based on a modified PCR, a specific DNA tag was covalently linked to the 5'-end of each primer by a nine-carbon linker to produce a sticky end. One of the sticky ends of the PCR products bound to gold nano-particles, while the other sticky end was captured onto a nitrocellulose membrane of lateral flow strips. The lateral flow strip showed a great sensitivity, which detected mutations in as low as 10 tumor cells. The positive rate and accuracy of the lateral flow strip for blood samples were over 92 and 96 %, respectively. The lateral flow strip provides an easy method for sensitive detection of gene mutations based on allele specific-PCR.

  2. 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.

  3. Control of thrombotic thrombocytopenic purpura by sirolimus in a child with juvenile myelomonocytic leukemia and somatic N-RAS mutation.

    Science.gov (United States)

    Maschan, Michael; Bobrynina, Vlasta; Khachatryan, Lili; Kalinina, Irina; Solopova, Galina; Avdonin, Pavel; Nasedkina, Tatiana; Novichkova, Galina; Maschan, Alexei

    2014-10-01

    We describe an infant who developed juvenile myelomonocytic leukemia (JMML) at the age of 6 months. Myeloproliferation was effectively controlled by low-dose cytosine arabinoside and 13-cis retinoic acid therapy. Two years after therapy for JMML was stopped, at the age of 5 years, the patient developed autoimmune thrombotic thrombocytopenic purpura (TTP). TTP was transiently controlled by plasma exchange, prednisolone, rituximab, and cyclophosphamide, but relapsed within a short time. Long-term control of TTP was established by sirolimus. Somatic N-RAS G38A→Gly13Asp substitution was restricted to hematopoietic cells. The somatic N-RAS mutation may link myeloproliferation and autoimmunity. © 2014 Wiley Periodicals, Inc.

  4. A chronocoulometric LNA sensor for amplified detection of K-ras mutation based on site-specific DNA cleavage of restriction endonuclease.

    Science.gov (United States)

    Lin, Liqing; Liu, Ailin; Zhao, Chengfei; Weng, Shaohuang; Lei, Yun; Liu, Qicai; Lin, Xinhua; Chen, Yuanzhong

    2013-04-15

    An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5 fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. The higher level of complexity of K-Ras4B activation at the membrane

    Science.gov (United States)

    Jang, Hyunbum; Banerjee, Avik; Chavan, Tanmay S.; Lu, Shaoyong; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2016-01-01

    Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5′-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.—Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane. PMID:26718888

  6. The higher level of complexity of K-Ras4B activation at the membrane.

    Science.gov (United States)

    Jang, Hyunbum; Banerjee, Avik; Chavan, Tanmay S; Lu, Shaoyong; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2016-04-01

    Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.-Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane. © FASEB.

  7. A Panel of High Resolution Melting (HRM Technology-Based Assays with Direct Sequencing Possibility for Effective Mutation Screening of EGFR and K-ras Genes

    Directory of Open Access Journals (Sweden)

    D. A. M. Heideman

    2009-01-01

    Full Text Available Background: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies.

  8. The adaptor-like protein ROG-1 is required for activation of the Ras-MAP kinase pathway and meiotic cell cycle progression in Caenorhabditis elegans.

    Science.gov (United States)

    Matsubara, Yosuke; Kawasaki, Ichiro; Urushiyama, Seiichi; Yasuda, Tomoharu; Shirakata, Masaki; Iino, Yuichi; Shibuya, Hiroshi; Yamanashi, Yuji

    2007-03-01

    The Ras-MAP kinase pathway regulates varieties of fundamental cellular events. In Caenorhabditis elegans, this pathway is required for oocyte development; however, the nature of its up-stream regulators has remained elusive. Here, we identified a C. elegans gene, rog-1, which encodes the only protein having the IRS-type phosphotyrosine-binding (PTB) domain in the worms. ROG-1 has no obvious domain structure aside from the PTB domain, suggesting that it could serve as an adaptor down-stream of protein-tyrosine kinases (PTKs). RNA interference (RNAi)-mediated down-regulation of rog-1 mRNA significantly decreased brood size. rog-1(tm1031) truncation mutants showed a severe disruption in progression of developing oocytes from pachytene to diakinesis, as was seen in worms carrying a loss-of-function mutation in the let-60 Ras or mpk-1 MAP kinase gene. Furthermore, let-60 Ras-regulated activation of MPK-1 in the gonad is undetectable in rog-1(tm1031) mutants. Conversely, a gain-of-function mutation in the let-60 Ras gene rescues the brood size reduction and germ cell abnormality in rog-1(tm1031) worms. Consistently, rog-1 is preferentially expressed in the germ cells and its expression in the gonad is essential for oocyte development. Thus, ROG-1 is a key positive regulator of the Ras-MAP kinase pathway that permits germ cells to exit from pachytene.

  9. 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...

  10. Phloretin induces apoptosis in H-Ras MCF10A human breast tumor cells through the activation of p53 via JNK and p38 mitogen-activated protein kinase signaling.

    Science.gov (United States)

    Kim, Mi-Sung; Kwon, Jung Yeon; Kang, Nam Joo; Lee, Ki Won; Lee, Hyong Joo

    2009-08-01

    Mutations in Ras play a critical role in the development of human cancers, including breast cancer. We investigated the possible antiproliferative effects of the naturally occurring dihydrochalcone phloretin [2',4',6'-trihydroxy-3-(4-hydroxyphenyl)-propiophenone] on H-Ras-transformed MCF10A human breast epithelial (H-Ras MCF10A) cells. Phloretin suppressed H-Ras MCF10A cell proliferation in a dose-dependent manner and induced nuclear condensation in the cells, indicating that phloretin-induced cell death occurs mainly via the induction of apoptosis. Prominent upregulation of p53 and Bax and cleavage of poly (ADP)-ribose polymerase were also detected in the phloretin-treated cells. Finally, phloretin markedly increased caspase-3 activity as well as JNK and p38 mitogen-activated protein kinase signaling. Our findings suggest that the phloretin-induced apoptosis of breast tumor cells contributes to the chemopreventive potential of phloretin against breast cancer.

  11. 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.

  12. 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

  13. Antroquinonol blocks Ras and Rho signaling via the inhibition of protein isoprenyltransferase activity in cancer cells.

    Science.gov (United States)

    Ho, Ching-Liang; Wang, Jui-Ling; Lee, Cheng-Chung; Cheng, Hsiu-Yi; Wen, Wu-Che; Cheng, Howard Hao-Yu; Chen, Miles Chih-Ming

    2014-10-01

    Antroquinonol is the smallest anticancer molecule isolated from Antrodia camphorata thus far. The ubiquinone-like structure of Antroquinonol exhibits a broad spectrum of activity against malignancies in vivo and in vitro. However, the mechanism of action of Antroquinonol remains unclear. Here, we provide evidence that Antroquinonol plays a role in the inhibition of Ras and Ras-related small GTP-binding protein functions through the inhibition of protein isoprenyl transferase activity in cancer cells. Using cell line-based assays, we found that the inactive forms of Ras and Rho proteins were significantly elevated after treatment with Antroquinonol. We also demonstrated that Antroquinonol binds directly to farnesyltransferase and geranylgeranyltransferase-I, which are key enzymes involved in activation of Ras-related proteins, and inhibits enzymes activities in vitro. Furthermore, a molecular docking analysis illustrated that the isoprenoid moiety of Antroquinonol binds along the hydrophobic cavity of farnesyltransferase similar to its natural substrate, farnesyl pyrophosphate. In contrast, the ring structure of Antroquinonol lies adjacent to the Ras-CAAX motif-binding site on farnesyltransferase. The molecular docking study also showed a reasonable correlation with the IC50 values of Antroquinonol analogues. We also found that the levels of LC3B-II and the autophagosome-associated LC3 form were also significantly increased in H838 after Antroquinonol administration. In conclusion, Antroquinonol inhibited Ras and Ras-related GTP-binding protein activation through inhibition of protein isoprenyl transferase activity, leading to activation of autophagy and associated mode of cell death in cancer cells. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  14. Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

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    Jennifer S. Liu

    2012-11-01

    Full Text Available Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

  15. 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

  16. K-ras mutation promotes ionizing radiation-induced invasion and migration of lung cancer in part via the Cathepsin L/CUX1 pathway.

    Science.gov (United States)

    Wang, Long; Zhao, Yifan; Xiong, Yajie; Wang, Wenjuan; Fei, Yao; Tan, Caihong; Liang, Zhongqin

    2018-01-15

    K-ras mutation is involved in cancer progression including invasion and migration, but the underlying mechanism is not yet clear. Cathepsin L is a lysosomal cysteine protease and has recently been associated with invasion and migration in human cancers when it is overexpressed. Our recent studies have shown that ionizing radiation (IR) enhanced expression of cathepsin L and increased invasion and migration of tumor cells, but the molecular mechanism is still unclear. In the present study, the effects of K-ras mutation and IR induced invasion and migration of lung cancer as well as the underlying mechanisms were investigated both in vitro and in vivo. Firstly, the levels of cathepsin L and epithelial mesenchymal transition (EMT) marker proteins remarkably changed in A549 (K-ras mutant) after irradiation compared with H1299 (K-ras wild), thereby promoting invasion and migration. Additionally, cathepsin L and its downstream transcription factor CUX1/p110 were increased after irradiation in A549 transfected with CUX1/p200, and the proteolytic processing of CUX1 by cathepsin L was remarkably increased after co-transfection of CUX1/p200 and cathepsin L-lentivirus in H1299. In addition, delivery of a mutant K-ras (V12) into HEK 293 cells stimulated EMT after irradiation due to the accumulation of cathepsin L. Moreover, mutated K-ras was associated with IR-induced cathepsin L and EMT in BALB/c nude mice. Finally, the level of cathepsin L expression was higher in samples carrying a K-ras mutation than in wild-type K-ras samples and the mesenchymal markers were upregulated in the samples of mutant K-ras, whereas the epithelial marker E-cadherin was downregulated in non-small cell lung cancers tissues. In conclusion, the findings demonstrated that mutated K-ras promotes cathepsin L expression and plays a pivotal role in EMT of human lung cancer. The regulatory effect of IR-induced cathepsin L on lung cancer invasion and migration was partially attributed to the Cathepsin L

  17. H-ras transformation sensitizes volume-activated anion channels and increases migratory activity of NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Schneider, Linda; Klausen, Thomas K; Stock, Christian

    2008-01-01

    The expression of the H-ras oncogene increases the migratory activity of many cell types and thereby contributes to the metastatic behavior of tumor cells. Other studies point to an involvement of volume-activated anion channels (VRAC) in (tumor) cell migration. In this paper, we tested whether...... 35%. Consistent with higher VRAC activity in H-ras than in wild-type fibroblasts, more VRAC blocker is needed to achieve a comparable degree of inhibition of migration. We suggest that H-ras modulates the volume set point of VRAC and thus facilitates transient changes of cell volume required...

  18. Prognostic implications of c-Ki-ras2 mutations in patients with advanced colorectal cancer treated with 5-fluorouracil and interferon: a study of the eastern cooperative oncology group (EST 2292)

    Science.gov (United States)

    Wadler, S; Bajaj, R; Neuberg, D; Agarwal, V; Haynes, H; Benson, A B

    1997-01-01

    Mutations in c-Ki-ras2 (ras) occur in about 40% of patients with colorectal cancers and occur early in the pathogenesis of this disease. To evaluate the prognostic value of mutations in ras, the Eastern Cooperative Oncology Group (ECOG) conducted a retrospective study (EST 2292) to determine the frequency of mutations in patients with advanced colorectal cancer, and to determine whether ras mutations were associated with altered response to therapy and survival. Patients were enrolled from four studies: P-Z289, an ECOG phase II trial of 5-fluorouracil (5-FU) and interferon (IFN) in patients with advanced colorectal cancer; P-Z991, an ECOG phase I trial of 5-FU and IFN in patients with advanced malignancies; and two trials from the Albert Einstein College of Medicine in patients with advanced colorectal cancer treated with 5-FU and either IFN-alpha or IFN-beta. All patients had advanced colorectal carcinoma and had sufficient histologic material available for analysis for the presence and type of ras, using polymerase chain reaction and dot-blot analysis with sets of probes sufficient to detect all the common mutations of ras at codons 12, 13, and 61. Seventy-two patients were enrolled in this trial. Mutations in ras were detected in 25 (35%), including 17 (23%) in codon 12, four (6%) in codon 13, and four (6%) in codon 61. There was no correlation between the presence of a ras mutation and age, sex, Dukes' stage, histology, or tumor markers. Thirty-one of 72 patients (43%) responded to therapy with 5-FU and IFN, and 10 of 31 responders (32%) and 15 of 41 nonresponders (37%) had mutations in ras. There was no difference in response rates or overall survival between the groups with and without ras mutations. It is unlikely that ras mutations will have significant prognostic value for either response to therapy or survival in patients with colorectal carcinomas treated with 5-FU and IFN.

  19. The Ras superfamily G-proteins.

    Science.gov (United States)

    Tetlow, Ashley L; Tamanoi, Fuyuhiko

    2013-01-01

    The Ras superfamily G-proteins are monomeric proteins of approximately 21kDa that act as a molecular switch to regulate a variety of cellular processes. The structure of the Ras superfamily G-proteins, their regulators as well as posttranslational modification of these proteins leading to their membrane association have been elucidated. The Ras superfamily G-proteins interact at their effector domains with their downstream effectors via protein-protein interactions. Mutational activation or overexpression of the Ras superfamily G-proteins has been observed in a number of human cancer cases. Over the years, a variety of approaches to inhibit the Ras superfamily G-proteins have been developed. These different approaches are discussed in this volume. © 2013 Elsevier Inc. All rights reserved.

  20. Consequences of RAS and MAPK activation in the ovary: the good, the bad and the ugly

    Science.gov (United States)

    Fan, Heng-Yu; Liu, Zhilin; Mullany, Lisa K.; Richards, JoAnne S.

    2012-01-01

    This review summarizes studies providing evidence 1) that endogenous RAS activation regulates important physiological events during ovulation and luteinization 2) that expression of the mutant, active KRASG12D in granulosa cells in vivo causes abnormal follicle growth arrest leading to premature ovarian failure and 3) that KRASG12D expression in ovarian surface epithelial (OSE) cells renders them susceptible to the pathological outcome of transformation and tumor formation. These diverse effects of RAS highlight how critical its activation is linked to cell- and stage-specific events in the ovary that control normal processes and that can also lead to altered granulosa cell and OSE cell fates. PMID:22197887

  1. Imaging of Ras/Raf activity induced by low energy laser irradiation in living cell using FRET

    Science.gov (United States)

    Wang, Fang; Chen, Tong-Sheng; Xing, Da

    2005-01-01

    Ras/Raf signaling pathway is an important signaling pathway that governs cell proliferation, differential and apoptosis. Low-energy laser irradiation (LELI) was found to modulate various processes. Generally, cell proliferation is induced by low doses LELI and apoptosis is induced by high doses LELI. Mechanism of biological effect of LELI has not been clear. Recently, activation of MEK (mitogen-activated protein kinase) and ERK (extracellular-signal-regulated kinase), which are downstream protein kinases of Ras/Raf, are observed during LELI-induced cell proliferation by immunoprecipitation and western blot analysis. RaichuRas reporter consisting of fusions of H-ras, the Ras-binding domain of Raf (RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). Therefore, intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) was transfected with the plasmid (pRaichuRas) and then treated with LELI at dose of 60J/cm2. Effect of LELI on Ras/Raf in physiological condition of living cells was observed by fluorescence resonance energy transfer (FRET) technique during lung adenocarcinoma cell apoptosis induced by high dose (60J/cm2) LELI. Experimental results showed that after high dose LELI treatment, the binding of Ras and Raf decreases obviously, Ras/Raf signaling pathway deregulates and cell apoptosis occurs.

  2. 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...

  3. Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling

    International Nuclear Information System (INIS)

    Kolokoltsova, Olga A.; Domina, Aaron M.; Kolokoltsov, Andrey A.; Davey, Robert A.; Weaver, Scott C.; Watowich, Stanley J.

    2008-01-01

    Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection

  4. 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.

  5. Incidência de mutação no códon 12 do protoncogene K-ras em carcinoma de próstata humana em uma amostra da população brasileira The incidence of mutation in codon 12 of the k-ras proto-oncogene in human prostate carcinoma with a Brazilian population sample

    Directory of Open Access Journals (Sweden)

    José Raul Cisternas Gajardo

    2004-06-01

    Full Text Available Com o intuito de estudar a participação do gene ras ativado na tumorigênese humana, pesquisamos a freqüência de mutação pontual no códon 12 do gene K-ras em espécimes cirúrgicos de pacientes portadores de câncer de próstata. Foi utilizado um grupo controle de pacientes com hiperplasia prostática benigna (HPB. Os cortes destinados ao estudo foram submetidos a extração do DNA pelo método da proteinase K. A amplificação do fragmento isolado foi obtida pela reação em cadeia de polimerase seguida por clivagem, utilizando-se a enzima de restrição Mval. A eletroforese em gel de agarose permitiu a verificação da presença de mutações. Constatamos a presença de mutação no códon 12 do gene K-ras em dois dos 15 carcinomas de próstata estudados (13,3%, sendo que nenhuma em pacientes com HPB. A ocorrência de mutação de 13,3% na amostra da população brasileira analisada caracteriza uma incidência intermediária entre as populações japonesa e americana. É pouco provável que a mutação isolada do K-ras seja um evento significativo na carcinogênese prostática nesta população.Aiming to study the participation of activated ras gene on the human tumorogenesis, we have researched the frequency of a punctual mutation in codon 12 of the K-Ras oncogene in surgical specimens of patients with prostate cancer. We used control group of patients with benign prostatic hyperplasia. The pieces addressed to the study was submitted to the extraction of DNA by the proteina kinase method. The isolated fragment amplification was obtained using a polymerase chain reaction followed by clevage with Mval restriction enzime. The electrophoresis process allowed the verification of the mutation presence. We noticed the presence of mutation in codon 12 of the K-ras oncogene in two of 15 prostate carcinomas studied (13.3%. None of the patients with prostatic benign prostatic hyperplasia presented any mutation. The mutation incidence of 13.3% on

  6. 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.

  7. Activation of myc and ras oncogenes in radiation-induced rat skin carcinogenesis

    International Nuclear Information System (INIS)

    Sawey, M.J.

    1987-01-01

    The radiation-induced rat skin carcinogenesis model system was used to examine oncogene activation from both the ras and myc complementation groups. Six of twelve tumors tested were positive in the NIH3T3 transfection assay and the rat K-ras oncogene was found to be activated in representative transformed foci. Southern analysis of the tumor DNAs revealed evidence for c-myc gene amplification and extensive restriction fragment polymorphisms in 9 of the 12 tumors. The appearance of several new c-myc restriction fragments was seen after digestion with Eco RI, Hind III, and Bam HI, and may represent gene rearrangement or activation of a c-myc homologous gene. Rehybridization of the filters with N-myc, K-ras, and H-ras probes failed to detect any differences compared to normal rat DNA. In addition, when probes from other oncogenes known to be activated by gene amplification and translocation mechanisms, were hybridized to these RAD tumor DNAs, no differences in either band intensity nor restriction fragment pattern were found

  8. 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

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

    Science.gov (United States)

    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. Monitoring the effect of first line treatment in RAS/RAF mutated metastatic colorectal cancer by serial analysis of tumor specific DNA in plasma

    DEFF Research Database (Denmark)

    Brenner Thomsen, Caroline Emilie; Hansen, T F; Andersen, R F

    2018-01-01

    BACKGROUND: Precision medicine calls for an early indicator of treatment efficiency. Circulating tumor DNA (ctDNA) is a promising marker in this setting. Our prospective study explored the association between disease development and change of ctDNA during first line chemotherapy in patients...... with RAS/RAF mutated metastatic colorectal cancer (mCRC). METHODS: The study included 138 patients with mCRC receiving standard first line treatment. In patients with RAS/RAF mutated tumor DNA the same mutation was quantified in the plasma using droplet digital PCR. The fractional abundance of ct...... on continuous treatment. The first increase in ctDNA level occurred at a median of 51 days before radiologically confirmed progression. CONCLUSIONS: The results indicate that the ctDNA level holds potential as a clinically valuable marker in first line treatment of mCRC. A rapid decrease was associated...

  11. Ras induces NBT-II epithelial cell scattering through the coordinate activities of Rac and MAPK pathways.

    Science.gov (United States)

    Edme, Natacha; Downward, Julian; Thiery, Jean-Paul; Boyer, Brigitte

    2002-06-15

    Cell dissociation and cell migration are the two main components of epithelium-mesenchyme transitions (EMT). We previously demonstrated that Ras is required for the accomplishment of both of these processes during the EGF-induced EMT of the NBT-II rat carcinoma cell line in vitro. In this study, we examined the downstream targets of Ras that are responsible for the dissociation and motility of NBT-II cells. Overexpression of activated forms of c-Raf and MEK1 (a component of the mitogen-activated protein kinase pathway, MAPK) led to cell dissociation, as inferred by the loss of desmosomes from the cell periphery. By contrast, active PI3K, RalA and RalB did not induce desmosome breakdown. The MEK1 inhibitor PD098059 inhibited EGF- and Ras-induced cell dispersion, whereas the PI3K inhibitor LY294002 had no effect. Accordingly, among the partial loss-of-function mutants of Ras (RasV12) that were used to distinguish between downstream targets of Ras, we found that the Raf-specific Ras mutants RasV12S35 and RasV12E38 induced cell dissociation. The PI3K- and RalGDS-activating Ras mutants had, in contrast, no effect on cell dispersion. However, MEK1 was unable to promote cell motility, whereas RasV12S35 and RasV12E38 induced cell migration, suggesting that another Ras effector was responsible for cell motility. We found that the small GTPase Rac is necessary for EGF-mediated cell dispersion since overexpression of a dominant-negative mutant of Rac1 (Rac1N17) inhibited EGF-induced NBT-II cell migration. All stimuli that promoted cell migration also induced Rac activation. Finally, coexpression of active Rac1 and active MEK1 induced the motility of NBT-II cells, suggesting that Ras mediates NBT-II cell scattering through the coordinate activation of Rac and the Raf/MAPK pathway.

  12. Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover

    DEFF Research Database (Denmark)

    Hong, Xin; Nguyen, Thanh Hung; Chen, Qingfeng

    2014-01-01

    Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor...... the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of Ras(V12) depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1....

  13. Prognostic effects of TERT promoter mutations are enhanced by coexistence with BRAF or RAS mutations and strengthen the risk prediction by the ATA or TNM staging system in differentiated thyroid cancer patients.

    Science.gov (United States)

    Song, Young Shin; Lim, Jung Ah; Choi, Hoonsung; Won, Jae-Kyung; Moon, Jae Hoon; Cho, Sun Wook; Lee, Kyu Eun; Park, Young Joo; Yi, Ka Hee; Park, Do Joon; Seo, Jeong-Sun

    2016-05-01

    Recent reports suggest that mutations in the promoter of the gene encoding telomerase reverse transcriptase (TERT) affect thyroid cancer outcomes. In all, 551 patients with differentiated thyroid cancer (DTC) enrolled in this study. The median follow-up duration was 4.8 years (interquartile range, 3.4-10.6 years). TERT promoter mutations were detected in 25 DTCs (4.5%): 2.8% in neither BRAF-mutated nor RAS-mutated tumors, 4.8% in BRAF-mutated tumors, and 11.3% in RAS-mutated tumors. Moreover, they were frequently observed in American Thyroid Association (ATA) high-risk and TNM stage III/IV groups (9.1% and 12.9%, respectively). The coexistence of BRAF or RAS with TERT promoter mutations increased aggressive clinicopathologic features, recurrence (hazard ratio [HR] for BRAF, 4.64; 95% confidence interval [CI], 1.42-15.18; HR for RAS, 5.36; 95% CI, 1.20-24.02), and mortality (HR for BRAF, 15.13; 95% CI, 1.55-148.23; HR for RAS, 14.75; 95% CI, 1.30-167.00), even after adjustments for the age at diagnosis and sex, although the significance was lost after additional adjustments for pathologic characteristics. Furthermore, TERT promoter mutations significantly increased the risk of both recurrence and mortality in the ATA high-risk (HR for recurrence, 5.79; 95% CI, 2.07-16.18; HR for mortality, 16.16; 95% CI, 2.10-124.15) and TNM stage III/IV groups (HR for recurrence, 3.60; 95% CI, 1.19-10.85; HR for mortality, 9.06; 95% CI, 2.09-39.26). The coexistence of BRAF or RAS mutations enhanced the prognostic effects of TERT promoter mutations. Furthermore, TERT promoter mutations strengthened the predictions of mortality and recurrence by the ATA and TNM staging systems, particularly for high-risk patients with DTC. Cancer 2016;122:1370-1379. © 2016 American Cancer Society. © 2016 American Cancer Society.

  14. Screening for circulating RAS/RAF mutations by multiplex digital PCR

    DEFF Research Database (Denmark)

    Andersen, Rikke Fredslund; Jakobsen, Anders

    2016-01-01

    by technical challenges primarily due to the low levels of ctDNA in patients with localized disease and in patients responding to therapy. The approach presented here is a multiplex digital PCR method of screening for 31 mutations in the KRAS, NRAS, BRAF, and PIK3CA genes in the plasma. The upper level...

  15. Detection of up to 65% of Precancerous Lesions of the Human Colon and Rectum by Mutation Analysis of APC, K-Ras, B-Raf and CTNNB1

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, Mandy; Scholtka, Bettina, E-mail: scholtka@uni-potsdam.de [Chair of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur- Scheunert-Allee 114-116, 14558 Nuthetal (Germany); Gottschalk, Uwe [Maria Heimsuchung Caritas-Klinik Pankow, Breite Straße 46/47, 13187 Berlin (Germany); Faiss, Siegbert [III. Medizinische Abteilung - Gastroenterologie und Hepatologie, Asklepios Klinik Barmbek, Rubenkamp 220, 22291 Hamburg (Germany); Schatz, Daniela; Berghof-Jäger, Kornelia [BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam (Germany); Steinberg, Pablo, E-mail: scholtka@uni-potsdam.de [Chair of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur- Scheunert-Allee 114-116, 14558 Nuthetal (Germany); Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover (Germany)

    2010-12-29

    In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form. CTNNB1 and B-Raf were screened by PCR-single-strand conformation polymorphism analysis, K-Ras by restriction fragment length polymorphism analysis and the APC gene mutation cluster region (codons 1243–1567) by direct DNA sequencing. APC mutations were only detected in 10% of the serrated lesions but in 34% of the adenomas. Twenty percent of the serrated lesions and 14% of the adenomas carried a mutated K-Ras. B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas. CTNNB1 was altered in 12% of the adenomas, but not in serrated lesions. By using the above gene marker panel it could be shown that 65% of the serrated lesions and 61% of the adenomas carried at least one of the four genes in a mutated form. Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer.

  16. Detection of up to 65% of Precancerous Lesions of the Human Colon and Rectum by Mutation Analysis of APC, K-Ras, B-Raf and CTNNB1

    International Nuclear Information System (INIS)

    Schneider, Mandy; Scholtka, Bettina; Gottschalk, Uwe; Faiss, Siegbert; Schatz, Daniela; Berghof-Jäger, Kornelia; Steinberg, Pablo

    2010-01-01

    In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form. CTNNB1 and B-Raf were screened by PCR-single-strand conformation polymorphism analysis, K-Ras by restriction fragment length polymorphism analysis and the APC gene mutation cluster region (codons 1243–1567) by direct DNA sequencing. APC mutations were only detected in 10% of the serrated lesions but in 34% of the adenomas. Twenty percent of the serrated lesions and 14% of the adenomas carried a mutated K-Ras. B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas. CTNNB1 was altered in 12% of the adenomas, but not in serrated lesions. By using the above gene marker panel it could be shown that 65% of the serrated lesions and 61% of the adenomas carried at least one of the four genes in a mutated form. Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer

  17. Loss of RASSF1A Expression in Colorectal Cancer and Its Association with K-ras Status

    Directory of Open Access Journals (Sweden)

    Dan Cao

    2013-01-01

    Full Text Available Background. The RAS-association domain family 1 A (RASSF1A is a classical member of RAS effectors regulating cell proliferation and apoptosis. Loss of RASSF1A expression may shift the balance towards a growth-promoting effect without the necessity of activating K-ras mutations. Its potential association with K-ras mutations in colorectal cancer (CRC is unclear. Methods. RASSF1A expression was examined in normal mucosa, adenoma, and tumor tissues of colon and rectum, respectively. We examined the association of RASSF1A expression, mutations of K-ras, and EGFR status in 76 primary CRCs. The relationship between clinicopathological characteristics and RASSF1A expression was also analyzed. Results. RASSF1A expression level decreased progressively in normal mucosa, adenoma and, tumor tissues, and the loss of RASSF1A expression occurred more frequently in tumor tissues. Of 76 primary CRCs, loss of RASSF1A expression and/or K-ras mutations were detected in 77% cases. Loss of RASSF1A expression was more frequent in K-ras wild-type than in mutation cases (63% versus 32%, . Conclusions. Our study indicates that loss of RASSF1A may be involved in pathogenesis of CRC, its expression was found predominantly in K-ras wild-type CRCs, suggesting that it may be another way of affecting RAS signaling, in addition to K-ras mutations.

  18. Assessment of brain activation regulation in first graders via RAN / RAS test

    Directory of Open Access Journals (Sweden)

    Tatiana V. Akhutina

    2015-03-01

    Full Text Available RAN / RAS test (Rapid Automatized Naming / Rapid Alternating Stimulus has been used successfully used by many psychologists, primarily to predict the risk of dyslexia, as it includes a language component and requires good visual-verbal connections. However, The research demonstrates that the low speed of naming is an effective indicator of neurocognitive problems of information processing as a whole (learning difficulties in general, not just reading difficulties. This can be explained in two ways: disturbance of executive mental control and the difficulties of automatization: the difficulties of the transition from a controlled energy-consuming assignment to a less energy-consuming one. The second interpretation describes the problems of energy resources of cognitive functioning. It is similar to weak maintenance of cortical structures activation. However, using the test mentioned herewith for assessing functions of activation regulation has not been described previously. In terms of the Luria’s three functional units of the brain theory the RAN / RAS test can be considered as sensitive to the weakness of the first unit, whose function is to maintain the activity of cortical structures. So the aim of the research is to prove the possibility of assessing the activation regulation using the RAN / RAS test. This issue is relevant because neuropsychological tools for determining the weakness of Unit I functions are not quite sufficient, while the problem of “energetic” unit ranks first in the frequency of occurrence in children with learning disabilities.

  19. K-RAS mutations indicating primary resistance to crizotinib in ALK-rearranged adenocarcinomas of the lung: Report of two cases and review of the literature.

    Science.gov (United States)

    Mengoli, Maria Cecilia; Barbieri, Fausto; Bertolini, Federica; Tiseo, Marcello; Rossi, Giulio

    2016-03-01

    The paradigm of mutually exclusive alterations among oncogenic drivers in non-small-cell lung cancer (NSCLC) is challenged by the increasing evidence of detection of two or more driver alterations in the same tumor using highly-sensitive molecular assays. We report here two cases of ALK-rearranged adenocarcinomas harboring concomitant exon 2 K-RAS mutations (G13D and Q61H). The patients, a 49-year-old smoker man and a 59-year-old non-smoking woman, experienced a rapid disease progression and primary resistance to crizotinib. Search for similar cases in the literature reveals that concomitant K-RAS mutations and ALK rearrangement occur in a subset of NSCLC and seems to lead to resistance to crizotinib. Among 8 similar cases receiving crizotinib previously reported (4 in first line and 4 in second line), 1 had a partial response, 1 stable disease and 6 disease progression. One patient still had progression disease when switching to ceritinib. At the end, K-RAS mutations seem to represent a negative predictive marker in ALK-rearranged adenocarcinomas treated with ALK inhibitor. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. 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)

  1. Ras activation and symmetry breaking during Dictyostelium chemotaxis

    NARCIS (Netherlands)

    Kortholt, Arjan; Keizer-Gunnink, Ineke; Kataria, Rama; Van Haastert, Peter J. M.

    2013-01-01

    Central to chemotaxis is the molecular mechanism by which a shallow spatial gradient of chemoattractant induces symmetry breaking of activated signaling molecules. Previously, we have used Dictyostelium mutants to investigate the minimal requirements for chemotaxis, and identified a basal signaling

  2. Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon

    Science.gov (United States)

    Calcagno, Shelly R.; Li, Shuhua; Colon, Migdalisel; Kreinest, Pamela A.; Thompson, E. Aubrey; Fields, Alan P.; Murray, Nicole R.

    2014-01-01

    Oncogenic K-ras mutations are frequently observed in colon cancers and contribute to transformed growth. Oncogenic K-ras is detected in aberrant crypt foci (ACF), precancerous colonic lesions, demonstrating that acquisition of a K-ras mutation is an early event in colon carcinogenesis. Here, we investigate the role of oncogenic K-ras in neoplastic initiation and progression. Transgenic mice in which an oncogenic K-rasG12D allele is activated in the colonic epithelium by sporadic recombination (K-rasLA2 mice) develop spontaneous ACF that are morphologically indistinguishable from those induced by the colon carcinogen azoxymethane (AOM). Similar neoplastic changes involving the entire colon are induced in transgenic mice constitutively expressing K-rasG12D throughout the colon (LSL-K-rasG12D/Villin-Cre mice). However, the biochemistry and fate of K-ras-induced lesions differ depending upon their location within the colon in these mice. In the proximal colon, K-rasG12D induces increased expression of procarcinogenic protein kinase CβII (PKCβII), activation of the MEK/ERK signaling axis and increased epithelial cell proliferation. In contrast, in the distal colon, K-rasG12D inhibits expression of procarcinogenic PKCβII and induces apoptosis. Treatment of K-rasLA2 mice with AOM leads to neoplastic progression of small ACF to large, dysplastic microadenomas in the proximal, but not the distal colon. Thus, oncogenic K-ras functions differently in the proximal and distal colon of mice, inducing ACF capable of neoplastic progression in the proximal colon, and ACF with little or no potential for progression in the distal colon. Our data indicate that acquisition of a K-ras mutation is an initiating neoplastic event in proximal colon cancer development in mice. PMID:18271008

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

    Science.gov (United States)

    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.

  4. 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...

  5. Second primary or recurrence? Comparative patterns of p53 and K-ras mutations suggest that serous borderline ovarian tumors and subsequent serous carcinomas are unrelated tumors.

    Science.gov (United States)

    Ortiz, B H; Ailawadi, M; Colitti, C; Muto, M G; Deavers, M; Silva, E G; Berkowitz, R S; Mok, S C; Gershenson, D M

    2001-10-01

    The role of serous borderline ovarian tumors (BOTs) in the pathogenesis of serous ovarian carcinomas is unclear. Some authors have compared mutations in serous BOTs to those in serous ovarian carcinomas, but the data on two common oncogenes, p53 and K-ras, remain inconclusive. To further clarify the relationship between the two tumors, we performed mutational analysis on tumors from a set of eight patients who first presented with advanced-stage serous BOTs and later developed grade 1 serous carcinomas. Epithelium from eight advanced-stage serous BOTs and subsequent grade 1 papillary serous carcinomas was microdissected and retrieved using a PixCell laser-capture microscope. Stroma was dissected as an internal control. The DNA was extracted with proteinase K and analyzed by single-strand conformational polymorphism-PCR for p53 and K-ras mutations. Bands with altered motility were analyzed by direct cycle sequencing. Seven of eight patients demonstrated different mutations in the secondary tumor compared with the primary tumor. For three patients, p53 mutations were identified in the BOTs that were absent from the carcinomas, suggesting a nonclonal origin for the carcinomas. These findings are consistent with the hypothesis that advanced-stage serous BOTs represent a distinct pathological entity compared with grade 1 serous epithelial ovarian carcinoma.

  6. A negative-feedback loop regulating ERK1/2 activation and mediated by RasGPR2 phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jinqi [Departments of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Cook, Aaron A.; Bergmeier, Wolfgang [Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Sondek, John, E-mail: sondek@med.unc.edu [Departments of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States)

    2016-05-20

    The dynamic regulation of ERK1 and -2 (ERK1/2) is required for precise signal transduction controlling cell proliferation, differentiation, and survival. However, the underlying mechanisms regulating the activation of ERK1/2 are not completely understood. In this study, we show that phosphorylation of RasGRP2, a guanine nucleotide exchange factor (GEF), inhibits its ability to activate the small GTPase Rap1 that ultimately leads to decreased activation of ERK1/2 in cells. ERK2 phosphorylates RasGRP2 at Ser394 located in the linker region implicated in its autoinhibition. These studies identify RasGRP2 as a novel substrate of ERK1/2 and define a negative-feedback loop that regulates the BRaf–MEK–ERK signaling cascade. This negative-feedback loop determines the amplitude and duration of active ERK1/2. -- Highlights: •ERK2 phosphorylates the guanine nucleotide exchange factor RasGRP2 at Ser394. •Phosphorylated RasGRP2 has decreased capacity to active Rap1b in vitro and in cells. •Phosphorylation of RasGRP2 by ERK1/2 introduces a negative-feedback loop into the BRaf-MEK-ERK pathway.

  7. A vertically-stacked, polymer, microfluidic point mutation analyzer: Rapid, high accuracy detection of low-abundance K-ras mutations

    Science.gov (United States)

    Han, Kyudong; Lee, Tae Yoon; Nikitopoulos, Dimitris E.; Soper, Steven A.; Murphy, Michael C.

    2011-01-01

    Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well-trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user-base for these types of molecular assays, a vertically-stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employed a primary PCR coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers and ExoSAP-IT® purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in ~1 h, an 80% reduction in time compared to conventional bench-top instrumentation. Purifying the post-PCR products with the ExoSAP-IT® enzyme led to optimized LDR performance minimizing false positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25 μL sample, equivalent to DNA from 42 mutant cells. PMID:21771577

  8. 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.

  9. 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.

  10. 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

  11. Molecular Genetic Analysis of Ovarian Brenner Tumors and Associated Mucinous Epithelial Neoplasms: High Variant Concordance and Identification of Mutually Exclusive RAS Driver Mutations and MYC Amplification.

    Science.gov (United States)

    Tafe, Laura J; Muller, Kristen E; Ananda, Guruprasad; Mitchell, Talia; Spotlow, Vanessa; Patterson, Sara E; Tsongalis, Gregory J; Mockus, Susan M

    2016-03-01

    Benign ovarian Brenner tumors often are associated with mucinous cystic neoplasms, which are hypothesized to share a histogenic origin and progression, however, supporting molecular characterization is limited. Our goal was to identify molecular mechanisms linking these tumors. DNA from six Brenner tumors with paired mucinous tumors, two Brenner tumors not associated with a mucinous neoplasm, and two atypical proliferative (borderline) Brenner tumors was extracted from formalin-fixed, paraffin-embedded tumor samples and sequenced using a 358-gene next-generation sequencing assay. Variant calls were compared within tumor groups to assess somatic mutation profiles. There was high concordance of the variants between paired samples (40% to 75%; P tumor pairs showed KRAS hotspot driver mutations specifically in the mucinous tumor. In the two paired samples that lacked KRAS mutations, MYC amplification was detected in both of the mucinous and the Brenner components; MYC amplification also was detected in a third Brenner tumor. Five of the Brenner tumors had no reportable potential driver alterations. The two atypical proliferative (borderline) Brenner tumors both had RAS mutations. The high degree of coordinate variants between paired Brenner and mucinous tumors supports a shared origin or progression. Differences observed in affected genes and pathways, particularly involving RAS and MYC, may point to molecular drivers of a divergent phenotype and progression of these tumors. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  12. K-Ras-Independent Effects of the Farnesyl Transferase Inhibitor L-744,832 on Cyclin B1/Cdc2 Kinase Activity, G2/M Cell Cycle Progression and Apoptosis in Human Pancreatic Ductal Adenocarcinoma Cell

    Directory of Open Access Journals (Sweden)

    Si Young Song

    2000-05-01

    Full Text Available Pancreatic ductal adenocarcinoma is a highly lethal malignancy that is resistant to traditional cytotoxic therapy. High rates of activating codon 12 K-Ras mutations in this disease have generated considerable interest in the therapeutic application of novel farnesyl transferase inhibitors (FTIs. However, a comprehensive analysis of the effects of FTI treatment on pancreatic cancer cells has not been performed. Treatment of five different human pancreatic cancer cell lines with FTI L744,832 resulted in inhibition of anchorage-dependent growth, with wide variation in sensitivity among different lines. Effective growth inhibition by L-744,832 correlated with accumulation of cells with a tetraploid (4N DNA content and high levels of cyclin B1/cdc2 kinase activity, implying cell cycle arrest downstream from the DNA damage -inducible G2/M cell cycle checkpoint. In addition, sensitive cell lines underwent apoptosis as evidenced by changes in nuclear morphology and internucleosomal DNA fragmentation. L-744,832 at a concentration of 1 µM additively enhanced the cytotoxic effect of ionizing radiation, apparently by overriding G2/M checkpoint activation. The effects of FTI treatment on cell growth and cell cycle regulation were associated with changes in posttranslational processing of H-Ras and N-Ras, but not K-Ras. The results confirm the potential therapeutic efficacy of FTI treatment in pancreatic cancer, and suggest that farnesylated proteins other than K-Ras may act as important regulators of G2/M cell cycle kinetics.

  13. 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.

  14. Clonal origin of multiple lung cancers: K-ras and p53 mutations determined by nonradioisotopic single-strand conformation polymorphism analysis.

    Science.gov (United States)

    Lau, D H; Yang, B; Hu, R; Benfield, J R

    1997-08-01

    Disease stage is the most important factor in determining prognosis and treatment of lung cancer. Staging of lung cancer is complicated by presentation of multiple pulmonary malignant lesions with a similar histology. It is a dilemma to decide if these lesions are synchronous primaries arising from different malignant clones or metastases from a single clone. Lung cancer is associated with multiple genetic abnormalities including mutations of K-ras and p53, which are believed to occur prior to onset of metastasis. To determine the clonal origin of multiple pulmonary malginant nodules, we analyzed point-mutations of K-ras and p53 by microdissection, polymerase chain reactions (PCR), nonradioisotopic single-strand conformation polymorphism (SSCP) analysis, and DNA sequencing. Each pulmonary lesion was microdissected from paraffin slides. Genomic DNA was amplified by two sequential PCRs followed by electrophoresis in a minigel and silver staining. Deoxyribonucleic acid sequencing was performed if necessary to confirm a mutation found upon SSCP analysis. Applying this molecular approach, we were able to differentiate the clonal origins of multiple malignant nodules of the lung as exemplified by the two cases presented.

  15. 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

  16. Gene expression profiling by DNA microarray analysis in mouse embryonic fibroblasts transformed by rasV12 mutated protein and the E1A oncogene

    Directory of Open Access Journals (Sweden)

    Berthezene Patrice

    2003-03-01

    that, in human cancers, proteases, protease inhibitors and angiogenic factors could be regulated through a mechanism disconnected from ras activation. Conclusions This study established a first catalog of genes whose expression is altered upon fibroblast transformation by rasV12/E1A. This catalog is representative of the genome but not exhaustive, because only one third of expressed genes was examined. In addition, contribution to ras signaling of post-transcriptional and post-translational modifications was not addressed. Yet, the information gathered should be quite useful to future investigations on the molecular mechanisms of oncogenic transformation.

  17. Detection of codon 12 mutation in the k-ras oncogene in pancreatic tumors Detecção de mutação no códon 12 do oncogene K-ras em tumores pancreáticos

    Directory of Open Access Journals (Sweden)

    Márcia Saldanha Kubrusly

    1999-02-01

    Full Text Available Mutations at codons 12, 13, or 61 of the H-ras, K-ras, and N-ras have been detected in human neoplasias by a variety of techniques. Some of these techniques are very sensitive and can detect K-ras mutation in 90% of the cases of pancreatic adenocarcinomas. We analyzed 11 samples of pancreatic adenocarcinoma, three samples of pancreatic mucinous cystadenoma, and two samples without tumors in formalin-fixed paraffin embedded tissue sections. K-ras mutations at codon 12 were detected by a two-step PCR-enriched technique in all the samples of pancreatic adenocarcinoma, but not in cystadenoma or control samples. This technique may be useful for early detection of pancreatic cancer.Muitos dos oncogenes detectados em neoplasias malignas humanas pertencem à família do gene ras. Mutações nos códons 12, 13 ou 61 em um dos tres genes ras; H-ras, K-ras e N-ras, convertem esses genes em oncogenes ativos. Ensaios rápidos para detecção dessas mutações pontuais, tais como a reação em cadeia de polimertização têm sido desenvolvidos nas últimas décadas e usados para investigar o papel dos genes ras mutados na patogênese de tumores humanos. As mutações no gene ras podem ser encontradas numa variedade de tipos de tumores. Incidências mais altas aparecem em adenocarcinomas do pâncreas (90% e cólon (50%. Analisamos 11 amostras de tumores primários de pâncreas com diferentes metástases, três amostras de cistadenoma mucinoso e dois casos de ausência de tumor de material incluído em parafina, de onde extraímos o DNA para realização das amplificações. Os resultados mostraram que todos os casos de tumores apresentaram a banda de 135 pares de bases correspondente ao gene mutado e para os normais, a banda característica de 106 pares de bases. Nos três casos de cistadenoma mucinosos, não detectamos a banda de 135 pares de bases , apenas a banda de 106 pares de bases.

  18. Preoperative chemoradiation with capecitabine, irinotecan and cetuximab in rectal cancer: significance of pre-treatment and post-resection RAS mutations.

    Science.gov (United States)

    Gollins, Simon; West, Nick; Sebag-Montefiore, David; Myint, Arthur Sun; Saunders, Mark; Susnerwala, Shabbir; Quirke, Phil; Essapen, Sharadah; Samuel, Leslie; Sizer, Bruce; Worlding, Jane; Southward, Katie; Hemmings, Gemma; Tinkler-Hundal, Emma; Taylor, Morag; Bottomley, Daniel; Chambers, Philip; Lawrie, Emma; Lopes, Andre; Beare, Sandy

    2017-10-24

    The influence of EGFR pathway mutations on cetuximab-containing rectal cancer preoperative chemoradiation (CRT) is uncertain. In a prospective phase II trial (EXCITE), patients with magnetic resonance imaging (MRI)-defined non-metastatic rectal adenocarinoma threatening/involving the surgical resection plane received pelvic radiotherapy with concurrent capecitabine, irinotecan and cetuximab. Resection was recommended 8 weeks later. The primary endpoint was histopathologically clear (R0) resection margin. Pre-planned retrospective DNA pyrosequencing (PS) and next generation sequencing (NGS) of KRAS, NRAS, PIK3CA and BRAF was performed on the pre-treatment biopsy and resected specimen. Eighty-two patients were recruited and 76 underwent surgery, with R0 resection in 67 (82%, 90%CI: 73-88%) (four patients with clinical complete response declined surgery). Twenty-four patients (30%) had an excellent clinical or pathological response (ECPR). Using NGS 24 (46%) of 52 matched biopsies/resections were discrepant: ten patients (19%) gained 13 new resection mutations compared to biopsy (12 KRAS, one PIK3CA) and 18 (35%) lost 22 mutations (15 KRAS, 7 PIK3CA). Tumours only ever testing RAS wild-type had significantly greater ECPR than tumours with either biopsy or resection RAS mutations (14/29 [48%] vs 10/51 [20%], P=0.008), with a trend towards increased overall survival (HR 0.23, 95% CI 0.05-1.03, P=0.055). This regimen was feasible and the primary study endpoint was met. For the first time using pre-operative rectal CRT, emergence of clinically important new resection mutations is described, likely reflecting intratumoural heterogeneity manifesting either as treatment-driven selective clonal expansion or a geographical biopsy sampling miss.

  19. 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

  20. 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

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

    Science.gov (United States)

    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.

  2. Comparative study of mutations in SNP loci of K-RAS, hMLH1 and hMSH2 genes in neoplastic intestinal polyps and colorectal cancer.

    Science.gov (United States)

    Yan, Zhi-Hui; Cui, Li-Hong; Wang, Xiao-Hui; Li, Chao; He, Xing

    2014-12-28

    To clarify the molecular mechanism involved in pathogenesis of colorectal cancer as well as clinical significance of genetic analysis of histological samples. A total of 480 blood and tissue specimens were collected in our hospital from January 2011 to October 2012. In the observation group, there were 120 blood specimens and 120 intestinal tract tissue specimens collected from patients with neoplastic intestinal polyps. In the control group I there were 80 blood specimens and 80 intestinal tract tissue specimens collected from patients with colorectal cancer. In the control group II there were 40 blood specimens and 40 intestinal tract tissue specimens collected from healthy individuals. The gene segments were amplified using PCR and DNA gel electrophoresis along with DNA sequence analysis were employed for the detection of the following single nucleotide polymorphisms (SNPs): K-RAS codons 12 and 13; hMLH1 (human mutS homolog 1) gene missense mutation at Va1384Asp; hMSH2 (human mutS homolog 2) gene missense mutation at 2783C/A. The mutation rate of the SNP at Va1384Asp locus of the hMLH1 gene from blood and tissue specimens in the observation group showed no statistical difference from those in the control group I. The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene were significantly lower in the observation group than in the control group I (χ(2) = 15.476, 29.670, 10.811, 16.618, 33.538, 7.898, P polyps and colorectal cancer in terms of the mechanisms involved in the pathogenesis.

  3. Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling.

    Science.gov (United States)

    Dhawan, Neil S; Scopton, Alex P; Dar, Arvin C

    2016-09-01

    Deregulation of the Ras-mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes. Kinase suppressor of Ras (KSR) is a MAPK scaffold that is subject to allosteric regulation through dimerization with RAF. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras-MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.

  4. H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes

    DEFF Research Database (Denmark)

    Espada, J; Pérez-Moreno, M; Braga, V M

    1999-01-01

    keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and alpha-catenin and relocalization of beta-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong...

  5. 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.

  6. 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.

  7. Evolutionary Analyses of Entire Genomes Do Not Support the Association of mtDNA Mutations with Ras/MAPK Pathway Syndromes

    Science.gov (United States)

    Cerezo, María; Balboa, Emilia; Heredia, Claudia; Castro-Feijóo, Lidia; Rica, Itxaso; Barreiro, Jesús; Eirís, Jesús; Cabanas, Paloma; Martínez-Soto, Isabel; Fernández-Toral, Joaquín; Castro-Gago, Manuel; Pombo, Manuel; Carracedo, Ángel; Barros, Francisco

    2011-01-01

    Background There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS) and related disorders (such as LEOPARD, neurofibromatosis type 1), although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA) genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM), which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45), most of them classified as NS patients (n = 42). Methods/Principal Findings The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg) patterns of a typical Iberian dataset (including hgs H, T, J, and U). Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5) are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. Conclusions/Significance As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS. PMID:21526175

  8. Comparison of the novel quantitative ARMS assay and an enriched PCR-ASO assay for K-ras mutations with conventional cytology on endobiliary brush cytology from 312 consecutive extrahepatic biliary stenoses.

    NARCIS (Netherlands)

    Heek, N.T. van; Clayton, S.J.; Sturm, P.D.J.; Walker, J.; Gouma, D.J.; Noorduyn, L.A.; Offerhaus, G.J.; Fox, J.C.

    2005-01-01

    BACKGROUND: Extrahepatic biliary stenosis (EBS) has malignant and benign causes. Patients with EBS are at risk of having or developing malignancy. Accurate diagnostic tests for early detection and surveillance are needed. The sensitivity of biliary cytology for malignancy is low. K-ras mutation

  9. Comparison of the novel quantitative ARMS assay and an enriched PCR-ASO assay for K-ras mutations with conventional cytology on endobiliary brush cytology from 312 consecutive extrahepatic biliary stenoses

    NARCIS (Netherlands)

    van Heek, N. T.; Clayton, S. J.; Sturm, P. D. J.; Walker, J.; Gouma, D. J.; Noorduyn, L. A.; Offerhaus, G. J. A.; Fox, J. C.

    2005-01-01

    Background: Extrahepatic biliary stenosis (EBS) has malignant and benign causes. Patients with EBS are at risk of having or developing malignancy. Accurate diagnostic tests for early detection and surveillance are needed. The sensitivity of biliary cytology for malignancy is low. K-ras mutation

  10. 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

  11. Targeting Ras signaling in AML: RALB is a small GTPase with big potential.

    Science.gov (United States)

    Pomeroy, Emily J; Eckfeldt, Craig E

    2017-07-06

    Acute myeloid leukemia (AML) is a devastating malignancy for which novel treatment approaches are desperately needed. Ras signaling is an attractive therapeutic target for AML because a large proportion of AMLs have mutations in NRAS, KRAS, or genes that activate Ras signaling, and key Ras effectors are activated in virtually all AML patient samples. This has inspired efforts to develop Ras-targeted treatment strategies for AML. Due to the inherent difficulty and disappointing efficacy of targeting Ras proteins directly, many have focused on inhibiting Ras effector pathways. Inhibiting the major oncogenic Ras effectors, the mitogen-activated protein kinase (MAPK) and/or phosphatidylinositiol-3-kinase (PI3K) pathways, has generally demonstrated modest efficacy for AML. While this may be in part related to functional redundancy between these pathways, it is now clear that other Ras effectors have key oncogenic roles. Specifically, the Ras-like (Ral) GTPases have emerged as critical mediators of Ras-driven transformation and AML cell survival. Our group recently uncovered a critical role for RALB signaling in leukemic cell survival and a potential mediator of relapse following Ras-targeted therapy in AML. Furthermore, we found that RALB signaling is hyperactivated in AML patient samples, and inhibiting RALB has potent anti-leukemic activity in preclinical AML models. While key questions remain regarding the importance of RALB signaling across the genetically diverse spectrum of AML, the specific mechanism(s) that promotes leukemic cell survival downstream of RALB, and how to pharmacologically target RALB signaling effectively - RALB has emerged as a critical Ras effector and potential therapeutic target for AML.

  12. CT radiogenomic characterization of EGFR, K-RAS, and ALK mutations in non-small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Rizzo, Stefania; Rampinelli, Cristiano [European Institute of Oncology, Department of Radiology, Milan (Italy); Petrella, Francesco; Spaggiari, Lorenzo [European Institute of Oncology, Department of Thoracic Surgery, Milan (Italy); Buscarino, Valentina; De Maria, Federica [University of Milan, Department of Health Sciences, Milan (Italy); Raimondi, Sara [European Institute of Oncology, Department of Epidemiology and Biostatistics, Milan (Italy); Barberis, Massimo; Fumagalli, Caterina [European Institute of Oncology, Department of Pathology, Milan (Italy); Spitaleri, Gianluca; De Marinis, Filippo [European Institute of Oncology, Department of Thoracic Oncology, Milan (Italy); Bellomi, Massimo [European Institute of Oncology, Department of Radiology, Milan (Italy); University of Milan, Department of Health Sciences, Milan (Italy)

    2016-01-15

    To assess the association between CT features and EGFR, ALK, KRAS mutations in non-small cell lung cancer. Patients undergoing chest CT and testing for the above gene mutations were included. Qualitative evaluation of CTs included: lobe; lesion diameter; shape; margins; ground-glass opacity; density; cavitation; air bronchogram; pleural thickening; intratumoral necrosis; nodules in tumour lobe; nodules in non-tumour lobes; pleural retraction; location; calcifications; emphysema; fibrosis; pleural contact; pleural effusion. Statistical analysis was performed to assess association of features with each gene mutation. ROC curves for gene mutations were drawn; the corresponding area under the curve was calculated. P-values <0.05 were considered significant. Of 285 patients, 60/280 (21.43 %) were positive for EGFR mutation; 31/270 (11.48 %) for ALK rearrangement; 64/240 (26.67 %) for KRAS mutation. EGFR mutation was associated with air bronchogram, pleural retraction, females, non-smokers, small lesion size, and absence of fibrosis. ALK rearrangements were associated with age and pleural effusion. KRAS mutation was associated with round shape, nodules in non-tumour lobes, and smoking. This study disclosed associations between CT features and alterations of EGFR (air bronchogram, pleural retraction, small lesion size, absence of fibrosis), ALK (pleural effusion) and KRAS (round lesion shape, nodules in non-tumour lobes). (orig.)

  13. Primary murine CD4+ T cells fail to acquire the ability to produce effector cytokines when active Ras is present during Th1/Th2 differentiation.

    Directory of Open Access Journals (Sweden)

    Sujit V Janardhan

    Full Text Available Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.

  14. p53 mutations promote proteasomal activity.

    Science.gov (United States)

    Oren, Moshe; Kotler, Eran

    2016-07-27

    p53 mutations occur very frequently in human cancer. Besides abrogating the tumour suppressive functions of wild-type p53, many of those mutations also acquire oncogenic gain-of-function activities. Augmentation of proteasome activity is now reported as a common gain-of-function mechanism shared by different p53 mutants, which promotes cancer resistance to proteasome inhibitors.

  15. Activation of overexpressed receptors for insulin and epidermal growth factor interferes in mitogenic signaling without affecting the activation of p21ras

    NARCIS (Netherlands)

    Osterop, A.P.R.M.; Medema, R.H.; Ouwens, D.M.; Zon, G.C.M. van der; Möller, W.; Maassen, J.A.

    1994-01-01

    Activated receptors with a tyrosine kinase activity induce a variety of responses like changes in the differentiation and mitogenic status of cells. These responses are mediated in part by p21ras. Some of these activated receptors induce in certain cell types a pronounced, but transient, increase in

  16. Rck1 up-regulates pseudohyphal growth by activating the Ras2 and MAP kinase pathways independently in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chang, Miwha; Kang, Chang-Min; Park, Yong-Sung; Yun, Cheol-Won

    2014-02-21

    Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD+1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N

    2000-01-01

    transfected with expression plasmids encoding constitutively active forms of Ras, Raf, MAP kinase kinases MEK1 and 2, dominant negative forms of Ras and Raf, and the FAK-related nonkinase. Alternatively, PC12-E2 cells were submitted to treatment with antibodies to the fibroblast growth factor (FGF) receptor......, inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...

  18. 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...... in human thyroid carcinoma cell lines and tumor samples, our results, therefore, providing a new molecular profile of the genes involved in thyroid neoplastic transformation....

  19. HOXA3 promotes tumor growth of human colon cancer through activating EGFR/Ras/Raf/MEK/ERK signaling pathway.

    Science.gov (United States)

    Zhang, Xianxiang; Liu, Guangwei; Ding, Lei; Jiang, Tao; Shao, Shihong; Gao, Yuan; Lu, Yun

    2018-03-01

    Homeobox A3 (HOXA3), one of HOX transcription factors, regulates gene expression during embryonic development. HOXA3 expression has been reported to be associated with several cancers; however, its role in colon cancer and underlying mechanism are still unclear. The expression of HOXA3 in 232 paired of human colon tumor and adjacent non-tumorous tissues were measured by qPCR. The relationship between HOXA3 expression and clinical outcomes were analyzed by Kaplan-Meier survival curves analysis. Human colon cancer cell lines HT29 and HTC116 were transfected with HOXA3 siRNA, or HOXA3 expressing vector, and then cell proliferation and apoptosis were assessed, respectively. Western blot was performed to detect the activation of EGFR/Ras/Raf/MEK/ERK signaling pathway. Moreover, HOXA3-overexpressing and HOXA3-suppressing HT29 cells were subcutaneous injected into nod mice to confirm the regulation of HOXA3 on EGFR/Ras/Raf/MEK/ERK signaling in regulating tumor growth. HOXA3 was upregulated in colon tumor tissues and cell lines, and upregulated expression of HOXA3 was associated with low survival rate. Knockdown of HOXA3 suppressed cell viability and clone formation, while induced cell apoptosis. HOXA3 knockdown could not induce the increase of cell apoptosis on the condition of EGFR overexpression. In vivo xenograft studies, HOXA3-suppressing cells showed less tumorigenic. Moreover, HOXA3 knockdown suppressed the activation of EGFR/Ras/Raf/MEK/ERK signaling pathway. To conclude, this study indicated that HOXA3 might act as a promoter of human colon cancer formation by regulating EGFR/Ras/Raf/MEK/ERK signaling pathway. HOXA3 might be a potential therapeutic target for the treatment of colon cancer. © 2017 Wiley Periodicals, Inc.

  20. Immunohistochemical evalulation of activated Ras and Rac1 as potential downstream effectors of aquaporin-5 in breast cancer in vivo.

    Science.gov (United States)

    Jensen, Helene H; Login, Frédéric H; Park, Ji-Young; Kwon, Tae-Hwan; Nejsum, Lene N

    2017-11-25

    Aberrant levels of aquaporin-5 (AQP5) expression have been observed in several types of cancer, including breast cancer, where AQP5 overexpression is associated with metastasis and poor prognosis. In cultured cancer cells, AQP5 facilitates cell migration and activates Ras signaling. Both increased cell migration and Ras activation are associated with cancer metastasis, but so far it is unknown if AQP5 also affects these processes in vivo. Therefore, we investigated if high AQP5 expression in breast cancer tissue correlated with increased activation of Ras and of Rac1, which is a GTPase also involved in cell migration. This was accomplished by immunohistochemical analysis of invasive ductal carcinoma of breast tissue sections from human patients, followed by qualitative and quantitative correlation analysis between AQP5 and activated Ras and Rac1. Immunohistochemistry revealed that activation of Ras and Rac1 was positively correlated. There was, however, no correlation between high AQP5 expression and activation of Ras, whereas a nonsignificant, but positive, tendency between the levels of AQP5 and activated Rac1 levels was observed. In summary, this is the first report that correlates AQP5 expression levels to downstream signaling partners in breast cancer tissue sections. The results suggest Rac1 as a potential downstream signaling partner of AQP5 in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. 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.

  2. Parallel imaging of Drosophila embryos for quantitative analysis of genetic perturbations of the Ras pathway

    Directory of Open Access Journals (Sweden)

    Yogesh Goyal

    2017-07-01

    Full Text Available The Ras pathway patterns the poles of the Drosophila embryo by downregulating the levels and activity of a DNA-binding transcriptional repressor Capicua (Cic. We demonstrate that the spatiotemporal pattern of Cic during this signaling event can be harnessed for functional studies of mutations in the Ras pathway in human diseases. Our approach relies on a new microfluidic device that enables parallel imaging of Cic dynamics in dozens of live embryos. We found that although the pattern of Cic in early embryos is complex, it can be accurately approximated by a product of one spatial profile and one time-dependent amplitude. Analysis of these functions of space and time alone reveals the differential effects of mutations within the Ras pathway. Given the highly conserved nature of Ras-dependent control of Cic, our approach provides new opportunities for functional analysis of multiple sequence variants from developmental abnormalities and cancers.

  3. Transforming growth factor (TGF)-beta in conjunction with H-ras activation promotes malignant progression of MCF10A breast epithelial cells.

    Science.gov (United States)

    Kim, Eun-Sook; Kim, Mi-Sung; Moon, Aree

    2005-01-21

    To address how transforming growth factor (TGF)-beta and oncogenic H-ras signal transduction pathways interact with each other in the malignant progression of breast epithelial cells, we investigated the role of TGF-beta signaling pathway in invasive and migrative properties of H-ras-transformed MCF10A human breast epithelial cells in this study. Here we show that TGF-beta treatment significantly enhanced invasion and migration of H-ras MCF10A cells. H-ras-mediated activation of p38 MAPK and ERK-1/2 was stimulated by TGF-beta. TGF-beta increased expression of matrix metalloproteinase (MMP)-2 through transcriptional activation while TGF-beta-stimulated MMP-9 up-regulation did not occur at transcription level. Activation of p38 MAPK pathway was required for TGF-beta-induced cell migration, invasion and MMP-2/-9 up-regulation, indicating a critical role of p38 MAPK signaling in TGF-beta-promoted tumor progression of H-ras-activated cells. ERKs signaling was also crucial for TGF-beta-enhanced invasive and migrative phenotypes but the up-regulation of MMP-2/-9 was not dependent on ERKs activity. Taken together, we show that TGF-beta promotes H-ras-mediated cell migration and invasive phenotypes in which p38 MAPK and ERKs signaling pathways are involved. Our findings revealing how H-ras and TGF-beta signal pathways interact with each other in MCF10A human breast cells may provide an insight into molecular mechanisms for contribution of TGF-beta to a malignant progression of breast cancer in collaboration with activated H-ras.

  4. PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors

    Directory of Open Access Journals (Sweden)

    Schmidt Enrico K

    2004-05-01

    Full Text Available Abstract Background Erythropoietin is a multifunctional cytokine which regulates the number of erythrocytes circulating in mammalian blood. This is crucial in order to maintain an appropriate oxygen supply throughout the body. Stimulation of primary human erythroid progenitors (PEPs with erythropoietin (Epo leads to the activation of the mitogenic kinases (MEKs and Erks. How this is accomplished mechanistically remained unclear. Results Biochemical studies with human cord blood-derived PEPs now show that Ras and the class Ib enzyme of the phosphatidylinositol-3 kinase (PI3K family, PI3K gamma, are activated in response to minimal Epo concentrations. Surprisingly, three structurally different PI3K inhibitors block Ras, MEK and Erk activation in PEPs by Epo. Furthermore, Erk activation in PEPs is insensitive to the inhibition of Raf kinases but suppressed upon PKC inhibition. In contrast, Erk activation induced by stem cell factor, which activates c-Kit in the same cells, is sensitive to Raf inhibition and insensitive to PI3K and PKC inhibitors. Conclusions These unexpected findings contrast with previous results in human primary cells using Epo at supraphysiological concentrations and open new doors to eventually understanding how low Epo concentrations mediate the moderate proliferation of erythroid progenitors under homeostatic blood oxygen levels. They indicate that the basal activation of MEKs and Erks in PEPs by minimal concentrations of Epo does not occur through the classical cascade Shc/Grb2/Sos/Ras/Raf/MEK/Erk. Instead, MEKs and Erks are signal mediators of PI3K, probably the recently described PI3K gamma, through a Raf-independent signaling pathway which requires PKC activity. It is likely that higher concentrations of Epo that are induced by hypoxia, for example, following blood loss, lead to additional mitogenic signals which greatly accelerate erythroid progenitor proliferation.

  5. 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.

  6. 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.

  7. Inhibition of DNA synthesis by carvacrol in mouse myoblast cells bearing a human N-RAS oncogene.

    Science.gov (United States)

    Zeytinoglu, H; Incesu, Z; Baser, K H C

    2003-05-01

    Monoterpenes are dietary components found in the essential oils of a wide variety of plants. A number of these monoterpenes have antitumor activity. We have investigated the effects of carvacrol obtained by fractional distillation of Origanum onites L. essential oil, on DNA synthesis of N-ras transformed myoblast cells, CO25. Incubation of the cells with different doses of carvacrol prevented DNA synthesis in the growth medium and ras-activating medium, which contains dexamethasone. This result demonstrates that carvacrol inhibits growth of myoblast cells even after activation of mutated N-ras oncogene, suggesting the possibility that carvacrol may find application in cancer therapy.

  8. PTEN loss and activation of K-RAS and β-catenin cooperate to accelerate prostate tumourigenesis.

    Science.gov (United States)

    Jefferies, Matthew T; Cox, Adam C; Shorning, Boris Y; Meniel, Valerie; Griffiths, David; Kynaston, Howard G; Smalley, Matthew J; Clarke, Alan R

    2017-12-01

    Aberrant phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and WNT signalling are emerging as key events in the multistep nature of prostate tumourigenesis and progression. Here, we report a compound prostate cancer murine model in which these signalling pathways cooperate to produce a more aggressive prostate cancer phenotype. Using Cre-LoxP technology and the probasin promoter, we combined the loss of Pten (Pten fl/fl ), to activate the PI3K signalling pathway, with either dominant stabilized β-catenin [Catnb +/lox(ex3) ] or activated K-RAS (K-Ras +/V12 ) to aberrantly activate WNT and MAPK signalling, respectively. Synchronous activation of all three pathways (triple mutants) significantly reduced survival (median 96 days) as compared with double mutants [median: 140 days for Catnb +/lox(ex3) Pten fl/fl ; 182 days for Catnb +/lox(ex3) K-Ras +/V12 ; 238 days for Pten fl/fl K-Ras +/V12 ], and single mutants [median: 383 days for Catnb +/lox(ex3) ; 407 days for Pten fl/fl ], reflecting the accelerated tumourigenesis. Tumours followed a stepwise progression from mouse prostate intraepithelial neoplasia to invasive adenocarcinoma, similar to that seen in human disease. There was significantly elevated cellular proliferation, tumour growth and percentage of invasive adenocarcinoma in triple mutants as compared with double mutants and single mutants. Triple mutants showed not only activated AKT, extracellular-signal regulated kinase 1/2, and nuclear β-catenin, but also significantly elevated signalling through mechanistic target of rapamycin complex 1 (mTORC1). In summary, we show that combined deregulation of the PI3K, MAPK and WNT signalling pathways drives rapid progression of prostate tumourigenesis, and that deregulation of all three pathways results in tumours showing aberrant mTORC1 signalling. As mTORC1 signalling is emerging as a key driver of androgen deprivation therapy resistance, our findings are important for

  9. Mutations in APC, CTNNBI en K-ras genes and expression of hMLHI in sporadic colorectal carcinomas from the Netherlands Cohort Study

    NARCIS (Netherlands)

    Luchtenborg, M.; Weijenberg, M.P.; Wark, P.A.; Merdan Saritas, M.

    2005-01-01

    Background - The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of

  10. Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Gennaro Altamura

    2013-01-01

    Full Text Available Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1 and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

  11. Hyperglycemia promotes K-Ras-induced lung tumorigenesis through BASCs amplification.

    Directory of Open Access Journals (Sweden)

    Carla Micucci

    Full Text Available Oncogenic K-Ras represents the most common molecular change in human lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC. The presence of K-Ras mutation is associated with a poor prognosis, but no effective treatment strategies are available for K-Ras -mutant NSCLC. Epidemiological studies report higher lung cancer mortality rates in patients with type 2 diabetes. Here, we use a mouse model of K-Ras-mediated lung cancer on a background of chronic hyperglycemia to determine whether elevated circulating glycemic levels could influence oncogenic K-Ras-mediated tumor development. Inducible oncogenic K-Ras mouse model was treated with subtoxic doses of streptozotocin (STZ to induce chronic hyperglycemia. We observed increased tumor mass and higher grade of malignancy in STZ treated diabetic mice analyzed at 4, 12 and 24 weeks, suggesting that oncogenic K-Ras increased lung tumorigenesis in hyperglycemic condition. This promoting effect is achieved by expansion of tumor-initiating lung bronchio-alveolar stem cells (BASCs in bronchio-alveolar duct junction, indicating a role of hyperglycemia in the activity of K-Ras-transformed putative lung stem cells. Notably, after oncogene K-Ras activation, BASCs show upregulation of the glucose transporter (Glut1/Slc2a1, considered as an important player of the active control of tumor cell metabolism by oncogenic K-Ras. Our novel findings suggest that anti-hyperglycemic drugs, such as metformin, may act as therapeutic agent to restrict lung neoplasia promotion and progression.

  12. 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.

  13. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

    Science.gov (United States)

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-06-03

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

  14. Distinct biological properties of two RET isoforms activated by MEN 2A and MEN 2B mutations.

    Science.gov (United States)

    Rossel, M; Pasini, A; Chappuis, S; Geneste, O; Fournier, L; Schuffenecker, I; Takahashi, M; van Grunsven, L A; Urdiales, J L; Rudkin, B B; Lenoir, G M; Billaud, M

    1997-01-23

    Germline mutations of the RET proto-oncogene, which codes for a receptor tyrosine kinase, cause multiple endocrine neoplasia type 2A (MEN 2A) and 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). MEN 2 mutations have been shown to result in RET oncogenic activation. The RET gene encodes several isoforms whose biological properties, when altered by MEN 2 mutations, have not been thoroughly addressed yet. In this study, we have introduced a MEN 2A mutation (Cys634-->Arg) and the unique MEN 2B mutation (Met918-->Thr) in two RET isoforms of 1114 and 1072 amino acids which differ in the carboxy-terminus part. Herein, we report that each RET isoform activated by MEN 2A or MEN 2B mutation was transforming in fibroblasts and induced neuronal differentiation of pheochromocytoma PC12 cells. However, among the different RET-MEN 2 mutants, the long RET isoform activated by the MEN 2B mutation stimulated the most prominent neurite outgrowth in PC12 cells, while the short RET isoform counterpart elicited a very weak differentiation effect in PC12 cells. We further demonstrate that the morphological changes of PC12 cells caused by constitutively activated RET oncoproteins involved the engagement of a Ras-dependent pathway. These findings provide evidence that the biological properties of RET-MEN 2 mutants depend on the interplay between the RET isoforms and the nature of the activating MEN 2 mutation.

  15. Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc

    NARCIS (Netherlands)

    Kranenburg, O.; Verlaan, I.; Hordijk, P. L.; Moolenaar, W. H.

    1997-01-01

    Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known

  16. Role of the YAP Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma.

    Directory of Open Access Journals (Sweden)

    Katherine K Slemmons

    Full Text Available Rhabdomyosarcoma (RMS, a cancer characterized by features of skeletal muscle histogenesis, is the most common soft tissue sarcoma of childhood and adolescence. Survival for high-risk groups is less than 30% at 5 years. RMS also occurs during adulthood, with a lower incidence but higher mortality. Recently, mutational profiling has revealed a correlation between activating Ras mutations in the embryonal (eRMS and pleomorphic (pRMS histologic variants of RMS, and a poorer outcome for those patients. Independently, the YAP transcriptional coactivator, an oncoprotein kept in check by the Hippo tumor suppressor pathway, is upregulated in eRMS. Here we show that YAP promotes cell proliferation and antagonizes apoptosis and myogenic differentiation of human RMS cells bearing oncogenic Ras mutations in cell culture studies in vitro and in murine xenografts in vivo. Pharmacologic inhibition of YAP by the benzoporphyrin derivative verteporfin decreased cell proliferation and tumor growth in vivo. To interrogate the temporal contribution of YAP in eRMS tumorigenesis, we used a primary human cell-based genetic model of Ras-driven RMS. Constitutively active YAP functioned as an early genetic lesion, permitting bypass of senescence and priming myoblasts to tolerate subsequent expression of hTERT and oncogenic Ras, which were necessary and sufficient to generate murine xenograft tumors mimicking RMS in vivo. This work provides evidence for cooperation between YAP and oncogenic Ras in RMS tumorigenesis, laying the foundation for preclinical co-targeting of these pathways.

  17. 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.

  18. Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Paolo Cossu-Rocca

    Full Text Available Triple Negative Breast Cancer (TNBC accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

  19. Nobiletin induces inhibitions of Ras activity and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling to suppress cell proliferation in C6 rat glioma cells.

    Science.gov (United States)

    Aoki, Koichi; Yokosuka, Akihito; Mimaki, Yoshihiro; Fukunaga, Kohji; Yamakuni, Tohru

    2013-01-01

    Ras, a small G-protein, physiologically directs cell proliferation and cell cycle via regulation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade. Dysregulation of Ras/MEK/ERK signaling has been reported to cause tumorigenesis and gliomas. Nobiletin, a citrus flavonoid, has been shown to have anti-tumor cells action. However, it remains elusive whether nobiletin could affect Ras activity. In this study, we provide the first evidence that nobiletin suppresses the proliferation by inhibiting Ras activity in C6 glioma cells, a rat glioma cell line. First, Ras pull-down assay showed that nobiletin inhibits Ras activity in a concentration-dependent manner in C6 cells. Second, farnesyltransferase inhibitor I, a Ras inhibitor, and U0126, a MEK inhibitor, induced an inhibition of the cell proliferation in C6 cells, while the cell proliferation was inhibited by nobiletin as well. Third, western blotting revealed that nobiletin showed inhibitory effects on MEK and ERK phopsphorylation levels in a concentration-dependent manner. Finally, such an inhibitory effect on the level of ERK phosphorylation by nobiletin was appreciably prevented by Gö6976, a selective inhibitor of conventional protein kinase Cs (PKCs) showing Ca(2+)-sensitivity, while GF109203X, a general inhibitor for PKCs, and BAPTA, a cell-permeable Ca(2+) chelator, to a lesser extent, suppressed a reduction of the phosphorylation. These findings suggest that the proliferation of C6 cells is Ras- and MEK/ERK signaling-dependent, and that nobiletin suppresses the cell proliferation by inhibiting Ras activity and MEK/ERK signaling cascade probably via a Ca(2+)-sensitive PKC-dependent mechanism. Thus, the natural compound has potential to be a therapeutic agent for glioma.

  20. Gain-of-function SOS1 mutations cause a distinctive form of noonansyndrome

    Energy Technology Data Exchange (ETDEWEB)

    Tartaglia, Marco; Pennacchio, Len A.; Zhao, Chen; Yadav, KamleshK.; Fodale, Valentina; Sarkozy, Anna; Pandit, Bhaswati; Oishi, Kimihiko; Martinelli, Simone; Schackwitz, Wendy; Ustaszewska, Anna; Martin, Joes; Bristow, James; Carta, Claudio; Lepri, Francesca; Neri, Cinzia; Vasta,Isabella; Gibson, Kate; Curry, Cynthia J.; Lopez Siguero, Juan Pedro; Digilio, Maria Cristina; Zampino, Giuseppe; Dallapiccola, Bruno; Bar-Sagi, Dafna; Gelb, Brude D.

    2006-09-01

    Noonan syndrome (NS) is a developmental disordercharacterized by short stature, facial dysmorphia, congenital heartdefects and skeletal anomalies1. Increased RAS-mitogenactivated proteinkinase (MAPK) signaling due to PTPN11 and KRAS mutations cause 50 percentof NS2-6. Here, we report that 22 of 129 NS patients without PTPN11 orKRAS mutation (17 percent) have missense mutations in SOS1, which encodesa RAS-specific guanine nucleotide exchange factor (GEF). SOS1 mutationscluster at residues implicated in the maintenance of SOS1 in itsautoinhibited form and ectopic expression of two NS-associated mutantsinduced enhanced RAS activation. The phenotype associated with SOS1defects is distinctive, although within NS spectrum, with a highprevalence of ectodermal abnormalities but generally normal developmentand linear growth. Our findings implicate for the first timegain-of-function mutations in a RAS GEF in inherited disease and define anew mechanism by which upregulation of the RAS pathway can profoundlychange human development.

  1. 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.

  2. Histone deacetylase inhibitor trichostatin A and autophagy inhibitor chloroquine synergistically exert anti-tumor activity in H-ras transformed breast epithelial cells

    Science.gov (United States)

    Gao, Liang; Sun, Xin; Zhang, Qi; Chen, Xiaochen; Zhao, Tongwei; Lu, Liqing; Zhang, Jianbin; Hong, Yupeng

    2018-01-01

    Histone deacetylase inhibitors (HDACIs) cause oncogene-transformed mammalian cell death. Our previous study indicated that HDACIs activate forkhead box O1 (FOXO1) and induce autophagy in liver and colon cancer cells. However, whether FOXO1 is involved in HDACI-mediated oncogene-transformed mammalian cell death remains unclear. In the present study, H-ras transformed MCF10A cells were used to investigate the role of FOXO1 in this pathway. Results showed that trichostatin A (TSA), a HDACI, activated apoptosis in MCF10A-ras cells, but not in MCF10A cells. Furthermore, TSA activated FOXO1 via P21 upregulation, whereas the knockdown of FOXO1 reduced TSA-induced cell death. In addition, TSA induced autophagy in MCF10A and MCF10A-ras cells by blocking the mammailian target of rapamycin signaling pathway. Furthermore, autophagy inhibition lead to higher MCF10A-ras cell death by TSA, thus indicating that autophagy is essential in cell survival. Taken together, the present study demonstrated that TSA causes oncogene-transformed cell apoptosis via activation of FOXO1 and HDACI-mediated autophagy induction, which served as important cell survival mechanisms. Notably, the present findings imply that a combination of HDACIs and autophagy inhibitors produce a synergistic anticancer effect. PMID:29344672

  3. Targeted elimination of senescent Ras-transformed cells by suppression of MEK/ERK pathway.

    Science.gov (United States)

    Kochetkova, Elena Y; Blinova, Galina I; Bystrova, Olga A; Martynova, Marina G; Pospelov, Valery A; Pospelova, Tatiana V

    2017-11-14

    The Ras-Raf-MEK-ERK pathway plays a central role in tumorigenesis and is a target for anticancer therapy. The successful strategy based on the activation of cell death in Ras-expressing cells is associated with the suppression of kinases involved in Ras pathway. However, activation of cytoprotective autophagy overcomes antiproliferative effect of the inhibitors and develops drug resistance. We studied whether cellular senescence induced by HDAC inhibitor sodium butyrate in E1a+cHa-Ras -transformed rat embryo fibroblasts (ERas) and A549 human Ki-Ras mutated lung adenocarcinoma cells would enhance the tumor suppressor effect of MEK/ERK inhibition. Treatment of control ERas cells with PD0325901 for 24 h results in mitochondria damage and apoptotic death of a part of cellular population. However, the activation of AMPK-dependent autophagy overcomes pro-apoptotic effects of MEK/ERK inhibitor and results in restoration of the mitochondria and rescue of viability. Senescent ERas cells do not develop cytoprotective autophagy upon inhibition of MEK/ERK pathway due to spatial dissociation of lysosomes and autophagosomes in the senescent cells. Senescent cells are unable to form the autophagolysosomes and to remove the damaged mitochondria resulting in apoptotic death. Our data show that suppression of MEK/ERK pathway in senescent cells provides a new strategy for elimination of Ras-expressing cells.

  4. New insight into the dynamic properties and the active site architecture of H-Ras p21 revealed by X-ray crystallography at very high resolution

    Directory of Open Access Journals (Sweden)

    Klink Björn U

    2010-10-01

    Full Text Available Abstract Background In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes. Results X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(β,γ-imido-triphosphate, a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom. Conclusion For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.

  5. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N

    2000-01-01

    , inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...

  6. Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras.

    Science.gov (United States)

    Xu, Shenyuan; Long, Brian N; Boris, Gabriel H; Chen, Anqi; Ni, Shuisong; Kennedy, Michael A

    2017-12-01

    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.

  7. RAP/RAS workshop.

    Science.gov (United States)

    2013-01-01

    : RAP & RAS increases mix stiffness : : Most WMA additives decrease stiffness : : Tear-Off shingles are stiffer than Man-waste shingles : : Using multiple recycled bins improves consistency : : Finer RAS material improves consiste...

  8. 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.

  9. 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

    activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated......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......, 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...

  10. 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.

  11. 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.

  12. Radiosensitivity of small-cell lung cancer xenografts compared with activity of c-myc, N-myc, L-myc, c-raf-1 and K-ras proto-oncogenes

    DEFF Research Database (Denmark)

    Rygaard, K; Slebos, R J; Spang-Thomsen, M

    1991-01-01

    Oncogenes of the myc family c-raf-1 and K-ras have been reported to modulate radiosensitivity. We examined the possible relationship between in vivo radiosensitivity to single-dose irradiation with 3-10 Gy, and activity of these proto-oncogenes in 2 sets of small-cell lung cancer (SCLC) xenografts...... than CPH-54B, while, with respect to the 3 GLC tumours examined, GLC-16 was most sensitive, followed by GLC-14 and GLC-19. The CPH tumours expressed similar amounts of c-myc and c-raf-1 mRNA, and neither expressed N-myc or L-myc. GLC-14 expressed N-myc and c-raf-1 mRNA but no c-myc. GLC-16 and GLC-19...... expressed identical amounts of c-raf-1 and high levels of c-myc mRNA, but neither expressed N-myc or L-myc. None of the tumours was mutated at codon 12 or K-ras. Our results show that SCLC xenografts with different radiosensitivity may express identical amounts of some of the proto-oncogenes reported...

  13. Novel point mutations attenuate autotaxin activity

    Directory of Open Access Journals (Sweden)

    Stracke Mary L

    2009-02-01

    Full Text Available Abstract Background The secreted enzyme autotaxin (ATX stimulates tumor cell migration, tumorigenesis, angiogenesis, and metastasis. ATX hydrolyzes nucleotides, but its hydrolysis of lysophospholipids to produce lysophosphatidic acid (LPA accounts for its biological activities. ATX has been identified only as a constitutively active enzyme, and regulation of its activity is largely unexplored. In spite of its presence in plasma along with abundant putative substrate LPC, the product LPA is found in plasma at unexpectedly low concentrations. It is plausible that the LPA-producing activity of ATX is regulated by its expression and by access to substrate(s. For this reason studying the interaction of enzyme with substrate is paramount to understanding the regulation of LPA production. Results In this study we determine ATX hydrolytic activities toward several artificial and natural substrates. Two novel point mutations near the enzyme active site (H226Q and H434Q confer attenuated activity toward all substrates tested. The Vmax for LPC compounds depends upon chain length and saturation; but this order does not differ among wild type and mutants. However the mutant forms show disproportionately low activity toward two artificial substrates, pNpTMP and FS-3. The mutant forms did not significantly stimulate migration responses at concentrations that produced a maximum response for WT-ATX, but this defect could be rescued by inclusion of exogenous LPC. Conclusion H226Q-ATX and H434Q-ATX are the first point mutations of ATX/NPP2 demonstrated to differentially impair substrate hydrolysis, with hydrolysis of artificial substrates being disproportionately lower than that of LPC. This implies that H226 and H434 are important for substrate interaction. Assays that rely on hydrolyses of artificial substrates (FS-3 and pNpTMP, or that rely on hydrolysis of cell-derived substrate, might fail to detect certain mutated forms of ATX that are nonetheless capable of

  14. Argos induces programmed cell death in the developing Drosophila eye by inhibition of the Ras pathway.

    Science.gov (United States)

    Sawamoto, K; Taguchi, A; Hirota, Y; Yamada, C; Jin, M H; Okano, H

    1998-04-01

    We studied the role of Ras signaling in the regulation of cell death during Drosophila eye development. Overexpression of Argos, a diffusible inhibitor of the EGF receptor and Ras signaling, caused excessive cell death in developing eyes at pupal stages. The Argos-induced cell death was suppressed by coexpression of the anti-apoptotic genes p35, diap1, or diap2 in the eye as well as by the Df(3L)H99 chromosomal deletion that lacks three apoptosis-inducing genes, reaper, head involution defective (hid) and grim. Transient misexpression of the activated Ras1 protein (Ras1V12) later in pupal development suppressed the Argos-induced cell death. Thus, Argos-induced cell death seemed to have resulted from the suppression of the anti-apoptotic function of Ras. Conversely, cell death induced by overexpression of Hid was suppressed by gain-of-function mutations of the genes coding for MEK and ERK. These results support the idea that Ras signaling functions in two distinct processes during eye development, first triggering the recruitment of cells and later negatively regulating cell death.

  15. Activated Ras signaling pathways and reovirus oncolysis: an update on the mechanism of preferential reovirus replication in cancer cells

    Directory of Open Access Journals (Sweden)

    Jun eGong

    2014-06-01

    Full Text Available The development of wild-type, unmodified Type 3 Dearing (T3D strain reovirus as an anticancer agent has currently expanded to 32 clinical trials (both completed and ongoing involving reovirus in the treatment of cancer. It has been more than 30 years since the potential of reovirus as an anticancer agent was first identified in studies that demonstrated the preferential replication of reovirus in transformed cell lines but not in normal cells. Later investigations have revealed the involvement of activated Ras signaling pathways (both upstream and downstream and key steps of the reovirus infectious cycle in promoting preferential replication in cancer cells with reovirus-induced cancer cell death occurring through necrotic, apoptotic, and autophagic pathways. There is increasing evidence that reovirus-induced antitumor immunity involving both innate and adaptive responses also contributes to therapeutic efficacy though this discussion is beyond the scope of this article. Here we review our current understanding of the mechanism of oncolysis contributing to the broad anticancer activity of reovirus. Further understanding of reovirus oncolysis is critical in enhancing the clinical development and efficacy of reovirus.

  16. Activated Ras Signaling Pathways and Reovirus Oncolysis: An Update on the Mechanism of Preferential Reovirus Replication in Cancer Cells

    Science.gov (United States)

    Gong, Jun; Mita, Monica M.

    2014-01-01

    The development of wild-type, unmodified Type 3 Dearing strain reovirus as an anticancer agent has currently expanded to 32 clinical trials (both completed and ongoing) involving reovirus in the treatment of cancer. It has been more than 30 years since the potential of reovirus as an anticancer agent was first identified in studies that demonstrated the preferential replication of reovirus in transformed cell lines but not in normal cells. Later investigations have revealed the involvement of activated Ras signaling pathways (both upstream and downstream) and key steps of the reovirus infectious cycle in promoting preferential replication in cancer cells with reovirus-induced cancer cell death occurring through necrotic, apoptotic, and autophagic pathways. There is increasing evidence that reovirus-induced antitumor immunity involving both innate and adaptive responses also contributes to therapeutic efficacy though this discussion is beyond the scope of this article. Here, we review our current understanding of the mechanism of oncolysis contributing to the broad anticancer activity of reovirus. Further understanding of reovirus oncolysis is critical in enhancing the clinical development and efficacy of reovirus. PMID:25019061

  17. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network.

    Science.gov (United States)

    Cheng, Yougan; Othmer, Hans

    2016-05-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that [Formula: see text] cycling modulated by Ric8, a nonreceptor guanine exchange factor for [Formula: see text] in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both [Formula: see text] and Gβγ are essential for direction sensing, in that membrane-localized [Formula: see text], the activated GTP-bearing form of [Formula: see text], leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient 'memory' to eliminate the 'back-of-the wave' problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since

  18. ERK2-regulated TIMP1 Induces Hyperproliferation of K-RasG12D-Transformed Pancreatic Ductal Cells

    Directory of Open Access Journals (Sweden)

    Gregory P. Botta

    2013-04-01

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC commonly contains a mutation in K-RasG12D and is characterized by a desmoplastic reaction composed of deregulated, proliferating cells embedded in an abnormal extracellular matrix (ECM. Our previous observations imply that inhibiting the mitogen-activated protein kinase (MAPK-extracellular signal-regulated kinase (ERK2 kinase signal pathway reverses a matrix metalloproteinase 1-specific invasive phenotype. Here, we investigated the specific genes downstream of MAPK-ERK2 responsible for the hyperproliferative abilities of human and murine primary ductal epithelial cells (PDCs within an ECM. Compared with control, DNA synthesis and total cell proliferation was significantly increased in human PDCs harboring the PDAC common p53, Rb/p16INK4a, and K-RasG12D mutations. Both of these effects were readily reversed following small-molecule inhibition or lentiviral silencing of ERK2. Microarray analysis of PDCs in three-dimensional (3D culture revealed a unique, MAPK-influenced gene signature downstream of K-RasG12D. Unbiased hierarchical analysis permitted filtration of tissue inhibitor of matrix metalloproteinase 1 (TIMP1. Pancreatic cells isolated from Pdx1-Cre; LSL-K-rasG12D/+-mutated mice exhibit increased TIMP1 RNA transcription compared to wild-type littermate controls. Analyses of both 3D, in vitro human K-RasG12D PDCs and data mining of publicly annotated human pancreatic data sets correlatively indicate increased levels of TIMP1 RNA. While silencing TIMP1 did not significantly effect PDC proliferation, exogenous addition of human recombinant TIMP1 significantly increased proliferation but only in transformed K-RasG12D PDCs in 3D. Overall, TIMP1 is an upregulated gene product and a proliferative inducer of K-RasG12D-mutated PDCs through the ERK2 signaling pathway.

  19. Gα16 interacts with tetratricopeptide repeat 1 (TPR1 through its β3 region to activate Ras independently of phospholipase Cβ signaling

    Directory of Open Access Journals (Sweden)

    Ho Maurice KC

    2011-04-01

    Full Text Available Abstract Background G protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα16 as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1 binds to the same region as its canonical effector, phospholipase Cβ (PLCβ. Results We have examined the specificity of Gα16/TPR1 association by testing a series of chimeras between Gα16 and Gαz. TPR1 co-immunoprecipitated with Gα16 and more tightly with its constitutively active Gα16QL, but not Gαz. Progressive replacement of Gα16 sequence with the corresponding residues of Gαz eventually identified a stretch of six amino acids in the β3 region of Gα16 which are responsible for TPR1 interaction and the subsequent Ras activation. Insertion of these six residues into Gαz allowed productive TPR1-interaction. Since the β3 region only minimally contributes to interact with PLCβ, several chimeras exhibited differential abilities to stimulate PLCβ and Ras. The ability of the chimeras to activate downstream transcription factors such as signal transducer and activator of transcription 3 and nuclear factor κB appeared to be associated with PLCβ signaling. Conclusions Our results suggest that Gα16 can signal through TPR1/Ras and PLCβ simultaneously and independently. The β3 region of Gα16 is essential for interaction with TPR1 and the subsequent activation of Ras, but has relatively minor influence on the PLCβ interaction. Gα16 may utilize different structural domains to bind TPR1 and PLCβ.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Ras-dva is a novel Pit-1- and glucocorticoid-regulated gene in the embryonic anterior pituitary gland.

    Science.gov (United States)

    Ellestad, Laura E; Porter, Tom E

    2013-01-01

    Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5'-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5'-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

  2. Elevated Ras/protein kinase A activity in Saccharomyces cerevisiae reduces proliferation rate and lifespan by two different reactive oxygen species-dependent routes

    Czech Academy of Sciences Publication Activity Database

    Hlavatá, Lydie; Nachin, L.; Ježek, Petr; Nyström, T.

    2008-01-01

    Roč. 7, č. 2 (2008), s. 148-157 ISSN 1474-9718 R&D Projects: GA ČR GP303/05/P100; GA AV ČR IAA500110701 Institutional research plan: CEZ:AV0Z50110509 Keywords : Ras/protein kinase A activity * ROS * Saccharomyces cerevisiae Subject RIV: CE - Biochemistry Impact factor: 7.791, year: 2008

  3. Restoration of E-cadherin Cell-Cell Junctions Requires Both Expression of E-cadherin and Suppression of ERK MAP Kinase Activation in Ras-Transformed Breast Epithelial Cells

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    Quanwen Li

    2008-12-01

    Full Text Available E-cadherin is a main component of the cell-cell adhesion junctions that play a principal role in maintaining normal breast epithelial cell morphology. Breast and other cancers that have up-regulated activity of Ras are often found to have down-regulated or mislocalized E-cadherin expression. Disruption of E-cadherin junctions and consequent gain of cell motility contribute to the process known as epithelial-to-mesenchymal transition (EMT. Enforced expression of E-cadherin or inhibition of Ras-signal transduction pathway has been shown to be effective in causing reversion of EMT in several oncogene-transformed and cancer-derived cell lines. In this study, we investigated MCF10A human breast epithelial cells and derivatives that were transformed with either activated H-Ras or N-Ras to test for the reversion of EMT by inhibition of Ras-driven signaling pathways. Our results demonstrated that inhibition of mitogen-activated protein kinase (MAPK kinase, but not PI3-kinase, Rac, or myosin light chain kinase, was able to completely restore E-cadherin cell-cell junctions and epithelial morphology in cell lines with moderate H-Ras expression. In MCF10A cells transformed by a high-level expression of activated H-Ras or N-Ras, restoration of E-cadherin junction required both the enforced reexpression of E-cadherin and suppression of MAPK kinase. Enforced expression of E-cadherin alone did not induce reversion from the mesenchymal phenotype. Our results suggest that Ras transformation has at least two independent actions to disrupt E-cadherin junctions, with effects to cause both mislocalization of E-cadherin away from the cell surface and profound decrease in the expression of E-cadherin.

  4. Mutations in PIK3CA are infrequent in neuroblastoma

    International Nuclear Information System (INIS)

    Dam, Vincent; Morgan, Brian T; Mazanek, Pavel; Hogarty, Michael D

    2006-01-01

    Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported. Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain 'hot spots' where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. These data suggest that activating

  5. Septin mutations in human cancers

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    Elias T Spiliotis

    2016-11-01

    Full Text Available Septins are GTP-binding proteins that are evolutionarily and structurally related to the RAS oncogenes. Septin expression levels are altered in many cancers and new advances point to how abnormal septin expression may contribute to the progression of cancer. In contrast to the RAS GTPases, which are frequently mutated and actively promote tumorigenesis, little is known about the occurrence and role of septin mutations in human cancers. Here, we review septin missense mutations that are currently in the Catalog of Somatic Mutations in Cancer (COSMIC database. The majority of septin mutations occur in tumors of the large intestine, skin, endometrium and stomach. Over 25% of the annotated mutations in SEPT2, SEPT4 and SEPT9 belong to large intestine tumors. From all septins, SEPT9 and SEPT14 exhibit the highest mutation frequencies in skin, stomach and large intestine cancers. While septin mutations occur with frequencies lower than 3%, recurring mutations in several invariant and highly conserved amino acids are found across different septin paralogs and tumor types. Interestingly, a significant number of these mutations occur in the GTP-binding pocket and septin dimerization interfaces. Future studies may determine how these somatic mutations affect septin structure and function, whether they contribute to the progression of specific cancers and if they could serve as tumor-specific biomarkers.

  6. Mutations in the c-Kit Gene Disrupt Mitogen-Activated Protein Kinase Signaling during Tumor Development in Adenoid Cystic Carcinoma of the Salivary Glands

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    Osamu Tetsu

    2010-09-01

    Full Text Available The Ras/mitogen-activated protein kinase (MAPK pathway is considered to be a positive regulator of tumor initiation, progression, and maintenance. This study reports an opposite finding: we have found strong evidence that the MAPK pathway is inhibited in a subset of adenoid cystic carcinomas (ACCs of the salivary glands. ACC tumors consistently overexpress the receptor tyrosine kinase (RTK c-Kit, which has been considered a therapeutic target. We performed mutational analysis of the c-Kit gene (KIT in 17 cases of ACC and found that 2 cases of ACC had distinct missense mutations in KIT at both the genomic DNA and messenger RNA levels. These mutations caused G664R and R796G amino acid substitutions in the kinase domains. Surprisingly, the mutations were functionally inactive in cultured cells. We observed a significant reduction of MAPK (ERK1/2 activity in tumor cells, as assessed by immunohistochemistry. We performed further mutational analysis of the downstream effectors in the c-Kit pathway in the genes HRAS, KRAS, NRAS, BRAF, PIK3CA, and PTEN. This analysis revealed that two ACC tumors without KIT mutations had missense mutations in either KRAS or BRAF, causing S17N K-Ras and V590I B-Raf mutants, respectively. Our functional analysis showed that proteins with these mutations were also inactive in cultured cells. This is the first time that MAPK activity from the RTK signaling has been shown to be inhibited by gene mutations during tumor development. Because ACC seems to proliferate despite inactivation of the c-Kit signaling pathway, we suggest that selective inhibition of c-Kit is probably not a suitable treatment strategy for ACC.

  7. RASOnD - A comprehensive resource and search tool for RAS superfamily oncogenes from various species

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    Singh Tej P

    2011-07-01

    Full Text Available Abstract Background The Ras superfamily plays an important role in the control of cell signalling and division. Mutations in the Ras genes convert them into active oncogenes. The Ras oncogenes form a major thrust of global cancer research as they are involved in the development and progression of tumors. This has resulted in the exponential growth of data on Ras superfamily across different public databases and in literature. However, no dedicated public resource is currently available for data mining and analysis on this family. The present database was developed to facilitate straightforward accession, retrieval and analysis of information available on Ras oncogenes from one particular site. Description We have developed the RAS Oncogene Database (RASOnD as a comprehensive knowledgebase that provides integrated and curated information on a single platform for oncogenes of Ras superfamily. RASOnD encompasses exhaustive genomics and proteomics data existing across diverse publicly accessible databases. This resource presently includes overall 199,046 entries from 101 different species. It provides a search tool to generate information about their nucleotide and amino acid sequences, single nucleotide polymorphisms, chromosome positions, orthologies, motifs, structures, related pathways and associated diseases. We have implemented a number of user-friendly search interfaces and sequence analysis tools. At present the user can (i browse the data (ii search any field through a simple or advance search interface and (iii perform a BLAST search and subsequently CLUSTALW multiple sequence alignment by selecting sequences of Ras oncogenes. The Generic gene browser, GBrowse, JMOL for structural visualization and TREEVIEW for phylograms have been integrated for clear perception of retrieved data. External links to related databases have been included in RASOnD. Conclusions This database is a resource and search tool dedicated to Ras oncogenes. It has

  8. Bioinformatics of non small cell lung cancer and the ras proto-oncogene

    CERN Document Server

    Kashyap, Amita; Babu M, Naresh

    2015-01-01

    Cancer is initiated by activation of oncogenes or inactivation of tumor suppressor genes. Mutations in the K-ras proto-oncogene are responsible for 10–30% of adenocarcinomas. Clinical Findings point to a wide variety of other cancers contributing to lung cancer incidence. Such a scenario makes identification of lung cancer difficult and thus identifying its mechanisms can contribute to the society. Identifying unique conserved patterns common to contributing proto-oncogenes may further be a boon to Pharmacogenomics and pharmacoinformatics. This calls for ab initio/de novo drug discovery that in turn will require a comprehensive in silico approach of Sequence, Domain, Phylogenetic and Structural analysis of the receptors, ligand screening and optimization and detailed Docking studies. This brief involves extensive role of the RAS subfamily that includes a set of proteins, which cause an over expression of cancer-causing genes like M-ras and initiate tumour formation in lungs. SNP Studies and Structure based ...

  9. 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

    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.

  10. A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation

    Czech Academy of Sciences Publication Activity Database

    Smida, M.; Posevitz-Fejfar, A.; Hořejší, Václav; Schraven, B.; Lindquist, J.A.

    2007-01-01

    Roč. 110, č. 2 (2007), s. 596-605 ISSN 0006-4971 R&D Projects: GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : PAG * Ras * lipid rafts Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 10.896, year: 2007

  11. H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes

    DEFF Research Database (Denmark)

    Espada, J; Pérez-Moreno, M; Braga, V M

    1999-01-01

    The mechanisms underlying downregulation of the cadherin/catenin complexes and beta-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and beta-catenin distribution in epidermal ...

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

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    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.

  13. [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.

  14. Transcription factors down-stream of Ras as molecular indicators for targeting malignancies with oncolytic herpes virus.

    Science.gov (United States)

    Esfandyari, Tuba; Tefferi, Ayalew; Szmidt, Anna; Alain, Tommy; Zwolak, Pawel; Lasho, Terra; Lee, Patrick W; Farassati, Faris

    2009-12-01

    Overactivation in Ras signaling has been under intensive study as the molecular basis for development of cancer. Such overactivation can occur in the presence or absence of mutations in Ras gene resulting in activation of a series of down-stream effectors such as transcription factors. Different studies have shown the activation of Ras down-stream effectors in non-Hodgkin lymphoma (NHL) although mutations in Ras are not prevalent in this malignancy. Since overactivation in Ras signaling also increases permissiveness of cancer cells to infection by oncolytic versions of herpes simplex virus (e.g. R3616), we were interested in evaluating the value of transcription factors down-stream of Ras as molecular indicators for permissiveness to herpes therapy. In order to accomplish this, and also to assess the permissiveness of lymphoma cells to infection with R3616, we used NHL cell lines Daudi, Jurkat, NC37, Raji, Ramos and ST486. Once the levels of phosphorylation (activation) of extracellular-signal regulated kinase (ERK, a Ras effector pathway) and its down-stream transcription factor ELK were evaluated, Raji and NC37 showed a significant increase in the phosphorylation levels of both molecules while ATF2 (another transcription factor down-stream of p38-kinase pathway) seemed to be activated in all studied cells. Raji and NC37 cells were also most permissive cells to infection with R3616 while their permissiveness was decreased upon treatment of cells with an inhibitor of ELK-DNA binding portraying ERK/ELK as a suitable predictive indicator for selection of cancer cells with increased sensitivity to R3616. This study, therefore, for the first time documents permissiveness of lymphoma cells to oncolytic herpes viruses and introduces ELK as a suitable factor for predicting tumor susceptibility to these novel anticancer agents.

  15. Publishing Activity of RAS Economic Institutes amid New Challenges of Science Policy

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    Tret’yakova Ol’ga Valentinovna

    2017-01-01

    Full Text Available New challenges of science policy in Russia have determined a number of objectives which require scientific institutions to implement specific solutions aimed at improving research quality and efficiency. Since the evaluation of research results requires the use of different metrics, including bibliometrics, the main issue lies in the choice of indicators which can be used to conduct multi-analysis. The aim of this study is to implement the holistic approach to bibliometric assessment of scientific institutions and to analyze the performance of academic activity of economic institutions on the basis of the proposed criteria. The article summarizes the results of scientometric indicators monitoring, conducted at the Institute of Socio-Economic Development of Territories of the Russian Academy of Sciences. The analysis is based on a large set of publications of academic economic institutions for the period 2011–2015. The informational basis of the research includes data of the Russian Science Citation Index (RSCI. The institutions were ranked by a number of criteria: total number of publications in the RSCI and journals from the VAK list of Publications, number of citations. The author analyses the h-index value dynamics and indicators characterizing the number of publications considered as most-cited for the reference group in question. Conclusions and data obtained during analysis may be useful for studying development trends of economic science in Russia and assessing the performance of scientific organizations based on researching the dynamics of individual publication indicators and their comparison by reference group

  16. Absence of mutations in the coding sequence of the potential tumor suppressor 3pK in metastatic melanoma

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    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.

  17. Requirement for Ras/Rac1-Mediated p38 and c-Jun N-Terminal Kinase Signaling in Stat3 Transcriptional Activity Induced by the Src Oncoprotein

    Science.gov (United States)

    Turkson, James; Bowman, Tammy; Adnane, Jalila; Zhang, Yi; Djeu, Julie Y.; Sekharam, Madhavi; Frank, David A.; Holzman, Lawrence B.; Wu, Jie; Sebti, Said; Jove, Richard

    1999-01-01

    Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis. PMID:10523640

  18. Nuclear Ras2-GTP controls invasive growth in Saccharomyces cerevisiae.

    Science.gov (United States)

    Broggi, Serena; Martegani, Enzo; Colombo, Sonia

    2013-01-01

    Using an eGFP-RBD3 probe, which specifically binds Ras-GTP, we recently showed that the fluorescent probe was localized to the plasma membrane and to the nucleus in wild type cells growing exponentially on glucose medium, indicating the presence of active Ras in these cellular compartments. To investigate the nuclear function of Ras-GTP, we generated a strain where Ras2 is fused to the nuclear export signal (NES) from the HIV virus, in order to exclude this protein from the nucleus. Our results show that nuclear active Ras2 is required for invasive growth development in haploid yeast, while the expression of the NES-Ras2 protein does not cause growth defects either on fermentable or non-fermentable carbon sources and does not influence protein kinase A (PKA) activity related phenotypes analysed. Moreover, we show that the cAMP/PKA pathway controls invasive growth influencing the localization of active Ras. In particular, we show that PKA activity plays a role in the localization of active Ras and influences the ability of the cells to invade the agar: high PKA activity leads to a predominant nuclear accumulation of active Ras and induces invasive growth, while low PKA activity leads to plasma membrane localization of active Ras and to a defective invasive growth phenotype.

  19. Toll-like receptor 9 agonist IMO cooperates with cetuximab in K-ras mutant colorectal and pancreatic cancers.

    Science.gov (United States)

    Rosa, Roberta; Melisi, Davide; Damiano, Vincenzo; Bianco, Roberto; Garofalo, Sonia; Gelardi, Teresa; Agrawal, Sudhir; Di Nicolantonio, Federica; Scarpa, Aldo; Bardelli, Alberto; Tortora, Giampaolo

    2011-10-15

    K-Ras somatic mutations are a strong predictive biomarker for resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with colorectal and pancreatic cancer. We previously showed that the novel Toll-like receptor 9 (TLR9) agonist immunomodulatory oligonucleotide (IMO) has a strong in vivo activity in colorectal cancer models by interfering with EGFR-related signaling and synergizing with the anti-EGFR monoclonal antibody cetuximab. In the present study, we investigated, both in vitro and in vivo, the antitumor effect of IMO alone or in combination with cetuximab in subcutaneous colon and orthotopic pancreatic cancer models harboring K-Ras mutations and resistance to EGFR inhibitors. We showed that IMO was able to significantly restore the sensitivity of K-Ras mutant cancer cells to cetuximab, producing a marked inhibition of cell survival and a complete suppression of mitogen-activated protein kinase phosphorylation, when used in combination with cetuximab. IMO interfered with EGFR-dependent signaling, modulating the functional interaction between TLR9 and EGFR. In vivo, IMO plus cetuximab combination caused a potent and long-lasting cooperative antitumor activity in LS174T colorectal cancer and in orthotopic AsPC1 pancreatic cancer. The capability of IMO to restore cetuximab sensitivity was further confirmed by using K-Ras mutant colorectal cancer cell models obtained through homologous recombination technology. We showed that IMO markedly inhibits growth of K-Ras mutant colon and pancreatic cancers in vitro and in nude mice and cooperates with cetuximab via multiple mechanisms of action. Therefore, we propose IMO plus cetuximab as a therapeutic strategy for K-Ras wild-type as well for K-Ras mutant, cetuximab-resistant colorectal and pancreatic cancers. ©2011 AACR.

  20. Comparative analysis of radiosensitizers for K-RAS mutant rectal cancers.

    Directory of Open Access Journals (Sweden)

    Laura B Kleiman

    Full Text Available Approximately 40% of rectal cancers harbor activating K-RAS mutations, and these mutations are associated with poor clinical response to chemoradiotherapy. We aimed to identify small molecule inhibitors (SMIs that synergize with ionizing radiation (IR ("radiosensitizers" that could be incorporated into current treatment strategies for locally advanced rectal cancers (LARCs expressing mutant K-RAS. We first optimized a high-throughput assay for measuring individual and combined effects of SMIs and IR that produces similar results to the gold standard colony formation assay. Using this screening platform and K-RAS mutant rectal cancer cell lines, we tested SMIs targeting diverse signaling pathways for radiosensitizing activity and then evaluated our top hits in follow-up experiments. The two most potent radiosensitizers were the Chk1/2 inhibitor AZD7762 and the PI3K/mTOR inhibitor BEZ235. The chemotherapeutic agent 5-fluorouracil (5-FU, which is used to treat LARC, synergized with AZD7762 and enhanced radiosensitization by AZD7762. This study is the first to compare different SMIs in combination with IR for the treatment of K-RAS mutant rectal cancer, and our findings suggest that Chk1/2 inhibitors should be evaluated in new clinical trials for LARC.

  1. Mutations in SYNGAP1 Cause Intellectual Disability, Autism, and a Specific Form of Epilepsy by Inducing Haploinsufficiency

    DEFF Research Database (Denmark)

    Berryer, Martin H; Hamdan, Fadi F; Klitten, Laura L

    2013-01-01

    De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID...

  2. RGS6 Suppresses Ras-induced Cellular Transformation by Facilitating Tip60-mediated Dnmt1 Degradation and Promoting Apoptosis

    Science.gov (United States)

    Huang, Jie; Stewart, Adele; Maity, Biswanath; Hagen, Jussara; Fagan, Rebecca L.; Yang, Jianqi; Quelle, Dawn E.; Brenner, Charles; Fisher, Rory A.

    2014-01-01

    The RAS protooncogene plays a central role in regulation of cell proliferation, and point mutations leading to oncogenic activation of Ras occur in a large number of human cancers. Silencing of tumor suppressor genes by DNA methyltransferase 1 (Dnmt1) is essential for oncogenic cellular transformation by Ras, and Dnmt1 is over-expressed in numerous human cancers. Here we provide new evidence that the pleiotropic Regulator of G protein Signaling (RGS) family member RGS6 suppresses Ras-induced cellular transformation by facilitating Tip60-mediated degradation of Dmnt1 and promoting apoptosis. Employing mouse embryonic fibroblasts (MEFs) from wild type (WT) and RGS6−/− mice, we found that oncogenic Ras induced up-regulation of RGS6, which in turn blocked Ras-induced cellular transformation. RGS6 functions to suppress cellular transformation in response to oncogenic Ras by down regulating Dnmt1 protein expression leading to inhibition of Dnmt1-mediated anti-apoptotic activity. Further experiments showed that RGS6 functions as a scaffolding protein for both Dnmt1 and Tip60 and is required for Tip60-mediated acetylation of Dnmt1 and subsequent Dnmt1 ubiquitylation and degradation. The RGS domain of RGS6, known only for its GAP activity toward Gα subunits, was sufficient to mediate Tip60 association with RGS6. This work demonstrates a novel signaling action for RGS6 in negative regulation of oncogene-induced transformation and provides new insights into our understanding of the mechanisms underlying Ras-induced oncogenic transformation and regulation of Dnmt1 expression. Importantly, these findings identify RGS6 as an essential cellular defender against oncogenic stress and a potential therapeutic target for developing new cancer treatments. PMID:23995786

  3. Clustered Mutation Signatures Reveal that Error-Prone DNA Repair Targets Mutations to Active Genes.

    Science.gov (United States)

    Supek, Fran; Lehner, Ben

    2017-07-27

    Many processes can cause the same nucleotide change in a genome, making the identification of the mechanisms causing mutations a difficult challenge. Here, we show that clustered mutations provide a more precise fingerprint of mutagenic processes. Of nine clustered mutation signatures identified from >1,000 tumor genomes, three relate to variable APOBEC activity and three are associated with tobacco smoking. An additional signature matches the spectrum of translesion DNA polymerase eta (POLH). In lymphoid cells, these mutations target promoters, consistent with AID-initiated somatic hypermutation. In solid tumors, however, they are associated with UV exposure and alcohol consumption and target the H3K36me3 chromatin of active genes in a mismatch repair (MMR)-dependent manner. These regions normally have a low mutation rate because error-free MMR also targets H3K36me3 chromatin. Carcinogens and error-prone repair therefore redistribute mutations to the more important regions of the genome, contributing a substantial mutation load in many tumors, including driver mutations. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. 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

  5. XRP44X, an Inhibitor of Ras/Erk Activation of the Transcription Factor Elk3, Inhibits Tumour Growth and Metastasis in Mice.

    Directory of Open Access Journals (Sweden)

    Kostyantyn Semenchenko

    Full Text Available Transcription factors have an important role in cancer but are difficult targets for the development of tumour therapies. These factors include the Ets family, and in this study Elk3 that is activated by Ras oncogene /Erk signalling, and is involved in angiogenesis, malignant progression and epithelial-mesenchymal type processes. We previously described the identification and in-vitro characterisation of an inhibitor of Ras / Erk activation of Elk3 that also affects microtubules, XRP44X. We now report an initial characterisation of the effects of XRP44X in-vivo on tumour growth and metastasis in three preclinical models mouse models, subcutaneous xenografts, intra-cardiac injection-bone metastasis and the TRAMP transgenic mouse model of prostate cancer progression. XRP44X inhibits tumour growth and metastasis, with limited toxicity. Tumours from XRP44X-treated animals have decreased expression of genes containing Elk3-like binding motifs in their promoters, Elk3 protein and phosphorylated Elk3, suggesting that perhaps XRP44X acts in part by inhibiting the activity of Elk3. Further studies are now warranted to develop XRP44X for tumour therapy.

  6. [Effects of subchronic aluminum exposure on long-term potentiation and activities of RAS and extracellular regulated protein kinases in rats].

    Science.gov (United States)

    Song, J; Li, Z Y; Zhang, L; Niu, Q

    2017-05-20

    Objective: To study the effects of subchronic aluminum exposure on LTP and activities of RAS and ERK in rats in vivo. Methods: 24 Wistar rats were randomly divided into control group、low-dose group、medium-dose group and high-dose group, and received saline (control group) or Al (mal) (3) (15 μmol、kg、30 μmol、kg or 45 μmol/kg) via intraperitoneal injection (i.p.) for 8 weeks, respectively. The fEPSP in CA1 region were recorded by field potentiation technique in vivo and the hippocampal activities of RAS and ERK were examined by ELISA. Results: The fEPSP amplitudes of the control group were 1.90±0.19, 1.64±0.15 and 1.54±0.08 at 1, 30 and 60 min after HFS, respectively. The fEPSP amplitudes of the low-dose group were 1.40±0.06 at 60 min, which represented a statistically significant decrease compared to the control group ( P memory induced by aluminum.

  7. [Detection of RAS genes mutation using the Cobas®method in a private laboratory of pathology: Medical and economical study in comparison to a public platform of molecular biology of cancer].

    Science.gov (United States)

    Albertini, Anne-Flore; Raoux, Delphine; Neumann, Frédéric; Rossat, Stéphane; Tabet, Farid; Pedeutour, Florence; Duranton-Tanneur, Valérie; Kubiniek, Valérie; Vire, Olivier; Weinbreck, Nicolas

    In France, determination of the mutation status of RAS genes for predictive response to anti-EGFR targeted treatments is carried out by public platforms of molecular biology of cancer created by the French National Cancer Institute. This study aims to demonstrate the feasibility of these analyses by a private pathology laboratory (MEDIPATH) as per the requirements of accreditation. We retrospectively studied the mutation status of KRAS and NRAS genes in 163 cases of colorectal metastatic cancer using the Cobas ® technique. We compared our results to those prospectively obtained through pyrosequencing and allelic discrimination by the genetic laboratory of solid tumors at the Nice University Hospital (PACA-EST regional platform). The results of both series were identical: 98.7% positive correlation; negative correlation of 93.1%; overall correlation of 95.7% (Kappa=0.92). This study demonstrates the feasibility of molecular analysis in a private pathology laboratory. As this practice requires a high level of guarantee, its accreditation, according to the NF-EN-ISO15189 quality compliance French standard, is essential. Conducting molecular analysis in this context avoids the steps of routing the sample and the result between the pathology laboratory and the platform, which reduces the overall time of rendering the result. In conclusion, the transfer of some analysis from these platforms to private pathology laboratories would allow the platforms to be discharged from a part of routine testing and therefore concentrate their efforts to the development of new analyses constantly required to access personalized medicine. Copyright © 2017. Published by Elsevier Masson SAS.

  8. The Drosophila secreted protein Argos regulates signal transduction in the Ras/MAPK pathway.

    Science.gov (United States)

    Sawamoto, K; Okabe, M; Tanimura, T; Mikoshiba, K; Nishida, Y; Okano, H

    1996-08-25

    The Drosophila argos gene encodes a secreted protein with an EGF motif which acts as an inhibitor of cellular differentiation in multiple developmental processes. To investigate the cellular pathways regulated by Argos, we screened for mutations which could modify the phenotype caused by overexpression of argos. We show that the effects of argos overexpression on the eye and wing vein development are suppressed by gain-of-function mutations of the MAPKK/D-MEK gene (Dsor1/D-mek) and the MAPK/ERK-A gene (rolled) and were enhanced by loss-of-function mutations of Star. Loss-of-function mutations in components of the Ras/MAPK signaling cascade act as dominant suppressors of the phenotype caused by the argos null mutations. A loss-of-function argos mutation enhanced the overproduction of R7 neurons caused by gain-of-function alleles of Son of sevenless and Dsor1. Conversely, overexpression of argos inhibited formation of the extra R7 cells that was caused by high-level MAPK/ERK-A activity. A phenotype of the sev; argos double mutants revealed that sev is epistatic to argos. These results provide evidence that Argos negatively regulates signal transduction events in the Ras/MAPK cascade.

  9. Oncogenic K-Ras Signals through Epidermal Growth Factor Receptor and Wild-Type H-Ras to Promote Radiation Survival in Pancreatic and Colorectal Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Keith A. Cengel

    2007-04-01

    Full Text Available Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor o was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFRactivated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

  10. 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

  11. Norrie disease pedigree carrying the novel mutation C65Y, predicted to disrupt the cystine knot growth factor motif, analyzed by RasI restriction digestion

    Energy Technology Data Exchange (ETDEWEB)

    Strasberg, P.M.; Liede, H.A.; Stein, T. [Univ. of Toronto, Ontario (Canada)] [and others

    1994-09-01

    Norrie disease (MIM 310600; ND) is an X-linked (Xp11.2-11.3) neurodevelopmental disorder characterized by congenital blindness, retinal dysplasia with pseudoglioma formation, and often associated with progressive mental retardation and deafness. The ND gene, comprised of 3 exons, codes for an evolutionarily conserved protein of 133 amino acids. We have analyzed 8 pedigrees segregating Norrie disease. Although microdeletions have been detected in several typical ND patients, Southern blot analysis with probes L1.28, MAO-A, MAO-B, TIMP-3.9X, pTak8, and M27{beta} failed to detect such deletions in these 8 ND pedigrees. With the cloning of the ND gene, PCR analysis of all 3 exons likewise did not reveal any insertions or deletions. SSCP analysis ({sup 35}S-dNTP PCR) on PCR products of exon 3 showed a band shift for 1 patient. Repeat `cold` SSCP on minigels (3 inches x 4 inches) followed by liver staining was confirmatory. Direct sequencing revealed a G{r_arrow}A transition at nucleotide 610 corresponding to amino acid 65, changing Cys to Tyr. The mutation created an RsaI site, such that the uncut, normal, and mutant PCR products (using the same PCR primers) were 297 bp, 243 and 54 bp, and 177, 72 and 54 bp respectively. Affected males in the relevant pedigree had restricted PCR products of 177, 72 and 54 bp, carrier mothers 243, 177, 72, and 54 bp, and normals, including 30 unrelated individuals, 243 and 54 bp. Recent evidence indicates that the ND gene has a C-terminal domain homologous to that of TGF{beta}, thus identifying it as putative peptide growth factor, providing a monogenic disease model for the family of cystine knot growth factors. This is the first report of a mutation in Cys 2, critical for crosslinking to Cys 5 forming a disulphide bridge which holds the cystine knot growth factor tertiary structure together.

  12. BAD enables ceramide to signal apoptosis via Ras and Raf-1.

    Science.gov (United States)

    Basu, S; Bayoumy, S; Zhang, Y; Lozano, J; Kolesnick, R

    1998-11-13

    Prior investigations document that proliferative signaling cascades, under some circumstances, initiate apoptosis, although mechanisms that dictate the final outcome are largely unknown. In COS-7 cells, ceramide signals Raf-1 activation through Ras (Zhang, Y., Yao, B., Delikat, S., Bayoumy, S., Lin, X. H., Basu, S., McGinley, M., Chan-Hui, P. Y., Lichenstein, H., and Kolesnick, R. (1997) Cell 89, 63-72), but not apoptosis. However, expression of small amounts of the pro-apoptotic Bcl-2 family member, BAD, conferred ceramide-induced apoptosis onto COS-7 cells. Ceramide signaled apoptosis in BAD-expressing cells by a pathway involving sequentially kinase suppressor of Ras (KSR)/ceramide-activated protein kinase, Ras, c-Raf-1, and MEK1. Downstream, this pathway linked to BAD dephosphorylation at serine 136 by prolonged inactivation of Akt/PKB. Further, mutation of BAD at serine 136 abrogated ceramide signaling of apoptosis. The present study indicates that when ceramide signals through the Ras/Raf cascade, the availability of a single target, BAD, may dictate an apoptotic outcome.

  13. Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

    Science.gov (United States)

    Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

    1995-03-01

    Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

  14. Activating mutations in β-catenin in colon cancer cells alter their interaction with macrophages; the role of snail.

    Directory of Open Access Journals (Sweden)

    Pawan Kaler

    Full Text Available Tumor cells become addicted to both activated oncogenes and to proliferative and pro-survival signals provided by the abnormal tumor microenvironment. Although numerous soluble factors have been identified that shape the crosstalk between tumor cells and stroma, it has not been established how oncogenic mutations in the tumor cells alter their interaction with normal cells in the tumor microenvironment.We showed that the isogenic HCT116 and Hke-3 cells, which differ only by the presence of the mutant kRas allele, both stimulate macrophages to produce IL1β. In turn, macrophages enhanced Wnt signaling, proliferation and survival in both HCT116 and Hke-3 cells, demonstrating that signaling by oncogenic kRas in tumor cells does not impact their interaction with macrophages. HCT116 cells are heterozygous for β-catenin (HCT116(WT/MT, harboring one wild type (WT and one mutant (MT allele, but isogenic lines that carry only the WT (HCT116(WT or MT β-catenin allele (HCT116(MT have been generated. We showed that macrophages promoted Wnt signaling in cells that carry the MT β-catenin allele, but not in HCT116(WT cells. Consistent with this observation, macrophages and IL1β failed to stabilize Snail in HCT116(WT cells, and to protect these cells from TRAIL-induced apoptosis. Finally, we demonstrated that HCT116 cells expressing dominant negative TCF4 (dnTCF4 or HCT116 cells with silenced Snail failed to stimulate IL1β production in macrophages, demonstrating that tumor cells activate macrophages via a Wnt-dependent factor.Our data demonstrate that oncogenic β-catenin mutations in tumor cells, and subsequent activation of Wnt signaling, not only trigger cell-intrinsic alterations, but also have a significant impact on the crosstalk of tumor cells with the tumor associated macrophages.

  15. 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...... and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent...... response to contact inhibition, a separate back-up mechanism enforced cell cycle arrest at higher cell density....

  16. Mutation of NRAS is a Rare Genetic Event in Ovarian Low-Grade Serous Carcinoma

    DEFF Research Database (Denmark)

    Xing, Deyin; Rahmanto, Yohan Suryo; Zeppernick, Felix

    2017-01-01

    Activating mutations involving the members of the RAS signaling pathway, including KRAS, NRAS, and BRAF, have been reported in ovarian low-grade serous carcinoma and its precursor lesion, serous borderline tumor (SBT). Whether additional genetic alterations in the RAS oncogene family accumulate...... during the progression of serous borderline tumor (SBT) to invasive low grade serous carcinoma (LGSC) remains largely unknown. While mutations of KRAS and BRAF occur at a very early stage of progression, even preceding the development of SBT, additional driving events, such as NRAS mutations, have been...

  17. Impairment of K-Ras signaling networks and increased efficacy of epidermal growth factor receptor inhibitors by a novel synthetic miR-143.

    Science.gov (United States)

    Akao, Yukihiro; Kumazaki, Minami; Shinohara, Haruka; Sugito, Nobuhiko; Kuranaga, Yuki; Tsujino, Takuya; Yoshikawa, Yuki; Kitade, Yukio

    2018-03-02

    Despite considerable research on K-Ras inhibitors, none had been established until now. We synthesized nuclease-resistant synthetic miR-143 (miR-143#12), which strongly silenced K-Ras, its effector signal molecules AKT and ERK, and the K-Ras activator Sos1. We examined the anti-proliferative effect of miR-143#12 and the mechanism in human colon cancer DLD-1 cell (G13D) and other cell types harboring K-Ras mutations. Cell growth was markedly suppressed in a concentration-dependent manner by miR-143#12 (IC 50 : 1.32 nmol L -1 ) with a decrease in the K-Ras mRNA level. Interestingly, this mRNA level was also downregulated by either a PI3K/AKT or MEK inhibitor, which indicates a positive circuit of K-Ras mRNA expression. MiR-143#12 silenced cytoplasmic K-Ras mRNA expression and impaired the positive circuit by directly targeting AKT and ERK mRNA. Combination treatment with miR-143#12 and a low-dose EGFR inhibitor induced a synergistic inhibition of growth with a marked inactivation of both PI3K/AKT and MAPK/ERK signaling pathways. However, silencing K-Ras by siR-KRas instead of miR-143#12 did not induce this synergism through the combined treatment with the EGFR inhibitor. Thus, miR-143#12 perturbed the K-Ras expression system and K-Ras activation by silencing Sos1 and, resultantly, restored the efficacy of the EGFR inhibitors. The in vivo results also supported those of the in vitro experiments. The extremely potent miR-143#12 enabled us to understand K-Ras signaling networks and shut them down by combination treatment with this miRNA and EGFR inhibitor in K-Ras-driven colon cancer cell lines. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. MECP2 Is a Frequently Amplified Oncogene with a Novel Epigenetic Mechanism That Mimics the Role of Activated RAS in Malignancy

    DEFF Research Database (Denmark)

    Neupane, Manish; Clark, Allison P.; Landini, Serena

    2016-01-01

    for efficient transformation. These observations suggest that MECP2 is a commonly amplified oncogene with an unusual epigenetic mode of action. MECP2 is a commonly amplified oncogene in human malignancies with a unique epigenetic mechanism of action. Cancer Discov; 6(1); 45-58. ©2015 AACR.This article......An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MECP2) as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers......, and many cancer cell lines have amplified, overexpressed MECP2 and are dependent on MECP2 expression for growth. MECP2 copy-number gain and RAS family member alterations are mutually exclusive in several cancer types. The MECP2 splicing isoforms activate the major growth factor pathways targeted...

  20. 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.

  1. 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

  2. Phase I expansion and pharmacodynamic study of the oral MEK inhibitor RO4987655 (CH4987655) in selected patients with advanced cancer with RAS-RAF mutations

    NARCIS (Netherlands)

    Zimmer, Lisa; Barlesi, Fabrice; Martinez-Garcia, Maria; Dieras, Veronique; Schellens, Jan H M; Spano, Jean-Philippe; Middleton, Mark R; Calvo, Emiliano; Paz-Ares, Luiz; Larkin, James; Pacey, Simon; Venturi, Miro; Kraeber-Bodéré, Françoise; Tessier, Jean J L; Eberhardt, Wilfried Ernst Erich; Paques, Michel; Guarin, Ernesto; Meresse, Valerie; Soria, Jean-Charles

    2014-01-01

    PURPOSE: This phase I expansion study assessed safety, pharmacodynamic effects, and antitumor activity of RO4987655, a pure MEK inhibitor, in selected patients with advanced solid tumor. EXPERIMENTAL DESIGN: We undertook a multicenter phase I two-part study (dose escalation and cohort expansion).

  3. 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.

  4. Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype

    Directory of Open Access Journals (Sweden)

    Pluk Helma

    2009-07-01

    Full Text Available Abstract Background The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression. Results Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity. Conclusion The high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.

  5. A Drosophila immune response against Ras-induced overgrowth

    Directory of Open Access Journals (Sweden)

    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.

  6. Mitochondrial STAT3 contributes to transformation of Barrett's epithelial cells that express oncogenic Ras in a p53-independent fashion.

    Science.gov (United States)

    Yu, Chunhua; Huo, Xiaofang; Agoston, Agoston T; Zhang, Xi; Theiss, Arianne L; Cheng, Edaire; Zhang, Qiuyang; Zaika, Alexander; Pham, Thai H; Wang, David H; Lobie, Peter E; Odze, Robert D; Spechler, Stuart J; Souza, Rhonda F

    2015-08-01

    Metaplastic epithelial cells of Barrett's esophagus transformed by the combination of p53-knockdown and oncogenic Ras expression are known to activate signal transducer and activator of transcription 3 (STAT3). When phosphorylated at tyrosine 705 (Tyr705), STAT3 functions as a nuclear transcription factor that can contribute to oncogenesis. STAT3 phosphorylated at serine 727 (Ser727) localizes in mitochondria, but little is known about mitochondrial STAT3's contribution to carcinogenesis in Barrett's esophagus, which is the focus of this study. We introduced a constitutively active variant of human STAT3 (STAT3CA) into the following: 1) non-neoplastic Barrett's (BAR-T) cells; 2) BAR-T cells with p53 knockdown; and 3) BAR-T cells that express oncogenic H-Ras(G12V). STAT3CA transformed only the H-Ras(G12V)-expressing BAR-T cells (evidenced by loss of contact inhibition, formation of colonies in soft agar, and generation of tumors in immunodeficient mice), and did so in a p53-independent fashion. The transformed cells had elevated levels of both mitochondrial (Ser727) and nuclear (Tyr705) phospho-STAT3. Introduction of a STAT3CA construct with a mutated tyrosine phosphorylation site into H-Ras(G12V)-expressing Barrett's cells resulted in high levels of mitochondrial phospho-STAT3 (Ser727) with little or no nuclear phospho-STAT3 (Tyr705), and the cells still formed tumors in immunodeficient mice. Thus tyrosine phosphorylation of STAT3 is not required for tumor formation in Ras-expressing Barrett's cells. We conclude that mitochondrial STAT3 (Ser727) can contribute to oncogenesis in Barrett's cells that express oncogenic Ras. These findings suggest that agents targeting STAT3 might be useful for chemoprevention in patients with Barrett's esophagus. Copyright © 2015 the American Physiological Society.

  7. PRC2 loss amplifies Ras-driven transcription and confers sensitivity to BRD4-based therapies

    DEFF Research Database (Denmark)

    De Raedt, Thomas; Beert, Eline; Pasmant, Eric

    2014-01-01

    Ras. We show that SUZ12 loss potentiates the effects of NF1 mutations by amplifying Ras-driven transcription through effects on chromatin. Importantly, however, SUZ12 inactivation also triggers an epigenetic switch that sensitizes these cancers to bromodomain inhibitors. Collectively, these studies...

  8. The association of clinicopathological features and survival in colorectal cancer patients with kras mutation status

    OpenAIRE

    Tulay Akman; Ilhan Oztop; Yasemin Baskin; Ilkay Tugba Unek; Necla Demir; Hulya Ellidokuz; Ahmet Ugur Yilmaz

    2016-01-01

    Background: KRAS mutations have a significant role in the consecutive activation of RAS.RAF.MEK.ERK pathway in colorectal cancer.Approximately 30.35% of sporadic colorectal cancers have KRAS mutation. While the predictive role of KRAS is commonly accepted at the present time, its prognostic role and association with different clinical and histopathological properties are currently unclear and inconsistent. The intent of this study, has been to evaluate the associations between KRAS gene mutat...

  9. Immunohistochemical assay for detection of K-ras protein expression in metastatic colorectal cancer

    International Nuclear Information System (INIS)

    Tag Elsabah, M.; Iman Adel, I.

    2013-01-01

    Background: The monoclonal antibodies (mAbs) that target the epidermal growth factor receptor (EGFR) had expanded the range of treatment options for metastatic colorectal cancer. However, such type of treatment was shown to be ineffective if there is K-ras mutation. In most previous studies K-ras gene mutation was mainly assessed by PCR. Aim: Our work is designed to detect K-ras protein expression by immunohistochemistry (IHC) aiming to reach a preliminary method that could be confirmed by PCR and considered an alternative way for the detection of K-ras aberration. We are also aiming to find a relation between K-ras protein expression and K-ras gene mutation. Materials and methods: Paraffin embedded tissue samples from 26 metastatic colorectal cancer (mCRC) patients were analyzed for K-ras protein expression by IHC using RaplA polyclonal antibody. Staining patterns were subjectively assessed and correlated with clinicopathological features. The results were statistically evaluated using the Chi-square test. Results: K-ras cytoplasmic positivity was observed in 42.3% of cases. The positivity was either strong in 26.9% or moderate in 15.4%. With respect to adenocarcinoma variants, 50% of cases were positive for K-ras protein expression while all mucinous and signet ring types were negative. The positivity was noted in 50% of moderately differentiated GII colorectal carcinomas as compared with 38.9% in poorly differentiated GIII. Positive staining was observed in 40% of cases with positive lymph node metastasis while in the absence of nodal metastasis the positivity was 45.5%. No significant correlation was found between clinicopathological parameters and K-ras staining results. Conclusion: IHC may compliment PCR in the detection of K-ras mutation.

  10. Promoter trans-activation of protooncogenes c-fos and c-myc, but not c-Ha-ras, by products of adenovirus early region 1A

    International Nuclear Information System (INIS)

    Sassone-Corsi, P.; Borrelli, E.

    1987-01-01

    The E1A (early region 1A) oncogene products of adenovirus type 2 trans-activate the other early viral transcription units, as well as some cellular promoters. Using a short-term cotransfection assay in murine NIH 3T3 fibroblasts, we show that c-fos and c-myc promoter activities are stimulated by the E1A proteins, whereas c-Ha-ras transcription is not affected. The product of E1A 13S mRNA is responsible for the trans-activation, whereas the 12S mRNA product has no effect. Analysis of the c-fos promoter sequences required for the E1A stimulation shows that responsive sequences are located between positions -402 and -240 upstream of the transcription initiation site. This same region also contains the c-fos serum-responsive element. Furthermore, transcription of the endogenous c-fos gene in HeLa cells is increased after E1A transfection

  11. Novel molecular targets for kRAS downregulation: promoter G-quadruplexes

    Science.gov (United States)

    2016-11-01

    monitor the effect of G4 formation, rather than mutating the promoter sequence. We screened the Diversity Set II and III from the National Cancer...Bgl I (kRAS-500 plasmid) or Nhe I (all kRAS-324 plasmids) and HIND III cut sites. kRAS-324 was con- structed by Operon; the kRAS-500, 324 mt Near, and...molecular targets for breast cancer therapeutics, Molecules ( Basel , Switz.) 18 (2013) 15019–15034. [38] T.C. He, A.B. Sparks, C. Rago, H. Hermeking

  12. Oncogenically active MYD88 mutations in human lymphoma

    Science.gov (United States)

    Ngo, Vu N.; Young, Ryan M.; Schmitz, Roland; Jhavar, Sameer; Xiao, Wenming; Lim, Kian-Huat; Kohlhammer, Holger; Xu, Weihong; Yang, Yandan; Zhao, Hong; Shaffer, Arthur L.; Romesser, Paul; Wright, George; Powell, John; Rosenwald, Andreas; Muller-Hermelink, Hans Konrad; Ott, German; Gascoyne, Randy D.; Connors, Joseph M.; Rimsza, Lisa M.; Campo, Elias; Jaffe, Elaine S.; Delabie, Jan; Smeland, Erlend B.; Fisher, Richard I.; Braziel, Rita M.; Tubbs, Raymond R.; Cook, J. R.; Weisenburger, Denny D.; Chan, Wing C.; Staudt, Louis M.

    2016-01-01

    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy1. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling2,3, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt’s lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, theMYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations

  13. Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

    Science.gov (United States)

    Lee, Hong-Won; Kyung, Taeyoon; Yoo, Janghyun; Kim, Tackhoon; Chung, Chaeuk; Ryu, Ji Young; Lee, Hanki; Park, Kihyun; Lee, Sangkyu; Jones, Walton D.; Lim, Dae-Sik; Hyeon, Changbong; Do Heo, Won; Yoon, Tae-Young

    2013-01-01

    Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein–protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein–protein interactions as they occur in unpurified cell extracts. By analysing single Ras–Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein–protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein–protein interaction level. PMID:23422673

  14. 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

    , 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...... 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....... importance, in c-rasH, of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-rasH transformation. Relative to the respective transforming activity of wild-type c-rasH and v-rasH, mutants with lesions in codons 102 and 103 were significantly less active in their c-rasH forms than...

  15. [Clinical relevance of the K-ras oncogene in colorectal cancer: experience in a Mexican population].

    Science.gov (United States)

    Cabrera-Mendoza, F; Gainza-Lagunes, S; Castañeda-Andrade, I; Castro-Zárate, A

    2014-01-01

    Colorectal cancer is frequent in the developed countries, with a cancer-specific mortality rate of 33%. Different biomarkers are associated with overall survival and the prediction of monoclonal treatment effectiveness. The presence of mutations in the K-ras oncogene alters the response to target therapy with cetuximab and could be an independent prognostic factor. To analyze the difference in survival between patients with mutated K-ras and those with K-ras wild-type status. Thirty-one clinical records were retrospectively analyzed of patients presenting with colorectal cancer that underwent K-ras sequencing through real-time polymerase chain reaction within the time frame of 2009 to 2012 at the Hospital de Alta Especialidad de Veracruz of the Instituto para la Salud y Seguridad Social de los Trabajadores del Estado (HAEV-ISSSTE). Survival analysis for patients with and without K-ras mutation was performed using the Kaplan Meier method. Contrast of covariates was performed using logarithmic transformations. No statistically significant difference was found in relation to survival in the patients with mutated K-ras vs. those with K-ras wild-type (P=.416), nor were significant differences found when analyzing the covariants and survival in the patients with mutated K-ras: ECOG scale (P=.221); age (less than, equal to or greater than 65years, P=.441); clinical stage according to the AJCC (P=.057), and primary lesion site (P=.614). No relation was found between the K-ras oncogene mutation and reduced survival, in contrast to what has been established in the international medical literature. Further studies that include both a larger number of patients and those receiving monoclonal treatment, need to be conducted. There were only 5 patients in the present study that received cetuximab, resulting in a misleading analysis. Copyright © 2013 Asociación Mexicana de Gastroenterología. Published by Masson Doyma México S.A. All rights reserved.

  16. Coordinating ERK signaling via the molecular scaffold Kinase Suppressor of Ras [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Danielle Frodyma

    2017-08-01

    Full Text Available Many cancers, including those of the colon, lung, and pancreas, depend upon the signaling pathways induced by mutated and constitutively active Ras. The molecular scaffolds Kinase Suppressor of Ras 1 and 2 (KSR1 and KSR2 play potent roles in promoting Ras-mediated signaling through the Raf/MEK/ERK kinase cascade. Here we summarize the canonical role of KSR in cells, including its central role as a scaffold protein for the Raf/MEK/ERK kinase cascade, its regulation of various cellular pathways mediated through different binding partners, and the phenotypic consequences of KSR1 or KSR2 genetic inactivation. Mammalian KSR proteins have a demonstrated role in cellular and organismal energy balance with implications for cancer and obesity. Targeting KSR1 in cancer using small molecule inhibitors has potential for therapy with reduced toxicity to the patient. RNAi and small molecule screens using KSR1 as a reference standard have the potential to expose and target vulnerabilities in cancer. Interestingly, although KSR1 and KSR2 are similar in structure, KSR2 has a distinct physiological role in regulating energy balance. Although KSR proteins have been studied for two decades, additional analysis is required to elucidate both the regulation of these molecular scaffolds and their potent effect on the spatial and temporal control of ERK activation in health and disease.

  17. 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

  18. Activation of growth factor secretion in tumorigenic states of breast cancer induced by 17β-estradiol or v-Ha-ras oncogene

    International Nuclear Information System (INIS)

    Dickson, R.B.; Kasid, A.; Huff, K.K.; Bates, S.E.; Knabbe, C.; Bronzert, D.; Gelmann, E.P.; Lippman, M.E.

    1987-01-01

    The MCF-7 human breast cancer cell line responds to estrogen stimulation in vitro by increased secretion of growth factors and proliferation and in vivo by tumor formation in the nude mouse. To test a possible role of growth factor secretion in expression of the tumorigenic phenotype, the authors stably transfected MCF-7 cells with the v-Ha-ras oncogene to produce the MCF-7ras cell line. The MCF-7ras cell line was tumorigenic in the absence of estrogens and secreted 3- to 5-fold elevated levels of a high molecular weight form of a type α transforming growth factor-like growth factor, type β transforming growth factor, and insulin-like growth factor I. MCF-7ras cells, in contrast to MCF-7, were less sensitive to further growth stimulation by estrogen, type α transforming growth factor, and insulin-like growth factor I and showed little change in receptor levels for these hormones. Conditioned medium from MCF-7ras cells as well as two of its component growth factors replaced estrogen in stimulating MCF-7 colony formation in vitro. A coordinate increase in growth factor secretion by human breast cancer may contribute to its escape from estrogen dependence

  19. Three faces of recombination activating gene 1 (RAG1) mutations.

    Science.gov (United States)

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam

    2015-12-01

    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation.

  20. Occurrence of mutations in the epidermal growth factor receptor gene in X-ray-induced rat lung tumors

    International Nuclear Information System (INIS)

    Kitahashi, Tsukasa; Takahashi, Mami; Yamada, Yutaka

    2008-01-01

    Epidermal growth factor receptor (EGFR) gene alterations have been found in human lung cancers. However, there is no information on the factors inducing EGFR mutations. In rodents, K-ras mutations are frequently found in many lung carcinogenesis models, but hitherto, Egfr mutations have not been reported. Their presence was therefore investigated in representative lung carcinogenesis models with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosobis(2-hydroxypropyl)amine (BHP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) and ethyl carbamate (urethane), as well as X-ray irradiation. With the chemical carcinogenesis models, no mutations were detected in Egfr, which is in clear contrast to the high rates observed in either codon 12 or 61 of K-ras (21/23 of the lung tumors induced with NNK, 4/5 with MelQx, 1/4 with urethane and 7/18 with BHP). However, in the X-ray-induced lung tumors, Egfr mutations with amino acid substitution were observed in exons 18 and 21 (4/12, 33%), but no activating mutation of K-ras was detected. In addition, one and four silent mutations were identified in K-ras (exon 1) and Egfr (exons 18, 20 and 21), respectively. Most mutations in both Egfr and K-ras were G/C→A/T transitions (7/8, 88% and 31/34, 91%, respectively). Although, the mutational patterns in equivalent human lesions were not completely coincident, this first report of Egfr mutations in an experimental lung tumor model suggests that X-rays or other factors producing oxygen radicals could cause EGFR mutations in some proportion of lung cancers in humans. (author)

  1. 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

    by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-rasH as well as for c-rasH, which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential......, 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...

  2. The role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway.

    Science.gov (United States)

    Heeren, Gino; Jarolim, Stefanie; Laun, Peter; Rinnerthaler, Mark; Stolze, Klaus; Perrone, Gabriel G; Kohlwein, Sepp D; Nohl, Hans; Dawes, Ian W; Breitenbach, Michael

    2004-11-01

    We show that the dominant activated allele of the yeast RAS gene, RAS2(ala18,val19), led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cell-specific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2(+) rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2(+) rho-zero strain to that of the wild-type cells. The double mutant RAS2(ala18,val19) rho-zero cells showed the same lifespan as the RAS2(ala18,val19) cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2(ala18,val19) mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.

  3. Transcriptional activation of the Axl and PDGFR-α by c-Met through a ras- and Src-independent mechanism in human bladder cancer

    International Nuclear Information System (INIS)

    Yeh, Chen-Yun; Tseng, Vincent S; Lee, Yuan-Chii G; Shen, Cheng-Huang; Chow, Nan-Haw; Liu, Hsiao-Sheng; Shin, Shin-Mei; Yeh, Hsuan-Heng; Wu, Tsung-Jung; Shin, Jyh-Wei; Chang, Tsuey-Yu; Raghavaraju, Giri; Lee, Chung-Ta; Chiang, Jung-Hsien

    2011-01-01

    A cross-talk between different receptor tyrosine kinases (RTKs) plays an important role in the pathogenesis of human cancers. Both NIH-Met5 and T24-Met3 cell lines harboring an inducible human c-Met gene were established. C-Met-related RTKs were screened by RTK microarray analysis. The cross-talk of RTKs was demonstrated by Western blotting and confirmed by small interfering RNA (siRNA) silencing, followed by elucidation of the underlying mechanism. The impact of this cross-talk on biological function was demonstrated by Trans-well migration assay. Finally, the potential clinical importance was examined in a cohort of 65 cases of locally advanced and metastatic bladder cancer patients. A positive association of Axl or platelet-derived growth factor receptor-alpha (PDGFR-α) with c-Met expression was demonstrated at translational level, and confirmed by specific siRNA knock-down. The transactivation of c-Met on Axl or PDGFR-α in vitro was through a ras- and Src-independent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. In human bladder cancer, co-expression of these RTKs was associated with poor patient survival (p < 0.05), and overexpression of c-Met/Axl/PDGFR-α or c-Met alone showed the most significant correlation with poor survival (p < 0.01). In addition to c-Met, the cross-talk with Axl and/or PDGFR-α also contributes to the progression of human bladder cancer. Evaluation of Axl and PDGFR-α expression status may identify a subset of c-Met-positive bladder cancer patients who may require co-targeting therapy

  4. RAS/MAPK activation is associated with reduced tumor-infiltrating lymphocytes in triple-negative breast cancer: therapeutic cooperation between MEK and PD-1/PD-L1 immune checkpoint inhibitors

    Science.gov (United States)

    Loi, Sherene; Dushyanthen, Sathana; Beavis, Paul A; Salgado, Roberto; Denkert, Carsten; Savas, Peter; Combs, Susan; Rimm, David L.; Giltnane, Jennifer M.; Estrada, Monica V.; Sánchez, Violeta; Sanders, Melinda E.; Cook, Rebecca S.; Pilkinton, Mark A.; Mallal, Simon A.; Wang, Kai; Miller, Vincent A.; Stephens, Phil J.; Yelensky, Roman; Doimi, Franco D.; Gómez, Henry; Ryzhov, Sergey V.; Darcy, Phillip K.; Arteaga, Carlos L.; Balko, Justin M.

    2015-01-01

    Purpose Tumor-infiltrating lymphocytes (TILs) in the residual disease (RD) of triple-negative breast cancers (TNBCs) after neoadjuvant chemotherapy (NAC) are associated with improved survival, but insight into tumor cell-autonomous molecular pathways affecting these features are lacking. Experimental Design We analyzed TILs in the RD of clinically and molecularly characterized TNBCs after NAC and explored therapeutic strategies targeting combinations of MEK inhibitors with PD-1/PD-L1-targeted immunotherapy in mouse models of breast cancer. Results Presence of TILs in the RD was significantly associated with improved prognosis. Genetic or transcriptomic alterations in Ras/MAPK signaling were significantly correlated with lower TILs. MEK inhibition up-regulated cell-surface major histocompatibility complex (MHC) expression and PD-L1 in TNBC cells both in vivo and in vitro. Moreover, combined MEK and PDL-1/PD-1 inhibition enhanced anti-tumor immune responses in mouse models of breast cancer. Conclusions These data suggest the possibility that Ras/MAPK pathway activation promotes immune-evasion in TNBC, and support clinical trials combining MEK- and PD-L1-targeted therapies. Furthermore, Ras/MAPK activation and MHC expression may be predictive biomarkers of response to immune checkpoint inhibitors. PMID:26515496

  5. 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

    2015-04-10

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. 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

  7. Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation.

    Science.gov (United States)

    Zhang, Leilei; He, Jie; Han, Bing; Lu, Linna; Fan, Jiayan; Zhang, He; Ge, Shengfang; Zhou, Yixiong; Jia, Renbing; Fan, Xianqun

    2016-01-01

    Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease.

  8. GCM2-Activating Mutations in Familial Isolated Hyperparathyroidism.

    Science.gov (United States)

    Guan, Bin; Welch, James M; Sapp, Julie C; Ling, Hua; Li, Yulong; Johnston, Jennifer J; Kebebew, Electron; Biesecker, Leslie G; Simonds, William F; Marx, Stephen J; Agarwal, Sunita K

    2016-11-03

    Primary hyperparathyroidism (PHPT) is a common endocrine disease characterized by parathyroid hormone excess and hypercalcemia and caused by hypersecreting parathyroid glands. Familial PHPT occurs in an isolated nonsyndromal form, termed familial isolated hyperparathyroidism (FIHP), or as part of a syndrome, such as multiple endocrine neoplasia type 1 or hyperparathyroidism-jaw tumor syndrome. The specific genetic or other cause(s) of FIHP are unknown. We performed exome sequencing on germline DNA of eight index-case individuals from eight unrelated kindreds with FIHP. Selected rare variants were assessed for co-segregation in affected family members and screened for in an additional 32 kindreds with FIHP. In eight kindreds with FIHP, we identified three rare missense variants in GCM2, a gene encoding a transcription factor required for parathyroid development. Functional characterization of the GCM2 variants and deletion analyses revealed a small C-terminal conserved inhibitory domain (CCID) in GCM2. Two of the three rare variants were recurrent, located in the GCM2 CCID, and found in seven of the 40 (18%) kindreds with FIHP. These two rare variants acted as gain-of-function mutations that increased the transcriptional activity of GCM2, suggesting that GCM2 is a parathyroid proto-oncogene. Our results demonstrate that germline-activating mutations affecting the CCID of GCM2 can cause FIHP. Published by Elsevier Inc.

  9. Noonan syndrome gain-of-function mutations in NRAS cause zebrafish gastrulation defects

    NARCIS (Netherlands)

    Runtuwene, V.J.; van Eekelen, M.J.L.; Overvoorde, J.; Rehmann, H.; Yntema, H.G.; Nillesen, W.M.; van Haeringen, A.; van der Burgt, I.; Burgering, B.; den Hertog, J.

    2011-01-01

    Noonan syndrome is a relatively common developmental disorder that is characterized by reduced growth, wide-set eyes and congenital heart defects. Noonan syndrome is associated with dysregulation of the Ras-mitogen-activated-protein-kinase (MAPK) signaling pathway. Recently, two mutations in NRAS

  10. Effect of cyclic hydrodynamic pressure-induced proliferation of human bladder smooth muscle through Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2.

    Science.gov (United States)

    Wu, Tao; Chen, Lin; Wei, Tangqiang; Wang, Yan; Xu, Feng; Wang, Kunjie

    2012-09-01

    To examine the role of Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 in the cyclic hydrodynamic pressure-induced proliferation of human bladder smooth muscle cells. Human bladder smooth muscle cells were exposed to cyclic hydrodynamic pressures in vitro with defined parameters (static, 100 cmH(2) O, 200 cmH(2) O and 300 cmH(2) O pressure) for 24 h. The proliferation of cells was assessed by flow cytometry. Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 messenger ribonucleic acid, and protein expression was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the Rac1 was determined with real-time polymerase chain reaction and Western blot technique with small interfering ribonucleic acid transfection and Rac1 inhibitor (NSC23766). The proliferation of human bladder smooth muscle cells was increased. Ras-related C3 botulinum toxin substrate 1, mitogen-activated protein kinase kinase 1/2 and extracellular regulated protein kinases 1/2 were activated by 200 and 300 cmH(2) O cyclic hydrodynamic pressure compared with static and 100 cmH(2) O pressure. The "knockdown" of activation of Rac1 using target small interfering ribonucleic acid transfection and Rac1 inhibitor (NSC23766) decreased proliferation of human bladder smooth muscle cells, and downregulated mitogen-activated protein kinase kinase 1/2, extracellular regulated protein kinases 1/2. The Rac1 pathway is activated in mechanotransduction and regulation of human bladder smooth muscle cell proliferation in response to cyclic hydrodynamic pressure. © 2012 The Japanese Urological Association.

  11. The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

    International Nuclear Information System (INIS)

    Kim, Han-Soo; Kim, Ju Won; Gang, Jingu; Wen, Jing; Koh, Sang Seok; Koh, Jong Sung; Chung, Hyun-Ho; Song, Si Young

    2006-01-01

    LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21 CIP1/WAF1 , RhoB and EGFR, that can explain the differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21 CIP1/WAF1 and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21 CIP1/WAF1 and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells

  12. Mediation of suppression of c-fos transcription in rasT24-transformed rat cells by a cis-acting repressor element.

    Science.gov (United States)

    Osei-Frimpong, J; Sepulveda, J; Rangdaeng, S; Lebovitz, R M

    1994-06-01

    Prolonged expression of activated ras mutants resulted in both neoplastic transformation and suppression of serum-induced c-fos expression in Rat1 fibroblasts. Expression of other serum-inducible genes, including c-jun and beta-actin, was not suppressed in ras-transformed Rat1 cells, indicating that these effects are specific for c-fos and that growth-factor signal transduction pathways remain essentially intact. Run-on transcription studies indicated that c-fos transcription was blocked at the level of initiation in these cells. Transient transfection studies using 360 bp from the wild-type c-fos promoter as well as a series of mutated c-fos promoter fragments linked to the chloramphenicol acetyltransferase gene indicated that repression of c-fos was mediated by approximately 49 bp immediately upstream of the dyad symmetry element (DSE). Deletion of this region, referred to as the upstream repressor region (URR), restored serum inducibility to the c-fos promoter in ras-transformed cells. In contrast, suppression of c-fos transcription was not affected by either deletion of 240 bp between the DSE and the TATA element or by base-substitution mutations that inactive the ternary complex factor and fos-AP-1-like binding sites. In addition, in vitro competition studies indicated that ras-transformed cells express one or more repressor factors that interact with as-yet-unidentified elements within the c-fos promoter (possibly the URR) and block serum induction of c-fos. These findings suggest that prolonged expression of activated ras results in the activation of one or more as-yet-unidentified proteins that suppress transcription of the c-fos gene by interacting with the URR.

  13. Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline.

    Science.gov (United States)

    Giannoulatou, Eleni; McVean, Gilean; Taylor, Indira B; McGowan, Simon J; Maher, Geoffrey J; Iqbal, Zamin; Pfeifer, Susanne P; Turner, Isaac; Burkitt Wright, Emma M M; Shorto, Jennifer; Itani, Aysha; Turner, Karen; Gregory, Lorna; Buck, David; Rajpert-De Meyts, Ewa; Looijenga, Leendert H J; Kerr, Bronwyn; Wilkie, Andrew O M; Goriely, Anne

    2013-12-10

    The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline.

  14. 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.

  15. Ras1(CA) overexpression in the posterior silk gland improves silk yield.

    Science.gov (United States)

    Ma, Li; Xu, Hanfu; Zhu, Jinqi; Ma, Sanyuan; Liu, Yan; Jiang, Rong-Jing; Xia, Qingyou; Li, Sheng

    2011-06-01

    Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm, Bombyx mori, but has reached a plateau during the last decades. For the first time, we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%, while increasing food consumption by only 20%. Ras activation by Ras1(CA) overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins, up-regulated fibroin mRNA levels, increased total DNA content, and stimulated endoreplication. Moreover, Ras1 activation increased cell and nuclei sizes, enriched subcellular organelles related to protein synthesis, and stimulated ribosome biogenesis for mRNA translation. We conclude that Ras1 activation increases cell size and protein synthesis in the posterior silk gland, leading to silk yield improvement.

  16. Studies on the Contribution of Cox-2 Expression in the Progression of Oral Squamous Cell Carcinoma and H-Ras Activation.

    Science.gov (United States)

    Moazeni-Roodi, Abdolkarim; Allameh, Abdolamir; Harirchi, Iraj; Motiee-Langroudi, Maziar; Garajei, Ata

    2017-04-01

    The aim of this study was to investigate the relationship between the H-ras and Cox-2 gene expression in tumors from Iranian Oral Squamous Cell Carcinoma (OSCC) patients. Fresh tumor biopsies removed from oral cavity were collected from 67 new cases. Total RNA was extracted from biopsies and processed for quantification of H-ras and Cox-2 specific RNA expression using real-time PCR (QPCR). In addition, 59 gingival biopsies from apparently normal individuals were processed for QPCR assays. The results showed that Cox-2 expression at mRNA levels was at minimal levels in normal gingival biopsies. However, there was a surge in Cox-2 expression in tumor tissues (11.5 fold, p Cox-2 expression was elevated depending on the tumor grade and there was a 1.7 fold increase (p = 0.003) in tumors diagnosed as MD/PD compared to that pathologically diagnosed as WD. This inflammatory marker was increased more significantly in smoker patients compared to non-smoker matching group. The H-ras expression at mRNA levels was significantly higher in OSCC samples compared to normal gingival (3 fold; p = 0.044). This expression was significantly higher in tumors diagnosed as MD/PD compared to WD (1.59 fold, p = 0.033). In conclusion, we found a correlation between H-ras expression and Cox-2 induction in OSCC tissue, suggesting that together these genes are contributing to cancer progression. Cox-2 is an early event in cancers of mucosal epithelial cells and a surge in Cox-2 expression in OSCC could be partly due to pro-inflammatory factors such as smoking.

  17. FLI-1 Flightless-1 and LET-60 Ras control germ line morphogenesis in C. elegans

    Directory of Open Access Journals (Sweden)

    Dentler William L

    2008-05-01

    Full Text Available Abstract Background In the C. elegans germ line, syncytial germ line nuclei are arranged at the cortex of the germ line as they exit mitosis and enter meiosis, forming a nucleus-free core of germ line cytoplasm called the rachis. Molecular mechanisms of rachis formation and germ line organization are not well understood. Results Mutations in the fli-1 gene disrupt rachis organization without affecting meiotic differentiation, a phenotype in C. elegans referred to here as the germ line morphogenesis (Glm phenotype. In fli-1 mutants, chains of meiotic germ nuclei spanned the rachis and were partially enveloped by invaginations of germ line plasma membrane, similar to nuclei at the cortex. Extensions of the somatic sheath cells that surround the germ line protruded deep inside the rachis and were associated with displaced nuclei in fli-1 mutants. fli-1 encodes a molecule with leucine-rich repeats and gelsolin repeats similar to Drosophila flightless 1 and human Fliih, which have been shown to act as cytoplasmic actin regulators as well as nuclear transcriptional regulators. Mutations in let-60 Ras, previously implicated in germ line development, were found to cause the Glm phenotype. Constitutively-active LET-60 partially rescued the fli-1 Glm phenotype, suggesting that LET-60 Ras and FLI-1 might act together to control germ line morphogenesis. Conclusion FLI-1 controls germ line morphogenesis and rachis organization, a process about which little is known at the molecular level. The LET-60 Ras GTPase might act with FLI-1 to control germ line morphogenesis.

  18. Src homology phosphotyrosyl phosphatase 2 mediates cisplatin-related drug resistance by inhibiting apoptosis and activating the Ras/PI3K/Akt1/survivin pathway in lung cancer cells.

    Science.gov (United States)

    Tang, Chunlan; Luo, Hu; Luo, Dan; Yang, Heping; Zhou, Xiangdong

    2018-02-01

    decreased in the H446, H446-SHP2-OE and H446/CDDP cells. However, upon Ras RNA interference, the SHP2 expression was not significantly changed, but the expression of Akt1, pAkt1 and survivin was significantly increased in the H446-SHP2-OE and H446/CDDP cells. In conclusion, SHP2 is a new cisplatin resistance-related phosphatase in lung cancer, which inhibits apoptosis by activating the Ras/PI3K/Akt1/survivin signaling pathway.

  19. Heterozygous Germline Mutations in the CBL Tumor-Suppressor Gene Cause a Noonan Syndrome-like Phenotype

    Science.gov (United States)

    Martinelli, Simone; De Luca, Alessandro; Stellacci, Emilia; Rossi, Cesare; Checquolo, Saula; Lepri, Francesca; Caputo, Viviana; Silvano, Marianna; Buscherini, Francesco; Consoli, Federica; Ferrara, Grazia; Digilio, Maria C.; Cavaliere, Maria L.; van Hagen, Johanna M.; Zampino, Giuseppe; van der Burgt, Ineke; Ferrero, Giovanni B.; Mazzanti, Laura; Screpanti, Isabella; Yntema, Helger G.; Nillesen, Willy M.; Savarirayan, Ravi; Zenker, Martin; Dallapiccola, Bruno; Gelb, Bruce D.; Tartaglia, Marco

    2010-01-01

    RAS signaling plays a key role in controlling appropriate cell responses to extracellular stimuli and participates in early and late developmental processes. Although enhanced flow through this pathway has been established as a major contributor to oncogenesis, recent discoveries have revealed that aberrant RAS activation causes a group of clinically related developmental disorders characterized by facial dysmorphism, a wide spectrum of cardiac disease, reduced growth, variable cognitive deficits, ectodermal and musculoskeletal anomalies, and increased risk for certain malignancies. Here, we report that heterozygous germline mutations in CBL, a tumor-suppressor gene that is mutated in myeloid malignancies and encodes a multivalent adaptor protein with E3 ubiquitin ligase activity, can underlie a phenotype with clinical features fitting or partially overlapping Noonan syndrome (NS), the most common condition of this disease family. Independent CBL mutations were identified in two sporadic cases and two families from among 365 unrelated subjects who had NS or suggestive features and were negative for mutations in previously identified disease genes. Phenotypic heterogeneity and variable expressivity were documented. Mutations were missense changes altering evolutionarily conserved residues located in the RING finger domain or the linker connecting this domain to the N-terminal tyrosine kinase binding domain, a known mutational hot spot in myeloid malignancies. Mutations were shown to affect CBL-mediated receptor ubiquitylation and dysregulate signal flow through RAS. These findings document that germline mutations in CBL alter development to cause a clinically variable condition that resembles NS and that possibly predisposes to malignancies. PMID:20619386

  20. A Novel Ras Inhibitor (MDC-1016 Reduces Human Pancreatic Tumor Growth in Mice

    Directory of Open Access Journals (Sweden)

    Gerardo G Mackenzie

    2013-10-01

    Full Text Available Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its persistent resistance to chemotherapy. The currently limited treatment options for pancreatic cancer underscore the need for more efficient agents. Because activating Kras mutations initiate and maintain pancreatic cancer, inhibition of this pathway should have a major therapeutic impact. We synthesized phospho-farnesylthiosalicylic acid (PFTS; MDC-1016 and evaluated its efficacy, safety, and metabolism in preclinical models of pancreatic cancer. PFTS inhibited the growth of human pancreatic cancer cells in culture in a concentration- and time-dependent manner. In an MIA PaCa-2 xenograft mouse model, PFTS at a dose of 50 and 100 mg/kg significantly reduced tumor growth by 62% and 65% (P < .05 vs vehicle control. Furthermore, PFTS prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. PFTS appeared to be safe, with the animals showing no signs of toxicity during treatment. Following oral administration, PFTS was rapidly absorbed, metabolized to FTS and FTS glucuronide, and distributed through the blood to body organs. Mechanistically, PFTS inhibited Ras-GTP, the active form of Ras, both in vitro and in vivo, leading to the inhibition of downstream effector pathways c-RAF/mitogen-activated protein-extracellular signal-regulated kinase (ERK kinase (MEK/ERK1/2 kinase and phosphatidylinositol 3-kinase/AKT. In addition, PFTS proved to be a strong combination partner with phospho-valproic acid, a novel signal transducer and activator of transcription 3 (STAT3 inhibitor, displaying synergy in the inhibition of pancreatic cancer growth. In conclusion, PFTS, a direct Ras inhibitor, is an efficacious agent for the treatment of pancreatic cancer in preclinical models, deserving further evaluation.

  1. Mutation of NRAS is a rare genetic event in ovarian low-grade serous carcinoma.

    Science.gov (United States)

    Xing, Deyin; Suryo Rahmanto, Yohan; Zeppernick, Felix; Hannibal, Charlotte G; Kjaer, Susanne K; Vang, Russell; Shih, Ie-Ming; Wang, Tian-Li

    2017-10-01

    Activating mutations involving the members of the RAS signaling pathway, including KRAS, NRAS, and BRAF, have been reported in ovarian low-grade serous carcinoma and its precursor lesion, serous borderline tumor (SBT). Whether additional genetic alterations in the RAS oncogene family accumulate during the progression of SBT to invasive low-grade serous carcinoma (LGSC) remains largely unknown. Although mutations of KRAS and BRAF occur at a very early stage of progression, even preceding the development of SBT, additional driving events, such as NRAS mutations, have been postulated to facilitate progression. In this study, we analyzed NRAS exon 3 mutational status in 98 cases that were diagnosed with SBT/atypical proliferative serous tumor, noninvasive LGSC, or invasive LGSC. Of the latter, NRAS Q61R (CAA to CGA) mutations were detected in only 2 of 56 (3.6%) cases. The same mutation was not detected in any of the SBTs (atypical proliferative serous tumors) or noninvasive LGSCs. Mutational analysis for hotspots in KRAS and BRAF demonstrated a wild-type pattern of KRAS and BRAF in one of the NRAS-mutated cases. Interestingly, another LGSC case with NRAS mutation harbored a concurrent BRAF V600L mutation. These findings indicate that, although recurrent NRAS mutations are present, their low prevalence indicates that NRAS plays a limited role in the development of LGSC. Further studies to identify other oncogenic events that drive LGSC progression are warranted. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Inhibitors: Rationale and Importance to Inhibiting These Pathways in Human Health

    Science.gov (United States)

    Chappell, William H.; Steelman, Linda S.; Long, Jacquelyn M.; Kempf, Ruth C.; Abrams, Stephen L.; Franklin, Richard A.; Bäsecke, Jörg; Stivala, Franca; Donia, Marco; Fagone, Paolo; Malaponte, Graziella; Mazzarino, Maria C.; Nicoletti, Ferdinando; Libra, Massimo; Maksimovic-Ivanic, Danijela; Mijatovic, Sanja; Montalto, Giuseppe; Cervello, Melchiorre; Laidler, Piotr; Milella, Michele; Tafuri, Agostino; Bonati, Antonio; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M.; McCubrey, James A.

    2011-01-01

    The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Integral components of these pathways, Ras, B-Raf, PI3K, and PTEN are also activated/inactivated by mutations. These pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of these pathways can contribute to chemotherapeutic drug resistance, proliferation of cancer initiating cells (CICs) and premature aging. This review will evaluate more recently described potential uses of MEK, PI3K, Akt and mTOR inhibitors in the proliferation of malignant cells, suppression of CICs, cellular senescence and prevention of aging. Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt/mTOR pathways play key roles in the regulation of normal and malignant cell growth. Inhibitors targeting these pathways have many potential uses from suppression of cancer, proliferative diseases as well as aging. PMID:21411864

  3. Induction of postmitotic neuroretina cell proliferation by distinct Ras downstream signaling pathways.

    Science.gov (United States)

    Peyssonnaux, C; Provot, S; Felder-Schmittbuhl, M P; Calothy, G; Eychène, A

    2000-10-01

    Ras-induced cell transformation is mediated through distinct downstream signaling pathways, including Raf, Ral-GEFs-, and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. In some cell types, strong activation of the Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) cascade leads to cell cycle arrest rather than cell division. We previously reported that constitutive activation of this pathway induces sustained proliferation of primary cultures of postmitotic chicken neuroretina (NR) cells. We used this model system to investigate the respective contributions of Ras downstream signaling pathways in Ras-induced cell proliferation. Three RasV12 mutants (S35, G37, and C40) which differ by their ability to bind to Ras effectors (Raf, Ral-GEFs, and the p110 subunit of PI 3-kinase, respectively) were able to induce sustained NR cell proliferation, although none of these mutants was reported to transform NIH 3T3 cells. Furthermore, they all repressed the promoter of QR1, a neuroretina growth arrest-specific gene. Overexpression of B-Raf or activated versions of Ras effectors Rlf-CAAX and p110-CAAX also induced NR cell division. The mitogenic effect of the RasC40-PI 3-kinase pathway appears to involve Rac and RhoA GTPases but not the antiapoptotic Akt (protein kinase B) signaling. Division induced by RasG37-Rlf appears to be independent of Ral GTPase activation and presumably requires an unidentified mechanism. Activation of either Ras downstream pathway resulted in ERK activation, and coexpression of a dominant negative MEK mutant or mKsr-1 kinase domain strongly inhibited proliferation induced by the three Ras mutants or by their effectors. Similar effects were observed with dominant negative mutants of Rac and Rho. Thus, both the Raf-MEK-ERK and Rac-Rho pathways are absolutely required for Ras-induced NR cell division. Activation of these two pathways by the three distinct Ras downstream effectors possibly relies on an autocrine or paracrine loop

  4. R and D activities on radiation induced mutation breeding

    International Nuclear Information System (INIS)

    Lapade, A.G.; Asencion, A.B.; Santos, I.S.; Grafia, A.O.; Veluz, AM.S.; Barrida, A.C.; Marbella, L.J.

    1996-01-01

    This paper summarizes the accomplishments, prospects and future plans of mutation breeding for crop improvement at the Philippine Nuclear Research Institute (PNRI). Mutation induction has become a proven way creating variation within a crop variety and inducing desired attributes that cannot be found in nature or have been lost during evolution. Several improved varieties with desirable traits were successfully developed through induced mutation breeding at our research institute. In rice, mutation breeding has resulted in the development of new varieties: (1) PARC 2, (2) Milagrosa mutant, (3) Bengawan mutant and (4) Azmil mutant. Mutation breeding in leguminous crops has led to the induction of an improved L 114 soybean mutant that is shorter that the original variety but yield about 40% more. Several PAEC mungbean varieties characterized with long pods that are non-shattering were also induced. In asexually propagated crops, an increase in yield and chlorophyll mutants were obtained in sweet potatos. Likewise, chlorophyll mutant which look-like 'ornamental bromeliads' and a mutant with reduced spines have been developed in pineapple Queen variety. At present, we have started a new project in mutation breeding in ornamentals. Tissue culture is being utilized in our mutation breeding program. In the near future, radiation induced mutagenesis coupled with in vitro culture techniques on protoplast culture and somatic hybridization will be integrated into our mutation breeding program to facilitate the production of new crop varieties. (author)

  5. A comparison of ARMS and mutation specific IHC for common activating EGFR mutations analysis in small biopsy and cytology specimens of advanced non small cell lung cancer

    OpenAIRE

    Wang, Xueqing; Wang, Guoqing; Hao, Yueyue; Xu, Yinhong; Zhang, Lihua

    2014-01-01

    We have compared mutation analysis by Amplification Refractory Mutation System (ARMS) and epidermal growth factor receptor (EGFR) mutant-specific antibodies for their ability to detect two common activating EGFR mutations in a cohort of 115 advanced non-small cell lung cancer (NSCLC), including cytology material, core biopsy, and bronchoscopic biopsies. Assessment of EGFR mutation status was performed by using antibodies and ARMS assay specific to the two major forms of mutant EGFR, exon 19 d...

  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

    transcription of mat1-Pm in response to M factor. Furthermore, an activated ras1val17 mutant exhibits a stronger induction of the mat1-Pm transcript. However, transcription still depends on nitrogen deprivation as well as on the presence of pheromone, showing that activation of the Ras1 protein alone does...

  7. Reduced signaling of PI3K-Akt and RAS-MAPK pathways is the key target for weight-loss-induced cancer prevention by dietary calorie restriction and/or physical activity.

    Science.gov (United States)

    Standard, Joseph; Jiang, Yu; Yu, Miao; Su, Xiaoyu; Zhao, Zhihui; Xu, Jianteng; Chen, Jie; King, Brenee; Lu, Lizhi; Tomich, John; Baybutt, Richard; Wang, Weiqun

    2014-12-01

    Weight control through either dietary calorie restriction (DCR) or exercise has been associated with cancer prevention in animal models. However, the underlying mechanisms are not fully defined. Bioinformatics using genomics, proteomics and lipidomics was employed to elucidate the molecular targets of weight control in a mouse skin cancer model. SENCAR mice were randomly assigned into four groups for 10 weeks: ad-libitum-fed sedentary control, ad-libitum-fed exercise (AE), exercise but pair-fed isocaloric amount of control (PE) and 20% DCR. Two hours after topical TPA treatment, skin epidermis was analyzed by Affymetrix for gene expression, DIGE for proteomics and lipidomics for phospholipids. Body weights were significantly reduced in both DCR and PE but not AE mice versus the control. Among 39,000 transcripts, 411, 67 and 110 genes were significantly changed in DCR, PE and AE, respectively. The expression of genes relevant to PI3K-Akt and Ras-MAPK signaling was effectively reduced by DCR and PE but not AE as measured through GenMAPP software. Proteomics analysis identified ~120 proteins, with 27 proteins significantly changed by DCR, including up-regulated apolipoprotein A-1, a key antioxidant protein that decreases Ras-MAPK activity. Of the total 338 phospholipids analyzed by lipidomics, 57 decreased by PE including 5 phophatidylinositol species that serve as PI3K substrates. Although a full impact has not been determined yet, it appears that the reduction of both Ras-MAPK and PI3K-Akt signaling pathways is a cancer preventive target that has been consistently demonstrated by three bioinformatics approaches. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Ras acylation, compartmentalization and signaling nanoclusters (Review)

    OpenAIRE

    HENIS, YOAV I.; HANCOCK, JOHN F.; PRIOR, IAN A.

    2008-01-01

    Ras proteins have become paradigms for isoform- and compartment-specific signaling. Recent work has shown that Ras isoforms are differentially distributed within cell surface signaling nanoclusters and on endomembranous compartments. The critical feature regulating Ras protein localization and isoform-specific functions is the C-terminal hypervariable region (HVR). In this review we discuss the differential post-translational modifications and reversible targeting functions of Ras isoform HVR...

  9. Different qualifiers of AUS/FLUS thyroid FNA have distinct BRAF, RAS, RET/PTC, and PAX8/PPARg alterations.

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    Bellevicine, Claudio; Sgariglia, Roberta; Migliatico, Ilaria; Vigliar, Elena; D'Anna, Melania; Nacchio, Maria Antonia; Serra, Nicola; Malapelle, Umberto; Bongiovanni, Massimo; Troncone, Giancarlo

    2018-02-22

    The Bethesda System for Reporting Thyroid Cytopathology category of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) includes fine-needle aspiration (FNA) specimens that cannot straightforwardly be classified as benign or malignant. To determine whether morphological subcategorization based on atypia qualifiers and molecular testing could improve malignancy risk stratification of AUS/FLUS patients, this study assessed the correlation between these qualifiers and the molecular alterations commonly harbored by thyroid neoplasms. A total of 162 AUS/FLUS cases were subcategorized by atypia qualifiers (Hürthle cell changes, architectural atypia, and cytologic atypia [CyA]) and were tested for BRAF, N-H-KRAS, RET/PTC, and paired box 8 (PAX8)/peroxisome proliferator activated receptor γ (PPARg) mutations. CyA was observed more frequently in mutation-positive AUS/FLUS (14 of 37 [37.84%]) than mutation-negative AUS/FLUS (20 of 125 [16.00%]; P < .0084), and it specifically harbored the BRAFV600E point mutation. Malignancy was confirmed in the available follow-up. Conversely, although RAS was the most frequent mutation identified in AUS/FLUS FNA specimens (26 of 37 cases [70.27%]; P < .0001), it was distributed across various AUS/FLUS subcategories and was not significantly associated with a specific atypia qualifier or malignant outcome according to the available follow-up. Rearrangements of both RET/PTC (n = 1) and PAX8/PPARg (n = 3) were rarely retrieved in the FNA samples. BRAF and RAS mutations are associated with different AUS/FLUS qualifiers and hence have different risks of malignancy. Consequently, a hybrid molecular and morphological subcategorization system could improve the malignancy risk stratification of thyroid FNA samples diagnosed as AUS/FLUS. Cancer Cytopathol 2018. © 2018 American Cancer Society. © 2018 American Cancer Society.

  10. 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.

  11. Time-resolved FTIR spectroscopy for monitoring protein dynamics exemplified by functional studies of Ras protein bound to a lipid bilayer

    International Nuclear Information System (INIS)

    Kötting, Carsten; Güldenhaupt, Jörn; Gerwert, Klaus

    2012-01-01

    Graphical abstract: The first time resolved FTIR investigation of a GTPase reaction of a protein anchored at a single lipid bilayer. Display Omitted Highlights: ► FTIR difference spectroscopy monitors protein dynamics with atomic detail. ► ATR–FTIR allows the measurement of a monolayer sample. ► Membrane proteins can be investigated near physiological conditions. ► The hydrolysis reaction of Ras was investigated in this condition for the first time. - Abstract: Time-resolved Fourier transform infrared (FTIR) difference spectroscopy is a valuable tool for monitoring the dynamics of protein reactions and interactions. Absorbance changes can be monitored with time resolutions down to nanoseconds and followed for time periods that range over nine orders of magnitude. Membrane proteins bound to solid supported lipid bilayers can be investigated in near physiological conditions with the attenuated total reflection (ATR) technique. Here, we review the basics of time-resolved FTIR with a focus on Ras, a GTPase that is mutated in 25% of human tumors. We show the first time-resolved measurements of membrane anchored Ras and observed the switching between its activated and its inactivated state. We compared those measurements with measurements of the truncated Ras in solution. We found that both the kinetics and the functional groups involved were very similar. This suggested that the membrane did not have a major influence on the hydrolysis reaction.

  12. Germline activating TYK2 mutations in pediatric patients with two primary acute lymphoblastic leukemia occurrences.

    Science.gov (United States)

    Waanders, E; Scheijen, B; Jongmans, M C J; Venselaar, H; van Reijmersdal, S V; van Dijk, A H A; Pastorczak, A; Weren, R D A; van der Schoot, C E; van de Vorst, M; Sonneveld, E; Hoogerbrugge, N; van der Velden, V H J; Gruhn, B; Hoogerbrugge, P M; van Dongen, J J M; Geurts van Kessel, A; van Leeuwen, F N; Kuiper, R P

    2017-04-01

    The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novo leukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silico modeling revealed that both mutations affect the conformation of this autoregulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.

  13. 05 Inkanyiso Ras 2.fm

    African Journals Online (AJOL)

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    and follow good moral values like in the Bible. Research approach .... teachings of John the Baptist and his advice to follow Jesus, the Christ (the Messiah) (John 1: 19, 23-26, 29-34; Ras, 1987). In other words, according to ..... A Greek-English Lexicon of the New Testament and other Early Christian Literature. Eighteenth.

  14. Plac8 Links Oncogenic Mutations to Regulation of Autophagy and Is Critical to Pancreatic Cancer Progression

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    Conan Kinsey

    2014-05-01

    Full Text Available Mutations in p53 and RAS potently cooperate in oncogenic transformation, and correspondingly, these genetic alterations frequently coexist in pancreatic ductal adenocarcinoma (PDA and other human cancers. Previously, we identified a set of genes synergistically activated by combined RAS and p53 mutations as frequent downstream mediators of tumorigenesis. Here, we show that the synergistically activated gene Plac8 is critical for pancreatic cancer growth. Silencing of Plac8 in cell lines suppresses tumor formation by blocking autophagy, a process essential for maintaining metabolic homeostasis in PDA, and genetic inactivation in an engineered mouse model inhibits PDA progression. We show that Plac8 is a critical regulator of the autophagic machinery, localizing to the lysosomal compartment and facilitating lysosome-autophagosome fusion. Plac8 thus provides a mechanistic link between primary oncogenic mutations and the induction of autophagy, a central mechanism of metabolic reprogramming, during PDA progression.

  15. Error-prone polymerase activity causes multinucleotide mutations in humans.

    Science.gov (United States)

    Harris, Kelley; Nielsen, Rasmus

    2014-09-01

    About 2% of human genetic polymorphisms have been hypothesized to arise via multinucleotide mutations (MNMs), complex events that generate SNPs at multiple sites in a single generation. MNMs have the potential to accelerate the pace at which single genes evolve and to confound studies of demography and selection that assume all SNPs arise independently. In this paper, we examine clustered mutations that are segregating in a set of 1092 human genomes, demonstrating that the signature of MNM becomes enriched as large numbers of individuals are sampled. We estimate the percentage of linked SNP pairs that were generated by simultaneous mutation as a function of the distance between affected sites and show that MNMs exhibit a high percentage of transversions relative to transitions, findings that are reproducible in data from multiple sequencing platforms and cannot be attributed to sequencing error. Among tandem mutations that occur simultaneously at adjacent sites, we find an especially skewed distribution of ancestral and derived alleles, with GC → AA, GA → TT, and their reverse complements making up 27% of the total. These mutations have been previously shown to dominate the spectrum of the error-prone polymerase Pol ζ, suggesting that low-fidelity DNA replication by Pol ζ is at least partly responsible for the MNMs that are segregating in the human population. We develop statistical estimates of MNM prevalence that can be used to correct phylogenetic and population genetic inferences for the presence of complex mutations. © 2014 Harris and Nielsen; Published by Cold Spring Harbor Laboratory Press.

  16. Proceedings from the 2009 genetic syndromes of the Ras/MAPK pathway: From bedside to bench and back.

    Science.gov (United States)

    Rauen, Katherine A; Schoyer, Lisa; McCormick, Frank; Lin, Angela E; Allanson, Judith E; Stevenson, David A; Gripp, Karen W; Neri, Giovanni; Carey, John C; Legius, Eric; Tartaglia, Marco; Schubbert, Suzanne; Roberts, Amy E; Gelb, Bruce D; Shannon, Kevin; Gutmann, David H; McMahon, Martin; Guerra, Carmen; Fagin, James A; Yu, Benjamin; Aoki, Yoko; Neel, Benjamin G; Balmain, Allan; Drake, Richard R; Nolan, Garry P; Zenker, Martin; Bollag, Gideon; Sebolt-Leopold, Judith; Gibbs, Jackson B; Silva, Alcino J; Patton, E Elizabeth; Viskochil, David H; Kieran, Mark W; Korf, Bruce R; Hagerman, Randi J; Packer, Roger J; Melese, Teri

    2010-01-01

    The RASopathies are a group of genetic syndromes caused by germline mutations in genes that encode components of the Ras/mitogen-activated protein kinase (MAPK) pathway. Some of these syndromes are neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, LEOPARD syndrome and Legius syndrome. Their common underlying pathogenetic mechanism brings about significant overlap in phenotypic features and includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, GI and ocular abnormalities, and a predisposition to cancer. The proceedings from the symposium "Genetic Syndromes of the Ras/MAPK Pathway: From Bedside to Bench and Back" chronicle the timely and typical research symposium which brought together clinicians, basic scientists, physician-scientists, advocate leaders, trainees, students and individuals with Ras syndromes and their families. The goals, to discuss basic science and clinical issues, to set forth a solid framework for future research, to direct translational applications towards therapy and to set forth best practices for individuals with RASopathies were successfully meet with a commitment to begin to move towards clinical trials.

  17. Repression of CD24 surface protein expression by oncogenic Ras is relieved by inhibition of Raf but not MEK or PI3K

    Directory of Open Access Journals (Sweden)

    Nikitha K. Pallegar

    2015-08-01

    Full Text Available CD24 is a dynamically regulated cell surface protein. High expression of CD24 leads to progression of lung, prostrate, colon and pancreatic cancers, among others. In contrast, low expression of CD24 leads to cell proliferation and metastasis of breast cancer stem cells (BCSCs. Activating mutations in Ras are found in 30% of all human cancers. Oncogenic Ras constitutively stimulates the Raf, PI3K and Ral GDS signaling pathways, leading to cellular transformation. Previous studies have shown that expression of oncogenic Ras in breast cancer cells generates CD24- cells from CD24+ cells. However, the molecular mechanisms involved in the generation of CD24- cells were not determined. Here, we demonstrate that oncogenic Ras (RasV12 expression suppresses CD24 mRNA, protein, and promoter levels when expressed in NIH/3T3 cells. Furthermore, activation of only the Raf pathway was sufficient to downregulate CD24 mRNA and protein expression to levels similar to those seen in with RasV12 expression. In contrast, activation of the PI3K pathway downregulated mRNA expression with a partial effect on protein expression whereas activation of the RalGDS pathway only partially affected protein expression. Surprisingly, inhibition of MEK with U0126 only partially restored CD24 mRNA expression but not surface protein expression. In contrast, inhibition of Raf with sorafenib did not restore CD24 mRNA expression but significantly increased the proportion of RasV12 cells expressing CD24. Therefore, the Raf pathway is the major repressor of CD24 mRNA and protein expression, with PI3K also able to substantially inhibit CD24 expression. Moreover, these data indicate that the levels of CD24 mRNA and surface protein are independently regulated. Although inhibition of Raf by sorafenib only partially restored CD24 expression, sorafenib should still be considered as a potential therapeutic strategy to alter CD24 expression in CD24- cells, such as BCSCs.

  18. Isoform-Specific Effects of Wild-Type Ras Genes on Carcinogen-Induced Lung Tumorigenesis in Mice.

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    Jamie D Weyandt

    Full Text Available The gene KRAS is commonly mutated in lung cancer to encode a constitutively active and oncogenic protein that is well established to initiate and maintain lung tumorigenesis. However, the remaining wild-type KRAS protein, or the other family members HRAS and NRAS, can still be activated in the presence of oncogenic KRAS. Moreover, loss of any one of these three genes has been shown to increase the sensitivity of mice to the carcinogen urethane, which induces Kras mutation-positive early lung lesions. To determine the contribution of progressively disrupting Hras and Nras genes on urethane lung tumorigenesis, mice with different combinations of wild-type and null alleles of Hras and Nras were exposed with urethane and tumor burden was assessed. As previously reported, loss of one allele of Hras increased the sensitivity of mice to this carcinogen, and this effect was further exacerbated by the loss of the second Hras allele. However, loss of one or both alleles of Nras failed to alter tumor burden, either in the absence or presence of Hras, after exposure to urethane. Additionally, no obvious difference between lung lesions in mice with wild-type versus null alleles was detected, suggesting that wild-type Ras proteins may exert a tumor suppressive effects at the time of initiation, although other interpretations are certainly possible. In summary, these data suggest that in some genetic backgrounds inactivation of different wild-type Ras genes can have different effects on urethane-induced lung tumorigenesis.

  19. A drosophila model for EGFR-Ras and PI3K-dependent human glioma.

    Directory of Open Access Journals (Sweden)

    Renee D Read

    2009-02-01

    Full Text Available Gliomas, the most common malignant tumors of the nervous system, frequently harbor mutations that activate the epidermal growth factor receptor (EGFR and phosphatidylinositol-3 kinase (PI3K signaling pathways. To investigate the genetic basis of this disease, we developed a glioma model in Drosophila. We found that constitutive coactivation of EGFR-Ras and PI3K pathways in Drosophila glia and glial precursors gives rise to neoplastic, invasive glial cells that create transplantable tumor-like growths, mimicking human glioma. Our model represents a robust organotypic and cell-type-specific Drosophila cancer model in which malignant cells are created by mutations in signature genes and pathways thought to be driving forces in a homologous human cancer. Genetic analyses demonstrated that EGFR and PI3K initiate malignant neoplastic transformation via a combinatorial genetic network composed primarily of other pathways commonly mutated or activated in human glioma, including the Tor, Myc, G1 Cyclins-Cdks, and Rb-E2F pathways. This network acts synergistically to coordinately stimulate cell cycle entry and progression, protein translation, and inappropriate cellular growth and migration. In particular, we found that the fly orthologs of CyclinE, Cdc25, and Myc are key rate-limiting genes required for glial neoplasia. Moreover, orthologs of Sin1, Rictor, and Cdk4 are genes required only for abnormal neoplastic glial proliferation but not for glial development. These and other genes within this network may represent important therapeutic targets in human glioma.

  20. Ras signaling influences permissiveness of malignant peripheral nerve sheath tumor cells to oncolytic herpes.

    Science.gov (United States)

    Farassati, Faris; Pan, Weihong; Yamoutpour, Farnaz; Henke, Susann; Piedra, Mark; Frahm, Silke; Al-Tawil, Said; Mangrum, Wells I; Parada, Luis F; Rabkin, Samuel D; Martuza, Robert L; Kurtz, Andreas

    2008-12-01

    Lack of expression of neurofibromin in neurofibromatosis 1 and its lethal derivative, malignant peripheral nerve sheath tumors (MPNSTs), is thought to result in the overactivation of the Ras signaling pathway. Our previous studies have shown that cells with overactivation in the Ras pathway are more permissive to infection with herpes simplex virus 1 and its mutant version R3616. In this study, we show that among five different mouse MPNST cell lines, only the ones with elevated levels of Ras signaling are highly permissive to infection with oncolytic herpes G207. Specific inhibitors of the Ras, ERK, and JNK pathways all reduced the synthesis of viral proteins in MPNST cells. The cell lines that contained lower levels of Ras and decreased activation of downstream signaling components underwent an enhancement in apoptosis upon exposure to G207. Additionally, mouse SW10 Schwann cells were able to become infected by parental herpes but were found to be resistant to G207. The immortalization of these cell lines with the expression of SV40 large T antigen increased the levels of Ras activation and permissiveness to oncolytic herpes. A Ras/Raf kinase inhibitor reduced the synthesis of both herpes simplex virus-1 and G207 proteins in SW10 cells. The results of this study, therefore, introduce Ras signaling as a divergent turning point for the response of MPNST cells to an assault by oncolytic herpes.

  1. Gain-Of-Function Mutational Activation of Human TRNA Synthetase Procytokine

    Energy Technology Data Exchange (ETDEWEB)

    Yang, X.L.; Kapoor, M.; Otero, F.J.; Slike, B.M.; Tsuruta, H.; Frausto, R.; Bates, A.; Ewalt, K.L.; Cheresh, D.A.; Schimmel, P.; /Scripps Res. Inst. /SLAC, SSRL

    2009-04-30

    Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.

  2. 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.

  3. 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

  4. De novo activating epidermal growth factor mutations (EGFR) in small-cell lung cancer.

    Science.gov (United States)

    Thai, Alesha; Chia, Puey L; Russell, Prudence A; Do, Hongdo; Dobrovic, Alex; Mitchell, Paul; John, Thomas

    2017-09-01

    In Australia, mutations in epidermal growth factor mutations (EGFR) occur in 15% of patients diagnosed with non-small-cell lung cancer and are found with higher frequency in female, non-smokers of Asian ethnicity. Activating mutations in the EGFR gene are rarely described in SCLC. We present two cases of de novo EGFR mutations in patients with SCLC detected in tissue and in plasma cell free DNA, both of whom were of Asian ethnicity and never-smokers. These two cases add to the growing body of evidence suggesting that screening for EGFR mutations in SCLC should be considered in patients with specific clinical features. © 2017 Royal Australasian College of Physicians.

  5. Activating mutation in MET oncogene in familial colorectal cancer

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    Schildkraut Joellen M

    2011-10-01

    Full Text Available Abstract Background In developed countries, the lifetime risk of developing colorectal cancer (CRC is 5%, and it is the second leading cause of death from cancer. The presence of family history is a well established risk factor with 25-35% of CRCs attributable to inherited and/or familial factors. The highly penetrant inherited colon cancer syndromes account for approximately 5%, leaving greater than 20% without clear genetic definition. Familial colorectal cancer has been linked to chromosome 7q31 by multiple affected relative pair studies. The MET proto-oncogene which resides in this chromosomal region is considered a candidate for genetic susceptibility. Methods MET exons were amplified by PCR from germline DNA of 148 affected sibling pairs with colorectal cancer. Amplicons with altered sequence were detected with high-resolution melt-curve analysis using a LightScanner (Idaho Technologies. Samples demonstrating alternative melt curves were sequenced. A TaqMan assay for the specific c.2975C >T change was used to confirm this mutation in a cohort of 299 colorectal cancer cases and to look for allelic amplification in tumors. Results Here we report a germline non-synonymous change in the MET proto-oncogene at amino acid position T992I (also reported as MET p.T1010I in 5.2% of a cohort of sibling pairs affected with CRC. This genetic variant was then confirmed in a second cohort of individuals diagnosed with CRC and having a first degree relative with CRC at prevalence of 4.1%. This mutation has been reported in cancer cells of multiple origins, including 2.5% of colon cancers, and in Conclusions Although the MET p.T992I genetic mutation is commonly found in somatic colorectal cancer tissues, this is the first report also implicating this MET genetic mutation as a germline inherited risk factor for familial colorectal cancer. Future studies on the cancer risks associated with this mutation and the prevalence in different at-risk populations will

  6. Differential binding to frequent HLA-A alleles of p21 RAS derived peptides bearing oncogenic substitutions at position 12 or 13.

    Science.gov (United States)

    Gouttefangeas, C; Demeur, C; Connan, F; Balayn, N; Choppin, J; Tilkin-Mariamé, A F

    1997-07-01

    RAS oncogenic proteins are frequently found mutated in human cancers, where they are known to be implicated in the tumoral process. Mutations occur preferentially at positions 12, 13 or 61. Identification of potential T cell epitopes is the first step to determine it RAS mutated proteins can generate tumor specific antigens which could be further used as targets for cancer immunotherapy protocols. We have investigated the capacity of synthetic wild-type and mutant RAS derived peptides encompassing positions 12 and 13 to bind to three frequent HLA-A alleles: HLA-A*0201, HLA-A*0301 and HLA-A*1101. Binding was evaluated by two methods using TAP-defective cell lines: a cytometric assay based on HLA molecules stabilization at the cell surface, and an assembly assay detecting interactions between solubilized HLA molecules and peptides. Positive HLA binding was observed for two sets of synthetic peptides, one specific for HLA-A*0201 allele (RAS 5-14), and the other one specific for HLA-A*0301 and HLA-A*1101 alleles (RAS 8-16). Interestingly, the different substitutions at positions 12 and 13 were not equivalent for HLA binding. These observations will be useful for the in vitro generation of restricted CD8+ T lymphocytes specific for mutated RAS proteins and recognizing tumoral cells expressing such RAS mutations.

  7. 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

  8. Kinetic characterization of apoptotic Ras signaling through Nore1-MST1 complex formation.

    Science.gov (United States)

    Koturenkiene, Agne; Makbul, Cihan; Herrmann, Christian; Constantinescu-Aruxandei, Diana

    2017-05-01

    Ras-mediated apoptotic signaling is expected to be mediated via Rassf-MST complexes, but the system has been poorly characterized in vitro until now. Here we demonstrate that active H-Ras, Nore1A and MST1 form a stable ternary complex in vitro without other external factors, Nore1A interacting simultaneously with H-Ras and MST1 via its RBD and SARAH domain, respectively. Moreover, our data show for the first time that the SARAH domain of Nore1A plays a role in the Nore1A binding to H-Ras. Finally, we analyze the relation between the electrostatic and hydrophobic forces and kinetic constants of the Nore1A - H-Ras complex.

  9. Effect of the G375C and G346E achondroplasia mutations on FGFR3 activation.

    Directory of Open Access Journals (Sweden)

    Lijuan He

    Full Text Available Two mutations in FGFR3, G380R and G375C are known to cause achondroplasia, the most common form of human dwarfism. The G380R mutation accounts for 98% of the achondroplasia cases, and thus has been studied extensively. Here we study the effect of the G375C mutation on the phosphorylation and the cross-linking propensity of full-length FGFR3 in HEK 293 cells, and we compare the results to previously published results for the G380R mutant. We observe identical behavior of the two achondroplasia mutants in these experiments, a finding which supports a direct link between the severity of dwarfism phenotypes and the level and mechanism of FGFR3 over-activation. The mutations do not increase the cross-linking propensity of FGFR3, contrary to previous expectations that the achondroplasia mutations stabilize the FGFR3 dimers. Instead, the phosphorylation efficiency within un-liganded FGFR3 dimers is increased, and this increase is likely the underlying cause for pathogenesis in achondroplasia. We further investigate the G346E mutation, which has been reported to cause achondroplasia in one case. We find that this mutation does not increase FGFR3 phosphorylation and decreases FGFR3 cross-linking propensity, a finding which raises questions whether this mutation is indeed a genetic cause for human dwarfism.

  10. Effect of the G375C and G346E achondroplasia mutations on FGFR3 activation.

    Science.gov (United States)

    He, Lijuan; Serrano, Christopher; Niphadkar, Nitish; Shobnam, Nadia; Hristova, Kalina

    2012-01-01

    Two mutations in FGFR3, G380R and G375C are known to cause achondroplasia, the most common form of human dwarfism. The G380R mutation accounts for 98% of the achondroplasia cases, and thus has been studied extensively. Here we study the effect of the G375C mutation on the phosphorylation and the cross-linking propensity of full-length FGFR3 in HEK 293 cells, and we compare the results to previously published results for the G380R mutant. We observe identical behavior of the two achondroplasia mutants in these experiments, a finding which supports a direct link between the severity of dwarfism phenotypes and the level and mechanism of FGFR3 over-activation. The mutations do not increase the cross-linking propensity of FGFR3, contrary to previous expectations that the achondroplasia mutations stabilize the FGFR3 dimers. Instead, the phosphorylation efficiency within un-liganded FGFR3 dimers is increased, and this increase is likely the underlying cause for pathogenesis in achondroplasia. We further investigate the G346E mutation, which has been reported to cause achondroplasia in one case. We find that this mutation does not increase FGFR3 phosphorylation and decreases FGFR3 cross-linking propensity, a finding which raises questions whether this mutation is indeed a genetic cause for human dwarfism.

  11. Inhibition of Ras farnesylation by lovastatin leads to downregulation of proliferation and migration in primary cultured human glioblastoma cells.

    Science.gov (United States)

    Bouterfa, H L; Sattelmeyer, V; Czub, S; Vordermark, D; Roosen, K; Tonn, J C

    2000-01-01

    Malignant astrocytomas are the most common primary intracranial human tumors. All therapeutic approaches are limited due to their high proliferative capacity and their ability to diffusely invade the brain. Amplification of tyrosine kinase receptors and their signaling pathways have been implicated as contributing to the molecular pathogenesis of astrocytomas, providing possible new targets for therapeutic intervention. In particular, astrocytomas, although lacking oncogenic Ras mutations, have elevated levels of activated Ras. Lovastatin, an inhibitor of the beta-hydroxy-beta-methylglutary CoA reductase (HMG-CoA-reductase), is currently used to treat patients with hypercholesterolemia. In addition, it inhibits isoprenylation of several members of the Ras superfamily of proteins and therefore has multiple cellular effects including the reduction of proliferation. In this study, we investigated the impact of lovastatin on two human glioma cell lines and on 15 primary cell cultures established from biopsies of patients with glioblastoma multiforme (GBM,) Proliferation of glioma cell lines and primary tumor cells was determined by cell counting and by using the MTT assay. The cell morphology was analyzed by staining of actin filaments with phalloidin. Apoptosis was measured using the TUNEL assay. To investigate the influence of this drug on glioma cell motility, tumor cell migration was investigated using three dimensional spheroid disintegration assays. In addition, tumor cell invasion was analyzed with a confrontational assay between tumor spheroids and rat brain aggregates. Inhibition of farnesyl biosynthesis using lovastatin led to a block in Ras mediated signaling, indicated by lower MAPK activity. Consequently, tumor cell proliferation was reduced up to 80%. Lovastatin appeared to decrease glioma viability by inducing apoptosis, as indicated by morphological changes and increase of TUNEL positive cells. Lovastatin acts through isoprenoid depletion, because

  12. A secondary RET mutation in the activation loop conferring resistance to vandetanib.

    Science.gov (United States)

    Nakaoku, Takashi; Kohno, Takashi; Araki, Mitsugu; Niho, Seiji; Chauhan, Rakhee; Knowles, Phillip P; Tsuchihara, Katsuya; Matsumoto, Shingo; Shimada, Yoko; Mimaki, Sachiyo; Ishii, Genichiro; Ichikawa, Hitoshi; Nagatoishi, Satoru; Tsumoto, Kouhei; Okuno, Yasushi; Yoh, Kiyotaka; McDonald, Neil Q; Goto, Koichi

    2018-02-12

    Resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic lung adenocarcinoma harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in a serine-to-phenylalanine substitution at codon 904 in the activation loop of the RET kinase domain. The S904F mutation confers resistance to vandetanib by increasing the ATP affinity and autophosphorylation activity of RET kinase. A reduced interaction with the drug is also observed in vitro for the S904F mutant by thermal shift assay. A crystal structure of the S904F mutant reveals a small hydrophobic core around F904 likely to enhance basal kinase activity by stabilizing an active conformer. Our findings indicate that missense mutations in the activation loop of the kinase domain are able to increase kinase activity and confer drug resistance through allosteric effects.

  13. Activities of different fluoroquinolones against Bacillus anthracis mutants selected in vitro and harboring topoisomerase mutations.

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    Grohs, Patrick; Podglajen, Isabelle; Gutmann, Laurent

    2004-08-01

    Three sets of mutants of Bacillus anthracis resistant to fluoroquinolones were selected on ciprofloxacin and moxifloxacin in a stepwise manner from a nalidixic acid-resistant but fluoroquinolone-susceptible plasmidless strain harboring a Ser85Leu GyrA mutation. A high level of resistance to fluoroquinolones could be obtained in four or five selection steps. In each case, ParC was the secondary target. However, in addition to the GyrA mutation, expression of high-level resistance required (i) in the first set of mutants, active drug efflux associated with a mutation in the QRDR of ParC; (ii) in the second set, two mutations in the QRDR of ParC associated with a mutation in GyrB; and (iii) in the third set, two QRDR mutations, one in ParC and one in GyrA. Interestingly, several selection steps occurred without obvious mutations in the QRDR of any topoisomerase, thereby implying the existence of other resistance mechanisms. Among the fluoroquinolones tested, garenoxacin showed the best activity.

  14. Mapping the Conformation Space of Wildtype and Mutant H-Ras with a Memetic, Cellular, and Multiscale Evolutionary Algorithm.

    Directory of Open Access Journals (Sweden)

    Rudy Clausen

    2015-09-01

    Full Text Available An important goal in molecular biology is to understand functional changes upon single-point mutations in proteins. Doing so through a detailed characterization of structure spaces and underlying energy landscapes is desirable but continues to challenge methods based on Molecular Dynamics. In this paper we propose a novel algorithm, SIfTER, which is based instead on stochastic optimization to circumvent the computational challenge of exploring the breadth of a protein's structure space. SIfTER is a data-driven evolutionary algorithm, leveraging experimentally-available structures of wildtype and variant sequences of a protein to define a reduced search space from where to efficiently draw samples corresponding to novel structures not directly observed in the wet laboratory. The main advantage of SIfTER is its ability to rapidly generate conformational ensembles, thus allowing mapping and juxtaposing landscapes of variant sequences and relating observed differences to functional changes. We apply SIfTER to variant sequences of the H-Ras catalytic domain, due to the prominent role of the Ras protein in signaling pathways that control cell proliferation, its well-studied conformational switching, and abundance of documented mutations in several human tumors. Many Ras mutations are oncogenic, but detailed energy landscapes have not been reported until now. Analysis of SIfTER-computed energy landscapes for the wildtype and two oncogenic variants, G12V and Q61L, suggests that these mutations cause constitutive activation through two different mechanisms. G12V directly affects binding specificity while leaving the energy landscape largely unchanged, whereas Q61L has pronounced, starker effects on the landscape. An implementation of SIfTER is made available at http://www.cs.gmu.edu/~ashehu/?q=OurTools. We believe SIfTER is useful to the community to answer the question of how sequence mutations affect the function of a protein, when there is an

  15. Mapping the Conformation Space of Wildtype and Mutant H-Ras with a Memetic, Cellular, and Multiscale Evolutionary Algorithm.

    Science.gov (United States)

    Clausen, Rudy; Ma, Buyong; Nussinov, Ruth; Shehu, Amarda

    2015-09-01

    An important goal in molecular biology is to understand functional changes upon single-point mutations in proteins. Doing so through a detailed characterization of structure spaces and underlying energy landscapes is desirable but continues to challenge methods based on Molecular Dynamics. In this paper we propose a novel algorithm, SIfTER, which is based instead on stochastic optimization to circumvent the computational challenge of exploring the breadth of a protein's structure space. SIfTER is a data-driven evolutionary algorithm, leveraging experimentally-available structures of wildtype and variant sequences of a protein to define a reduced search space from where to efficiently draw samples corresponding to novel structures not directly observed in the wet laboratory. The main advantage of SIfTER is its ability to rapidly generate conformational ensembles, thus allowing mapping and juxtaposing landscapes of variant sequences and relating observed differences to functional changes. We apply SIfTER to variant sequences of the H-Ras catalytic domain, due to the prominent role of the Ras protein in signaling pathways that control cell proliferation, its well-studied conformational switching, and abundance of documented mutations in several human tumors. Many Ras mutations are oncogenic, but detailed energy landscapes have not been reported until now. Analysis of SIfTER-computed energy landscapes for the wildtype and two oncogenic variants, G12V and Q61L, suggests that these mutations cause constitutive activation through two different mechanisms. G12V directly affects binding specificity while leaving the energy landscape largely unchanged, whereas Q61L has pronounced, starker effects on the landscape. An implementation of SIfTER is made available at http://www.cs.gmu.edu/~ashehu/?q=OurTools. We believe SIfTER is useful to the community to answer the question of how sequence mutations affect the function of a protein, when there is an abundance of experimental

  16. ERK/pERK expression and B-raf mutations in colon adenocarcinomas: correlation with clinicopathological characteristics

    Directory of Open Access Journals (Sweden)

    Levidou Georgia

    2012-02-01

    Full Text Available Abstract Background Colorectal (CRC carcinogenesis through various morphological stages has been linked to several genetic and epigenetic changes. The Raf/MEK/ERK (MAPK signal transduction cascade is an important mediator of a number of cellular fates. Methods In this study, we investigated the presence of B-raf and K-ras mutations in 94 consecutive cases of primary colon adenocarcinoma in correlation with the immunohistochemical expression of total and activated ERK and the expression of mismatch repair proteins (MMR hMLH1 and hMSH2 as well as their correlations with standard clinicopathological parameters. Results The immunostaining pattern for total and activated ERK was nuclear and cytoplasmic. hMLH1 and hMSH2 proteins were preserved in 45/63 (71.43% cases and 35/53 (66.04% cases respectively. Total ERK nuclear expression, was positively correlated with tumor stage (p = 0.049, whereas nuclear pERK expression was positively correlated with histological grade (p = 0.0113 and tumor stage (p = 0.0952, although the latter relationship was of marginal significance. DNA sequencing showed that 12 samples (12.7% had a mutation in B-RAF Exon 15 and none in Exon 11, whereas 22 (23.4% had a K-ras mutation. Disruption of the MAP kinase pathway-either through K-ras or B-raf mutation-was detected in 37% of all the examined cases, although the overexpression of total and activated ERK1/2 was not correlated with the mutational status of K-ras or B-raf genes. Finally, the preservation of hMLH1 or hMSH2 immunoexpression was not correlated with the presence of B-raf and/or K-ras mutations. Conclusions In this study, we present evidence that ERK activation occurs in a K-ras or B-raf -independent manner in the majority of primary colon cancer cases. Moreover, B-raf mutations are not associated with mismatch-repair deficiency through loss of hMLH1 or hMSH2 expression. Activated ERK could possibly be implicated in tumor invasiveness as well as in the acquisition of

  17. 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

  18. Impact of kinase activating and inactivating patient mutations on binary PKA interactions.

    Science.gov (United States)

    Röck, Ruth; Mayrhofer, Johanna E; Bachmann, Verena; Stefan, Eduard

    2015-01-01

    The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions.

  19. Controlling the activity of the Tec kinase Itk by mutation of the phenylalanine gatekeeper residue.

    Science.gov (United States)

    Joseph, Raji E; Andreotti, Amy H

    2011-01-18

    The regulatory spine is a set of conserved residues that are assembled and disassembled upon activation and inactivation of kinases. We recently identified the regulatory spine within the immunologically important Tec family kinases and have shown that in addition to the core spine residues within the kinase domain itself, contributions from the SH2-kinase linker region result in an extended spine structure for this kinase family. Disruption of the regulatory spine, either by mutation or by removal of the amino-terminal SH2-kinase linker region or by mutation of core spine residues, leads to inactivation of the Tec kinases. With a focus on the Tec family members, Itk and Btk, we now show that the gatekeeper residue is also critical for the assembly of the regulatory spine. Mutation of the bulky Itk F434 gatekeeper residue to alanine or glycine inactivates Itk. The activity of the Itk F434A mutant can be recovered by a secondary site mutation within the N-terminal lobe, specifically L432I. The Itk L432I mutation likely rescues the activity of the gatekeeper F434A mutation by promoting the assembly of the regulatory spine. We also show that mutation of the Itk and Btk gatekeeper residues to methionine is sufficient to activate the isolated kinase domains of Tec kinases in the absence of the amino-terminal SH2-kinase linker. Thus, shifting the conformational equilibrium between the assembled and disassembled states of the regulatory spine by changing the nature of the gatekeeper residue is key to regulating the activity of Tec kinases.

  20. Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1 genes

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    Olschwang Sylviane

    2008-10-01

    Full Text Available Abstract Background Chronic myelomonocytic leukemia (CMML is a hematological disease close to, but separate from both myeloproliferative disorders (MPD and myelodysplastic syndromes and may show either myeloproliferative (MP-CMML or myelodysplastic (MD-CMML features. Not much is known about the molecular biology of this disease. Methods We studied a series of 30 CMML samples (13 MP- and 11 MD-CMMLs, and 6 acutely transformed cases from 29 patients by using Agilent high density array-comparative genomic hybridization (aCGH and sequencing of 12 candidate genes. Results Two-thirds of samples did not show any obvious alteration of aCGH profiles. In one-third we observed chromosome abnormalities (e.g. trisomy 8, del20q and gain or loss of genes (e.g. NF1, RB1 and CDK6. RAS mutations were detected in 4 cases (including an uncommon codon 146 mutation in KRAS and PTPN11 mutations in 3 cases. We detected 11 RUNX1 alterations (9 mutations and 2 rearrangements. The rearrangements were a new, cryptic inversion of chromosomal region 21q21-22 leading to break and fusion of RUNX1 to USP16. RAS and RUNX1 alterations were not mutually exclusive. RAS pathway mutations occurred in MP-CMMLs (~46% but not in MD-CMMLs. RUNX1 alterations (mutations and cryptic rearrangement occurred in both MP and MD classes (~38%. Conclusion We detected RAS pathway mutations and RUNX1 alterations. The latter included a new cryptic USP16-RUNX1 fusion. In some samples, two alterations coexisted already at this early chronic stage.

  1. Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1 genes

    International Nuclear Information System (INIS)

    Gelsi-Boyer, Véronique; Bentires-Alj, Mohamed; Olschwang, Sylviane; Vey, Norbert; Mozziconacci, Marie-Joëlle; Birnbaum, Daniel; Chaffanet, Max; Trouplin, Virginie; Adélaïde, José; Aceto, Nicola; Remy, Virginie; Pinson, Stephane; Houdayer, Claude; Arnoulet, Christine; Sainty, Danielle

    2008-01-01

    Chronic myelomonocytic leukemia (CMML) is a hematological disease close to, but separate from both myeloproliferative disorders (MPD) and myelodysplastic syndromes and may show either myeloproliferative (MP-CMML) or myelodysplastic (MD-CMML) features. Not much is known about the molecular biology of this disease. We studied a series of 30 CMML samples (13 MP- and 11 MD-CMMLs, and 6 acutely transformed cases) from 29 patients by using Agilent high density array-comparative genomic hybridization (aCGH) and sequencing of 12 candidate genes. Two-thirds of samples did not show any obvious alteration of aCGH profiles. In one-third we observed chromosome abnormalities (e.g. trisomy 8, del20q) and gain or loss of genes (e.g. NF1, RB1 and CDK6). RAS mutations were detected in 4 cases (including an uncommon codon 146 mutation in KRAS) and PTPN11 mutations in 3 cases. We detected 11 RUNX1 alterations (9 mutations and 2 rearrangements). The rearrangements were a new, cryptic inversion of chromosomal region 21q21-22 leading to break and fusion of RUNX1 to USP16. RAS and RUNX1 alterations were not mutually exclusive. RAS pathway mutations occurred in MP-CMMLs (~46%) but not in MD-CMMLs. RUNX1 alterations (mutations and cryptic rearrangement) occurred in both MP and MD classes (~38%). We detected RAS pathway mutations and RUNX1 alterations. The latter included a new cryptic USP16-RUNX1 fusion. In some samples, two alterations coexisted already at this early chronic stage

  2. Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn's disease.

    Science.gov (United States)

    Mao, Liming; Kitani, Atsushi; Similuk, Morgan; Oler, Andrew J; Albenberg, Lindsey; Kelsen, Judith; Aktay, Atiye; Quezado, Martha; Yao, Michael; Montgomery-Recht, Kim; Fuss, Ivan J; Strober, Warren

    2018-02-06

    In these studies we evaluated the contribution of the NLRP3 inflammasome to Crohn's disease (CD) in a kindred containing individuals having a missense mutation in CARD8, a protein known to inhibit this inflammasome. Whole exome sequencing and PCR studies identified that the affected individuals had a V44I mutation in a single allele of the T60 isoform of CARD8. The serum levels of IL-1β in the affected individuals were increased compared with that in healthy controls and their peripheral monocytes produced increased amounts of IL-1β when stimulated by NLRP3 activators. Immunoblot studies probing the basis of these findings showed that mutated T60 CARD8 fails to down-regulate the NLRP3 inflammasome because it does not bind to NLRP3 and inhibit its oligomerization. In addition, these studies showed that mutated T60 CARD8 exerts a dominant negative effect by its capacity to bind to and form oligomers with unmutated T60 or T48 CARD8 that impede their binding to NLRP3. Finally, inflammasome activation studies revealed that intact but not mutated CARD8 prevents NLRP3 deubiquitination and serine dephosphorylation. CD due to a CARD8 mutation was not effectively treated by anti-TNF-α, but did respond to IL-1β inhibitors. Thus, patients with anti-TNF-α-resistant CD may respond to this treatment option.

  3. RAS signalling through PI3-Kinase controls cell migration via modulation of Reelin expression.

    Science.gov (United States)

    Castellano, Esther; Molina-Arcas, Miriam; Krygowska, Agata Adelajda; East, Philip; Warne, Patricia; Nicol, Alastair; Downward, Julian

    2016-04-13

    RAS signalling through phosphoinositide 3-kinase (PI3-Kinase) has been shown to have an essential role in tumour initiation and maintenance. RAS also regulates cell motility and tumour invasiveness, but the role of direct RAS binding to PI3-Kinase in this remains uncertain. Here, we provide evidence that disruption of RAS interaction with PI3-Kinase p110α decreases cell motility and prevents activation of Rac GTPase. Analysis of gene expression in cells lacking RAS interaction with p110α reveals increased levels of the extracellular matrix glycoprotein Reelin and activation of its downstream pathway resulting in upregulation of E-cadherin expression. Induction of the Reelin/E-cadherin axis is also observed in Kras mutant lung tumours that are regressing due to blockade of RAS interaction with PI3-Kinase. Furthermore, loss of Reelin correlates with decreased survival of lung and breast cancer patients. Reelin thus plays a role in restraining RAS and PI3-kinase promotion of cell motility and potentially tumour metastasis.

  4. Mutations in the catalytic loop HRD motif alter the activity and function of Drosophila Src64.

    Directory of Open Access Journals (Sweden)

    Taylor C Strong

    Full Text Available The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele.

  5. Sorafenib paradoxically activates the RAS/RAF/ERK pathway in polyclonal human NK cells during expansion and thereby enhances effector functions in a dose and time-dependent manner.

    Science.gov (United States)

    Lohmeyer, Julian; Nerreter, Thomas; Dotterweich, Julia; Einsele, Hermann; Seggewiss-Bernhardt, Ruth

    2018-03-24

    Natural killer (NK) cells play a major role in host immunity against leukemia and lymphoma. However, clinical trials applying NK cells have not been as efficient as hoped for. Patients treated with RAF inhibitors exhibit increased tumor infiltration by immune cells suggesting that a combination of RAF inhibitors with immunotherapy might be beneficial. As MAPKs such as CRAF regulate NK cell functions, we performed an in vitro investigation on the potential of clinically relevant short acting tyrosine kinase inhibitors (TKIs) as potential adjuvants for NK cell therapy: NK cells from healthy human blood donors were thus treated with sorafenib, sunitinib or the pan-RAF inhibitor ZM336372 during ex vivo expansion. Functional outcomes assessed after washout of the drugs included cytokine production, degranulation, cytotoxicity, apoptosis induction and signal transduction with/without target cell contact. Paradoxically, sorafenib enhanced NK cell effector functions in a time- and dose- dependent manner by raising the steady-state-activation level. Of note, this did not lead to NK cell exhaustion, but enhanced activity against target cells such as K562 or Daudis mediated via the RAS/RAF/ERK pathway but not via AKT. Our data shall pave the path to develop a rationale for the considered use of RAF inhibitors such as sorafenib for pre-activation in NK cell-based adoptive immune therapy. This article is protected by copyright. All rights reserved. © 2018 British Society for Immunology.

  6. Activating mutations of GNAS in canine cortisol-secreting adrenocortical tumors.

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    Kool, M M J; Galac, S; Spandauw, C G; Kooistra, H S; Mol, J A

    2013-01-01

    Cushing's syndrome or hypercortisolism is a common endocrinopathy in dogs. In approximately 15% of cases, the disorder is caused by adrenocorticotropin (ACTH)-independent hypersecretion of cortisol by an adrenocortical tumor (AT). Without other explanation, the cortisol hypersecretion has been referred to as autonomous. To investigate whether ACTH-independent hypersecretion of cortisol may be associated with aberrant activation of the melanocortin 2 receptor (MC2R)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway. All analyses were performed on 44 cortisol-secreting ATs (14 adenomas and 30 carcinomas) derived from dogs diagnosed with ACTH-independent hypercortisolism. Mutation analysis was performed of genes encoding the stimulatory G protein alpha subunit (GNAS), MC2R, and PKA regulatory subunit 1A (PRKAR1A) in all ATs. Approximately one-third of all ATs harbored an activating mutation of GNAS. Missense mutations, known to result in constitutive activation, were present in codon 201 in 11 ATs, in codon 203 (1 AT), and in codon 227 (3 ATs). No functional mutations were found in MC2R and PRKAR1A. Activation of cAMP signaling is a frequent event in canine cortisol-secreting ATs and may play a crucial role in both ACTH-independent cortisol production and tumor formation. To the best of our knowledge, this is the first report of potentially causative mutations in canine cortisol-secreting ATs. Copyright © 2013 by the American College of Veterinary Internal Medicine.

  7. 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

    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 and inor......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...

  8. Computational identification of harmful mutation regions to the activity of transposable elements.

    Science.gov (United States)

    Jin, Lingling; McQuillan, Ian; Li, Longhai

    2017-11-17

    Transposable elements (TEs) are interspersed DNA sequences that can move or copy to new positions within a genome. TEs are believed to promote speciation and their activities play a significant role in human disease. In the human genome, the 22 AluY and 6 AluS TE subfamilies have been the most recently active, and their transposition has been implicated in many inherited human diseases and in various forms of cancer. Therefore, understanding their transposition activity is very important and identifying the factors that affect their transpositional activity is of great interest. Recently, there has been some work done to quantify the activity levels of active Alu TEs based on variation in the sequence. Given this activity data, an analysis of TE activity based on the position of mutations is conducted. A method/simulation is created to computationally predict so-called harmful mutation regions in the consensus sequence of a TE; that is, mutations that occur in these regions decrease the transpositional activity dramatically. The methods are applied to the most active subfamily, AluY, to identify the harmful regions, and seven harmful regions are identified within the AluY consensus with q-values less than 0.05. A supplementary simulation also shows that the identified harmful regions covering the AluYa5 RNA functional regions are not occurring by chance. This method is then applied to two additional TE families: the Alu family and the L1 family, to computationally detect the harmful regions in these elements. We use a computational method to identify a set of harmful mutation regions. Mutations within the identified harmful regions decrease the transpositional activity of active elements. The correlation between the mutations within these regions and the transpositional activity of TEs are shown to be statistically significant. Verifications are presented using the activity of AluY elements and the secondary structure of the AluYa5 RNA, providing evidence that the

  9. Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Uhlin, Ulla; Munch-Petersen, Birgitte

    2012-01-01

    ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket......'-deoxythymidine (AZT) compared with the natural substrate thymidine. The crystal structure of the T163S-mutated HuTK1 reveals a less ordered conformation of the ligand thymidine triphosphate compared with the wild-type structure but the cause of the changed specificity towards AZT is not obvious. Based on its...... highly increased AZT activity relative to thymidine activity this TK1 mutant could be suitable for suicide gene therapy....

  10. Altered glucose metabolism in Harvey-ras transformed MCF10A cells.

    Science.gov (United States)

    Zheng, Wei; Tayyari, Fariba; Gowda, G A Nagana; Raftery, Daniel; McLamore, Eric S; Porterfield, D Marshall; Donkin, Shawn S; Bequette, Brian; Teegarden, Dorothy

    2015-02-01

    Metabolic reprogramming that alters the utilization of glucose including the "Warburg effect" is critical in the development of a tumorigenic phenotype. However, the effects of the Harvey-ras (H-ras) oncogene on cellular energy metabolism during mammary carcinogenesis are not known. The purpose of this study was to determine the effect of H-ras transformation on glucose metabolism using the untransformed MCF10A and H-ras oncogene transfected (MCF10A-ras) human breast epithelial cells, a model for early breast cancer progression. We measured the metabolite fluxes at the cell membrane by a selective micro-biosensor, [(13)C6 ]glucose flux by (13)C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of glucose metabolism enzymes by quantitative PCR. Results from these studies indicated that MCF10A-ras cells exhibited enhanced glycolytic activity and lactate production, decreased glucose flux through the tricarboxylic acid (TCA) cycle, as well as an increase in the utilization of glucose in the pentose phosphate pathway (PPP). These results provide evidence for a role of H-ras oncogene in the metabolic reprogramming of MCF10A cells during early mammary carcinogenesis. © 2013 Wiley Periodicals, Inc.

  11. IRF1 Downregulation by Ras/MEK Is Independent of Translational Control of IRF1 mRNA.

    Science.gov (United States)

    Komatsu, Yumiko; Derwish, Leena; Hirasawa, Kensuke

    2016-01-01

    Oncogenic activation of Ras/MEK downregulates the expression of interferon regulatory factor 1 (IRF1), which is a prerequisite for oncolytic viruses to replicate in cancer cells [1]. Moreover, restoration of IRF1 expression is essential to induce apoptosis of cancer cells treated with a MEK inhibitor [2]. However, the molecular mechanisms that underlie IRF1 downregulation by Ras/MEK remain unclear. In this study, we determined whether Ras/MEK activation modulates IRF1 expression at its translational level. MEK inhibition increased the activity of IRF1 promoter construct in Ras transformed NIH3T3 cells and wild type MEF, but not in IRF1 deficient MEF, indicating that IRF1 protein is required for the transcriptional activation of IRF1. By conducting reporter analysis using IRF1 5'- and 3'- UTR constructs, we determined that cis elements on 5'- and 3'-UTR of IRF1 mRNA are not involved in the IRF1 regulation by Ras/MEK. We further compared the recruitment of ribosomes to IRF1 mRNA in RasV12 cells treated with or without the MEK inhibitor by conducting polysome analysis. No difference was observed in the polysomal distribution of IRF1 mRNA between RasV12 cells treated with and without the MEK inhibitor. These results suggest that regulation of IRF1 translation is independent of IRF1 downregulation by Ras/MEK.

  12. XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Tainer, John; Fan, Li; Fuss, Jill O.; Cheng, Quen J.; Arvai, Andrew S.; Hammel, Michal; Roberts, Victoria A.; Cooper, Priscilla K.; Tainer, John A.

    2008-06-02

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  13. 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

  14. Cantú Syndrome Resulting from Activating Mutation in the KCNJ8 Gene

    OpenAIRE

    Cooper, Paige E.; Reutter, Heiko; Woelfle, Joachim; Engels, Hartmut; Grange, Dorothy K.; van Haaften, Gijs; van Bon, Bregje W.; Hoischen, Alexander; Nichols, Colin G.

    2014-01-01

    ATP-sensitive potassium (KATP) channels, composed of inward-rectifying potassium channel subunits (Kir6.1 and Kir6.2, encoded by KCNJ8 and KCNJ11, respectively) and regulatory sulfonylurea receptor (SUR1 and SUR2, encoded by ABCC8 and ABCC9, respectively), couple metabolism to excitability in multiple tissues. Mutations in ABCC9 cause Cantú syndrome, a distinct multi-organ disease, potentially via enhanced KATP channel activity. We screened KCNJ8 in an ABCC9 mutation-negative patient who also...

  15. Mutational analysis of the active site of human insulin-regulated aminopeptidase

    DEFF Research Database (Denmark)

    Laustsen, P G; Vang, S; Kristensen, T

    2001-01-01

    -length transmembrane form of human IRAP was expressed in HEK293 cells and recombinant wild-type IRAP was shown to have biochemical and enzymatic properties similar to those reported for native IRAP and the soluble serum form of IRAP. Mutational analysis using single amino-acid substitutions in the GAMEN motif (G428A...... acids N-terminal to the Zn(2+)-coordination sequence element distinguishes the gluzincin aminopeptidases from other gluzincins. To investigate the importance of the G428AMEN and H464ELAH-(18X)-E487 motifs for the activity of IRAP, mutational analysis was carried out. cDNA encoding the full...

  16. Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity

    Directory of Open Access Journals (Sweden)

    Korvala Johanna

    2012-04-01

    Full Text Available Abstract Background Primary osteoporosis is a rare childhood-onset skeletal condition whose pathogenesis has been largely unknown. We have previously shown that primary osteoporosis can be caused by heterozygous missense mutations in the Low-density lipoprotein receptor-related protein 5 (LRP5 gene, and the role of LRP5 is further investigated here. Methods LRP5 was analyzed in 18 otherwise healthy children and adolescents who had evidence of osteoporosis (manifested as reduced bone mineral density i.e. BMD, recurrent peripheral fractures and/or vertebral compression fractures but who lacked the clinical features of osteogenesis imperfecta (OI or other known syndromes linked to low BMD. Also 51 controls were analyzed. Methods used in the genetic analyses included direct sequencing and multiplex ligation-dependent probe amplification (MLPA. In vitro studies were performed using luciferase assay and quantitative real-time polymerase chain reaction (qPCR to examine the effect of two novel and three previously identified mutations on the activity of canonical Wnt signaling and on expression of tryptophan hydroxylase 1 (Tph1 and 5-hydroxytryptamine (5-Htr1b. Results Two novel LRP5 mutations (c.3446 T > A; p.L1149Q and c.3553 G > A; p.G1185R were identified in two patients and their affected family members. In vitro analyses showed that one of these novel mutations together with two previously reported mutations (p.C913fs, p.R1036Q significantly reduced the activity of the canonical Wnt signaling pathway. Such reductions may lead to decreased bone formation, and could explain the bone phenotype. Gut-derived Lrp5 has been shown to regulate serotonin synthesis by controlling the production of serotonin rate-limiting enzyme, Tph1. LRP5 mutations did not affect Tph1 expression, and only one mutant (p.L1149Q reduced expression of serotonin receptor 5-Htr1b (p Conclusions Our results provide additional information on the role of LRP5 mutations and their

  17. Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity.

    Science.gov (United States)

    Korvala, Johanna; Jüppner, Harald; Mäkitie, Outi; Sochett, Etienne; Schnabel, Dirk; Mora, Stefano; Bartels, Cynthia F; Warman, Matthew L; Deraska, Donald; Cole, William G; Hartikka, Heini; Ala-Kokko, Leena; Männikkö, Minna

    2012-04-10

    Primary osteoporosis is a rare childhood-onset skeletal condition whose pathogenesis has been largely unknown. We have previously shown that primary osteoporosis can be caused by heterozygous missense mutations in the Low-density lipoprotein receptor-related protein 5 (LRP5) gene, and the role of LRP5 is further investigated here. LRP5 was analyzed in 18 otherwise healthy children and adolescents who had evidence of osteoporosis (manifested as reduced bone mineral density i.e. BMD, recurrent peripheral fractures and/or vertebral compression fractures) but who lacked the clinical features of osteogenesis imperfecta (OI) or other known syndromes linked to low BMD. Also 51 controls were analyzed. Methods used in the genetic analyses included direct sequencing and multiplex ligation-dependent probe amplification (MLPA). In vitro studies were performed using luciferase assay and quantitative real-time polymerase chain reaction (qPCR) to examine the effect of two novel and three previously identified mutations on the activity of canonical Wnt signaling and on expression of tryptophan hydroxylase 1 (Tph1) and 5-hydroxytryptamine (5-Htr1b). Two novel LRP5 mutations (c.3446 T > A; p.L1149Q and c.3553 G > A; p.G1185R) were identified in two patients and their affected family members. In vitro analyses showed that one of these novel mutations together with two previously reported mutations (p.C913fs, p.R1036Q) significantly reduced the activity of the canonical Wnt signaling pathway. Such reductions may lead to decreased bone formation, and could explain the bone phenotype. Gut-derived Lrp5 has been shown to regulate serotonin synthesis by controlling the production of serotonin rate-limiting enzyme, Tph1. LRP5 mutations did not affect Tph1 expression, and only one mutant (p.L1149Q) reduced expression of serotonin receptor 5-Htr1b (p LRP5 mutations and their effects on the development of juvenile-onset primary osteoporosis, and hence the pathogenesis of the disorder. The

  18. Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs.

    Science.gov (United States)

    Suzukawa, Keisuke; Yamagami, Takeshi; Ohnuma, Takayuki; Hirakawa, Hideki; Kuhara, Satoru; Aso, Yoichi; Ishiguro, Masatsune

    2003-02-01

    We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

  19. Mild thyroid peroxidase deficiency caused by TPO mutations with residual activity: Correlation between clinical phenotypes and enzymatic activity.

    Science.gov (United States)

    Narumi, Satoshi; Fox, Larry A; Fukudome, Keisuke; Sakaguchi, Zenichi; Sugisawa, Chiho; Abe, Kiyomi; Kameyama, Kaori; Hasegawa, Tomonobu

    2017-11-29

    Thyroid peroxidase (TPO) deficiency, caused by biallelic TPO mutations, is a well-established genetic form of congenital hypothyroidism (CH). More than 100 patients have been published, and the patients have been diagnosed mostly in the frame of newborn screening (NBS) programs. Correlation between clinical phenotypes and TPO activity remains unclear. Here, we report clinical and molecular findings of two unrelated TPO mutation-carrying mildly hypothyroid patients. The two patients were born at term after an uneventful pregnancy and delivery, and were NBS negative. They sought medical attention due to goiter at age 8 years. Evaluation of the thyroid showed mild elevation of serum TSH levels, normal or slightly low serum T 4 levels, high serum T 3 to T 4 molar ratio, high serum thyroglobulin levels, and high thyroidal 123 I uptake. We performed next-generation sequencing-based genetic screening, and found that one patient was compound heterozygous for two novel TPO mutations (p.Asp224del; c.820-2A>G), and the other was homozygous for a previously known mutation (p.Trp527Cys). In vitro functional analyses using HEK293 cells showed that the two amino acid-altering mutations (p.Asp224del and p.Trp527Cys) caused partial loss of the enzymatic activity. In conclusion, we report that TPO mutations with residual activity are associated with mild TPO deficiency, which is clinically characterized by marked goiter, mild TSH elevation, high serum T 3 to T 4 molar ratio, and high serum thyroglobulin levels. Our findings illuminate the hitherto under-recognized correlation between clinical phenotypes and residual enzymatic activity among patients with TPO deficiency.

  20. 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

  1. Structural Mutations that Probe the Interactions between the Catalytic and Dianion Activation Sites at Triosephosphate Isomerase‡

    Science.gov (United States)

    Zhai, Xiang; Amyes, Tina L.; Wierenga, Rik K.; Loria, J. Patrick; Richard, John P.

    2013-01-01

    Triosephosphate isomerase (TIM) catalyzes the isomerization of dihydroxyacetone phosphate to form D-glyceraldehyde 3-phosphate. The effects of two structural mutations at TIM on the kinetic parameters for catalysis of the reaction of the truncated substrate glycolaldehyde (GA) and the activation of this reaction by phosphite dianion are reported. The P168A mutation results in similar 50-fold and 80-fold decreases, respectively, in (kcat/Km)E and (kcat/Km)E•HPi for deprotonation of GA catalyzed by free TIM and by the TIM•HPO32− complex. The mutation has little effect on the observed and intrinsic phosphite dianion binding energy, or on the magnitude of phosphite dianion activation of TIM for catalysis of deprotonation of GA. A loop 7 replacement mutant (L7RM) of TIM from chicken muscle was prepared by substitution of the archaeal sequence 208-TGAG for 208-YGGS. The L7RM exhibits a 25-fold decrease in (kcat/Km)E and a larger 170-fold decrease in (kcat/Km)E•HPi for reactions of GA. The mutation has little effect on the observed and intrinsic phosphodianion binding energy, and only a modest effect on phosphite dianion activation of TIM. The observation that both the P168A and loop 7 replacement mutations affect mainly the kinetic parameters for TIM-catalyzed deprotonation, but result in much smaller changes in the parameters for enzyme activation by phosphite dianion provide support for the conclusion that catalysis of proton transfer and dianion activation of TIM take place at separate, weakly interacting, sites in the protein catalyst. PMID:23909928

  2. The second activating glucokinase mutation (A456V)

    DEFF Research Database (Denmark)

    Christesen, Henrik B T; Jacobsen, Bendt B; Odili, Stella

    2002-01-01

    and chlorothiazide was effective. His mother never had clinical hypoglycemic symptoms, even though her fasting blood glucose ranged from 2.9 to 3.5 mmol/l. Increases in serum insulin, C-peptide, and proinsulin in response to an OGTT suggested a lower threshold for glucose-stimulated insulin release (GSIR). Screening...... concentration of glucose needed to achieve the half-maximal rate of phosphorylation) from 8.04 (wild-type) to 2.53 mmol/l. The mutant's Hill coefficient was decreased, and its maximal specific activity k(cat) was increased. Mathematical modeling predicted a markedly lowered GSIR threshold of 1.5 mmol...

  3. Characterization of the MKS1 gene, a new negative regulator of the Ras-cyclic AMP pathway in Saccharomyces cerevisiae.

    Science.gov (United States)

    Matsuura, A; Anraku, Y

    1993-04-01

    In order to isolate genes that function downstream of the Ras-cAMP pathway in Saccharomyces cerevisiae, a YEp13-based genomic library was screened for clones that inhibit growth of cells with diminished A-kinase activity. One such gene, MKS1, was found to encode a hydrophilic 52 kDa protein that shares weak homology with the yeast SPT2/SIN1 gene product. Three lines of evidence suggest that the MKS1 gene product is a negative regulator downstream of the Ras-cAMP pathway: (i) overexpression of MKS1 inhibits growth of cyr1 disruptant cells on YPD medium containing a low concentration of cAMP; (ii) overexpression of MKS1 does not affect TPK1 expression; and (iii) the temperature-sensitive cyr1-230 mutation is partially suppressed by mks1 disruption. The mks1 mutant shows similar phenotypes to gal11/spt13, i.e., it cannot grow on YPGal containing ethidium bromide at 25 degrees C, or on YPGly or SGal at 37 degrees C. The mks1 gal11 double mutant shows more marked phenotypic changes than the single mutants. These results suggest that MKS1 is involved in transcriptional regulation of several genes by cAMP.

  4. Low concentrations of hydrogen peroxide or nitrite induced of Paracoccidioides brasiliensis cell proliferation in a Ras-dependent manner.

    Science.gov (United States)

    Haniu, Ana Eliza Coronel Janu; Maricato, Juliana Terzi; Mathias, Pedro Paulo Moraes; Castilho, Daniele Gonçalves; Miguel, Rodrigo Bernardi; Monteiro, Hugo Pequeno; Puccia, Rosana; Batista, Wagner Luiz

    2013-01-01

    Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM), should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS) and nitrogen (RNS) species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. In lower eukaryotes, Byr2 kinase represents a Ras target. The present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H2O2 (0.1 mM) or NO2 (0.1-0.25 µM) stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD) fused with GST (RBD-Byr2-GST) to detect the Ras active form. After stimulation with low concentrations of H2O2 or NO2, the Ras active form was observed in fungal extracts. Besides, NO2 induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. In conclusion, low concentrations of H2O2 or NO2 stimulated P. brasiliensis proliferation through Ras activation.

  5. Low concentrations of hydrogen peroxide or nitrite induced of Paracoccidioides brasiliensis cell proliferation in a Ras-dependent manner.

    Directory of Open Access Journals (Sweden)

    Ana Eliza Coronel Janu Haniu

    Full Text Available Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM, should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS and nitrogen (RNS species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. In lower eukaryotes, Byr2 kinase represents a Ras target. The present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H2O2 (0.1 mM or NO2 (0.1-0.25 µM stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD fused with GST (RBD-Byr2-GST to detect the Ras active form. After stimulation with low concentrations of H2O2 or NO2, the Ras active form was observed in fungal extracts. Besides, NO2 induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. In conclusion, low concentrations of H2O2 or NO2 stimulated P. brasiliensis proliferation through Ras activation.

  6. 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)

  7. Epilepsy in RAS/MAPK syndrome: two cases of cardio-facio-cutaneous syndrome with epileptic encephalopathy and a literature review.

    Science.gov (United States)

    Adachi, Masao; Abe, Yu; Aoki, Yoko; Matsubara, Yoichi

    2012-01-01

    We report two individual cases of cardio-facio-cutaneous (CFC) syndrome with severe neurological impairment consisting of infantile spasms with hypsarrhythmia and refractory epilepsy with multifocal epileptic paroxysms such as modified hypsarrhythmia. Both cases shared diffuse brain atrophy and severely delayed myelination on neuroimaging. Genetic analysis revealed individual heterozygous mutations in the KRAS (phenotype of CFC/Noonan syndrome) and BRAF genes (phenotype of CFC syndrome). Neurological impairment in cases with mutations in the RAS/MAPK (mitogen activated protein kinase) signal pathway may be more severe, and could be linked to some forms of refractory epilepsy, especially epileptic encephalopathy that includes infantile spasms. Copyright © 2011 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

  8. Effect of point mutations on Herbaspirillum seropedicae NifA activity

    Directory of Open Access Journals (Sweden)

    B. Aquino

    2015-08-01

    Full Text Available NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

  9. Effect of point mutations on Herbaspirillum seropedicae NifA activity

    Energy Technology Data Exchange (ETDEWEB)

    Aquino, B.; Stefanello, A.A.; Oliveira, M.A.S.; Pedrosa, F.O.; Souza, E.M.; Monteiro, R.A.; Chubatsu, L.S. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

    2015-07-10

    NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

  10. A restricted spectrum of NRAS mutations causes Noonan syndrome

    NARCIS (Netherlands)

    Cirstea, Ion C.; Kutsche, Kerstin; Dvorsky, Radovan; Gremer, Lothar; Carta, Claudio; Horn, Denise; Roberts, Amy E.; Lepri, Francesca; Merbitz-Zahradnik, Torsten; Koenig, Rainer; Kratz, Christian P.; Pantaleoni, Francesca; Dentici, Maria L.; Joshi, Victoria A.; Kucherlapati, Raju S.; Mazzanti, Laura; Mundlos, Stefan; Patton, Michael A.; Silengo, Margherita Cirillo; Rossi, Cesare; Zampino, Giuseppe; Digilio, Cristina; Stuppia, Liborio; Seemanova, Eva; Pennacchio, Len A.; Gelb, Bruce D.; Dallapiccola, Bruno; Wittinghofer, Alfred; Ahmadian, Mohammad R.; Tartaglia, Marco; Zenker, Martin

    Noonan syndrome, a developmental disorder characterized by congenital heart defects, reduced growth, facial dysmorphism and variable cognitive deficits, is caused by constitutional dysregulation of the RAS-MAPK signaling pathway. Here we report that germline NRAS mutations conferring enhanced

  11. A CLINICAL CASE OF SYNGAP1 GENE MUTATION IN A GIRL WITH EPILEPSY, MENTAL RETARDATION, AUTISM, AND MOTOR DISORDERS

    OpenAIRE

    M. Yu. Bobylova; M. B. Mironov; M. O. Abramov; A. V. Kulikov; M. V. Kazakova; L. Yu. Glukhova; E. I. Barletova; K. Yu. Mukhin

    2015-01-01

    The introduction of the latest genetic techniques into practice could discover a basis for the comorbidity of genetic epilepsies and behavioral disturbances with cognitive impairments. Some chromosomal syndromes are characterized by a specific electroencephalogram (EEG) pattern, the type of seizures, and the variant of the course of epilepsy. This paper describes a case of synaptic RAS GTP-ase-activating protein 1 (SYNGAP1) gene mutation in a 9-year-old female patient with eyelid myoclonic ep...

  12. The protist Trichomonas vaginalis harbors multiple lineages of transcriptionally active Mutator-like elements

    Directory of Open Access Journals (Sweden)

    Pereira Gonçalo AG

    2009-07-01

    Full Text Available Abstract Background For three decades the Mutator system was thought to be exclusive of plants, until the first homolog representatives were characterized in fungi and in early-diverging amoebas earlier in this decade. Results Here, we describe and characterize four families of Mutator-like elements in a new eukaryotic group, the Parabasalids. These Trichomonas vaginalis Mutator- like elements, or TvMULEs, are active in T. vaginalis and patchily distributed among 12 trichomonad species and isolates. Despite their relatively distinctive amino acid composition, the inclusion of the repeats TvMULE1, TvMULE2, TvMULE3 and TvMULE4 into the Mutator superfamily is justified by sequence, structural and phylogenetic analyses. In addition, we identified three new TvMULE-related sequences in the genome sequence of Candida albicans. While TvMULE1 is a member of the MuDR clade, predominantly from plants, the other three TvMULEs, together with the C. albicans elements, represent a new and quite distinct Mutator lineage, which we named TvCaMULEs. The finding of TvMULE1 sequence inserted into other putative repeat suggests the occurrence a novel TE family not yet described. Conclusion These findings expand the taxonomic distribution and the range of functional motif of MULEs among eukaryotes. The characterization of the dynamics of TvMULEs and other transposons in this organism is of particular interest because it is atypical for an asexual species to have such an extreme level of TE activity; this genetic landscape makes an interesting case study for causes and consequences of such activity. Finally, the extreme repetitiveness of the T. vaginalis genome and the remarkable degree of sequence identity within its repeat families highlights this species as an ideal system to characterize new transposable elements.

  13. Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma.

    Science.gov (United States)

    Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B; Wu, Chia-Chin; Akdemir, Kadir C; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T; Welch, Heidi C E; Garraway, Levi A; Chin, Lynda

    2016-03-01

    PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2(E824)*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57(KIP2)). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

  14. Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

    KAUST Repository

    Lissanu Deribe, Yonathan

    2016-03-01

    PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

  15. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    Science.gov (United States)

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase.

  16. Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

    Energy Technology Data Exchange (ETDEWEB)

    B McCray; E Skordalakes; J Taylor

    2011-12-31

    Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.

  17. Manipulation of EphB2 regulatory motifs and SH2 binding sites switches MAPK signaling and biological activity.

    Science.gov (United States)

    Tong, Jiefei; Elowe, Sabine; Nash, Piers; Pawson, Tony

    2003-02-21

    Signaling by the Eph family of receptor tyrosine kinases (RTKs) is complex, because they can interact with a variety of intracellular targets, and can potentially induce distinct responses in different cell types. In NG108 neuronal cells, activated EphB2 recruits p120RasGAP, in a fashion that is associated with down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway and neurite retraction. To pursue the role of the Ras-MAPK pathway in EphB2-mediated growth cone collapse, and to explore the biochemical and biological functions of Eph receptors, we sought to re-engineer the signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM) RasGAP binding sites but incorporated a Grb2 binding motif at an alternate RasGAP binding site within the kinase domain had little effect on basal Erk MAPK activation. In contrast, elimination of all RasGAP binding sites, accompanied by the addition of a Grb2 binding site within the kinase domain, led to an increase in phospho-Erk levels in NG108 cells following ephrin-B1 stimulation. Functional assays indicated a correlation between neurite retraction and the ability of the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data suggest that EphB2 can be designed to repress, stabilize, or activate the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2 domain binding sites and support the notion that Erk MAPK regulation plays a significant role in axon guidance. The behavior of EphB2 variants with mutations in the JM region and kinase domains suggests an intricate pattern of regulation and target recognition by Eph receptors.

  18. dpl-1 DP and efl-1 E2F act with lin-35 Rb to antagonize Ras signaling in C. elegans vulval development.

    Science.gov (United States)

    Ceol, C J; Horvitz, H R

    2001-03-01

    The synthetic multivulva (synMuv) genes define two functionally redundant pathways that antagonize RTK/Ras signaling during Caenorhabditis elegans vulval induction. The synMuv gene lin-35 encodes a protein similar to the mammalian tumor suppressor pRB and has been proposed to act as a transcriptional repressor. Studies using mammalian cells have shown that pRB can prevent cell cycle progression by inhibiting DP/E2F-mediated transcriptional activation. We identified C. elegans genes that encode proteins similar to DP or E2F. Loss-of-function mutations in two of these genes, dpl-1 DP and efl-1 E2F, caused the same vulval abnormalities as do lin-35 Rb loss-of-function mutations. We propose that rather than being inhibited by lin-35 Rb, dpl-1 DP and efl-1 E2F act with lin-35 Rb in transcriptional repression to antagonize RTK/Ras signaling during vulval development.

  19. Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

    Directory of Open Access Journals (Sweden)

    Pathan AAK

    2016-05-01

    Full Text Available Akbar Ali Khan Pathan,1,2,* Bhavana Panthi,3,* Zahid Khan,1 Purushotham Reddy Koppula,4–6 Mohammed Saud Alanazi,1 Sachchidanand,3 Narasimha Reddy Parine,1 Mukesh Chourasia3,* 1Genome Research Chair (GRC, Department of Biochemistry, College of Science, King Saud University, 2Integrated Gulf Biosystems, Riyadh, Kingdom of Saudi Arabia; 3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Hajipur, India; 4Department of Internal Medicine, School of Medicine, 5Harry S. Truman Memorial Veterans Affairs Hospital, 6Department of Radiology, School of Medicine, Columbia, MO, USA *These authors contributed equally to this work Objective: Kirsten rat sarcoma (K-Ras protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods: In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results: Interestingly, the designed compounds exhibit a binding preference for the

  20. Combined Inactivation of MYC and K-Ras oncogenes reverses tumorigenesis in lung adenocarcinomas and lymphomas.

    Directory of Open Access Journals (Sweden)

    Phuoc T Tran

    2008-05-01

    Full Text Available Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as "oncogene-addiction." However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment.To examine how the MYC and K-ras(G12D oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-ras(G12D to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-ras(G12D- or MYC/K-ras(G12D-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-ras(G12D-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-ras(G12D resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-ras(G12D in maintenance of lung tumors, we found that the down-stream mediators of K-ras(G12D signaling, Stat3 and Stat5, are dephosphorylated following conditional K-ras(G12D but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-ras(G12D. Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation.Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic pathways is more likely to be effective in the

  1. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    Science.gov (United States)

    Ledoux, Sarah; Guthrie, Christine

    2016-06-03

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity

    Science.gov (United States)

    Salvat, Regina S.; Parker, Andrew S.; Kirsch, Jack R.; Brooks, Seth A.

    2017-01-01

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide–MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 109 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening. PMID:28607051

  3. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

    Science.gov (United States)

    Salvat, Regina S; Verma, Deeptak; Parker, Andrew S; Kirsch, Jack R; Brooks, Seth A; Bailey-Kellogg, Chris; Griswold, Karl E

    2017-06-27

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 10 9 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

  4. Distinct roles of the RasGAP family proteins in C. elegans associative learning and memory.

    Science.gov (United States)

    Gyurkó, M Dávid; Csermely, Péter; Sőti, Csaba; Steták, Attila

    2015-10-15

    The Ras GTPase activating proteins (RasGAPs) are regulators of the conserved Ras/MAPK pathway. Various roles of some of the RasGAPs in learning and memory have been reported in different model systems, yet, there is no comprehensive study to characterize all gap genes in any organism. Here, using reverse genetics and neurobehavioural tests, we studied the role of all known genes of the rasgap family in C. elegans in associative learning and memory. We demonstrated that their proteins are implicated in different parts of the learning and memory processes. We show that gap-1 contribute redundantly with gap-3 to the chemosensation of volatile compounds, gap-1 plays a major role in associative learning, while gap-2 and gap-3 are predominantly required for short- and long-term associative memory. Our results also suggest that the C. elegans Ras orthologue let-60 is involved in multiple processes during learning and memory. Thus, we show that the different classes of RasGAP proteins are all involved in cognitive function and their complex interplay ensures the proper formation and storage of novel information in C. elegans.

  5. Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity.

    Science.gov (United States)

    Dolinska, Monika B; Kus, Nicole J; Farney, S Katie; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

    2017-01-01

    Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  6. Epidermal Growth Factor Receptor (EGFR gene copy number (GCN correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH and chromogenic in situ hybridization (CISH analysis

    Directory of Open Access Journals (Sweden)

    Scartozzi Mario

    2009-08-01

    Full Text Available Abstract Background K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Methods Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Results Forty-four patients were available for analysis. We observed a partial remission in 9 (60% and 2 (9% cases with a FISH EGFR GCN ≥ 2.6 and Conclusion FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.

  7. PROGRANULIN MUTATIONS AFFECTS BRAIN OSCILLATORY ACTIVITY IN FRONTO-TEMPORAL DEMENTIA

    Directory of Open Access Journals (Sweden)

    Davide Vito Moretti

    2016-02-01

    Full Text Available Background: mild cognitive impairment (MCI is a clinical stage indicating a prodromal phase of dementia. This practical concept could be used also for fronto-temporal dementia (FTD. Progranulin (PGRN has been recently recognized as a useful diagnostic biomarker for fronto-temporal lobe degeneration (FTLD due to GRN null mutations. Electroencephalography (EEG is a reliable tool in detecting brain networks changes. The working hypothesis of the present study is that EEG oscillations could detect different modifications among FTLD stages (FTD-MCI versus overt FTD as well as differences between GRN mutation carriers versus non carriers in patients with overt FTD. Methods: EEG in all patients and PGRN dosage in patients with a clear FTD were detected. The cognitive state has been investigated through mini mental state examination (MMSE. Results: MCI-FTD showed a significant lower spectral power in both alpha and theta oscillations as compared to overt FTD. GRN mutations carriers affected by FTLD show an increase in high alpha and decrease in theta oscillations as compared to non-carriers.Conclusion: EEG frequency rhythms are sensible to different stage of FTD and could detect changes in brain oscillatory activity affected by GRN mutations

  8. New hyperekplexia mutations provide insight into glycine receptor assembly, trafficking, and activation mechanisms

    DEFF Research Database (Denmark)

    Bode, Anna; Wood, Sian-Elin; Mullins, Jonathan G L

    2013-01-01

    Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperek......Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage...... to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated...... a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X...

  9. Mutation of Chinese hamster cells by near-UV activation of promutagens

    International Nuclear Information System (INIS)

    Barnhart, B.J.; Cox, S.H.

    1980-01-01

    A tissue-culture assay for mutagenesis and cytotoxicity incorporating near ultraviolet (NUV) light activation of polyaromatic hydrocarbons (PAH) has been developed. Cultures of Chinese hamster cells (line CHO) growing in suspension culture were inoculated with benzo[a]pyrene (B[a]P), 7,12-dimethylbenzanthracene (DMBA) or shale-oil retort-water and exposed to light from a high-pressure mercury lamp fitted with a Corning NUV bandpass filter. This light source both permitted activation of PAH and the shale-oil water and precluded detectable damage to DNA. Neither the PAH nor the NUV alone had any effect on cell survival or mutation frequencies but the chemicals plus NUV were extremely effective in producing mutations to 6-thioguanine resistance (hgprt gene). (orig.)

  10. Dexras1 a unique ras-GTPase interacts with NMDA receptor activity and provides a novel dissociation between anxiety, working memory and sensory gating.

    Science.gov (United States)

    Carlson, G C; Lin, R E; Chen, Y; Brookshire, B R; White, R S; Lucki, I; Siegel, S J; Kim, S F

    2016-05-13

    Dexras1 is a novel GTPase that acts at a confluence of signaling mechanisms associated with psychiatric and neurological disease including NMDA receptors, NOS1AP and nNOS. Recent work has shown that Dexras1 mediates iron trafficking and NMDA-dependent neurodegeneration but a role for Dexras1 in normal brain function or psychiatric disease has not been studied. To test for such a role, mice with germline knockout (KO) of Dexras1 were assayed for behavioral abnormalities as well as changes in NMDA receptor subunit protein expression. Because Dexras1 is up-regulated during stress or by dexamethasone treatment, we included measures associated with emotion including anxiety and depression. Baseline anxiety-like measures (open field and zero maze) were not altered, nor were depression-like behavior (tail suspension). Measures of memory function yielded mixed results, with no changes in episodic memory (novel object recognition) but a significant decrement on working memory (T-maze). Alternatively, there was an increase in pre-pulse inhibition (PPI), without concomitant changes in either startle amplitude or locomotor activity. PPI data are consistent with the direction of change seen following exposure to dopamine D2 antagonists. An examination of NMDA subunit expression levels revealed an increased expression of the NR2A subunit, contrary to previous studies demonstrating down-regulation of the receptor following antipsychotic exposure (Schmitt et al., 2003) and up-regulation after exposure to isolation rearing (Turnock-Jones et al., 2009). These findings suggest a potential role for Dexras1 in modulating a selective subset of psychiatric symptoms, possibly via its interaction with NMDARs and/or other disease-related binding-partners. Furthermore, data suggest that modulating Dexras1 activity has contrasting effects on emotional, sensory and cognitive domains. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. An activating mutation of interferon regulatory factor 4 (IRF4) in adult T cell leukemia.

    Science.gov (United States)

    Cherian, Mathew A; Olson, Sydney; Sundaramoorthi, Hemalatha; Cates, Kitra; Cheng, Xiaogang; Harding, John; Martens, Andrew; Challen, Grant A; Tyagi, Manoj; Ratner, Lee; Rauch, Daniel

    2018-03-14

    The human T cell leukemia virus-1 (HTLV-1) oncoprotein Tax drives cell proliferation and resistance to apoptosis early in the pathogenesis of adult T-cell leukemia (ATL). Subsequently, likely as a result of specific immuno-editing, Tax expression is downregulated and functionally replaced by somatic driver mutations of the host genome. Both amplification and point mutations of interferon regulatory factor 4 (IRF4) have been previously detected in ATL, and the K59R mutation is the most common single-nucleotide variation in IRF4 and is found exclusively in ATL. Here high throughput whole-exome sequencing revealed recurrent activating genetic alterations in the T cell receptor, CD28, and NF-kB pathways. Moreover, we found that IRF4, which is transcriptionally activated downstream of these pathways, is frequently mutated in ATL. IRF4 RNA, protein, and IRF4 transcriptional targets are uniformly elevated in HTLV transformed cells and ATL cell lines, and IRF4 was bound to genomic regulatory DNA of many of these transcriptional targets in HTLV-1 transformed cell lines. We further noted that the K59R IRF4 mutant is expressed at higher levels in the nucleus than is wild-type IRF4, and is transcriptionally more active. Expression of both wild-type and the K59R mutant of IRF4 from a constitutive promoter in retrovirally transduced murine bone marrow cells increased the abundance of T lymphocytes but not myeloid cells or B lymphocytes in mice. IRF4 may represent a therapeutic target in ATL since ATL cells select for a mutant of IRF4 with higher nuclear expression and transcriptional activity, and over-expression of IRF4 induces the expansion of T lymphocytes in vivo. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Clinicopathologic features and outcomes of patients with lung adenocarcinomas harboring BRAF mutations in the Lung Cancer Mutation Consortium.

    Science.gov (United States)

    Villaruz, Liza C; Socinski, Mark A; Abberbock, Shira; Berry, Lynne D; Johnson, Bruce E; Kwiatkowski, David J; Iafrate, A John; Varella-Garcia, Marileila; Franklin, Wilbur A; Camidge, D Ross; Sequist, Lecia V; Haura, Eric B; Ladanyi, Mark; Kurland, Brenda F; Kugler, Kelly; Minna, John D; Bunn, Paul A; Kris, Mark G

    2015-02-01

    The advent of effective targeted therapy for BRAF(V600E) -mutant lung adenocarcinomas necessitates further exploration of the unique clinical features and behavior of advanced-stage BRAF-mutant lung adenocarcinomas. Data were reviewed for patients with advanced lung adenocarcinomas enrolled in the Lung Cancer Mutation Consortium whose tumors underwent testing for mutations in epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), human epidermal growth factor receptor 2 (HER2), AKT1, BRAF, dual-specificity mitogen-activated protein kinase kinase 1 (MEK1), neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA); for anaplastic lymphoma kinase (ALK) translocations; and for MET amplification. Twenty-one BRAF mutations were identified in 951 patients with adenocarcinomas (2.2%; 95% confidence interval [CI], 1.4%-3.4%): 17 (81%; 95% CI, 60%-92%) were BRAF(V600E) mutations, and 4 were non-BRAF(V600E) mutations. Among the 733 cases tested for all 10 genes, BRAF mutations were more likely to occur than most other genotypic abnormalities in current or former smokers (BRAF vs sensitizing EGFR, 82% vs 36%, mid-P mutations, 49%, mid-P = .02; BRAF vs patients with more than 1 oncogenic driver [doubleton], 46%, mid-P = .04.) The double-mutation rate was 16% among patients with BRAF mutations but 5% among patients with other genomic abnormalities (mid-P = .045). Differences were not found in survival between patients with BRAF mutations and those with other genomic abnormalities (P > .20). BRAF mutations occurred in 2.2% of advanced-stage lung adenocarcinomas, were most commonly V600E, and were associated with distinct clinicopathologic features in comparison with other genomic subtypes and with a high mutation rate in more than 1 gene. These findings underscore the importance of comprehensive genomic profiling in assessing patients with

  13. Enzyme-activity mutations detected in mice after paternal fractionated irradiation

    International Nuclear Information System (INIS)

    Charles, D.J.; Pretsch, W.

    1986-01-01

    (101/E1 X C3H/E1)F 1 -hybrid male mice were exposed in a 24-h fractionation interval to either 3.0 + 3.0-Gy or 5.1 + 5.1-Gy X-irradiation, and mated to untreated Test-stock females. The offspring were examined for mutations at 7 recessive specific loci and for activity alterations of erythrocyte enzymes controlled presumably by 12 loci. No enzyme-activity mutant was found in 3610 F 1 -offspring of the control group. In the experimental groups, no mutant was detected in 533 (3.0 + 3.0 Gy) and 173 (5.1 + 5.1 Gy) offspring from postspermatogonial germ cells treated. After treatment of spermatogonia, 1 mutant in 3388 F 1 -offspring of the 3.0 + 3.0-Gy group, and 5 mutants in 3187 F 1 offspring of the 5.1 + 5.1-Gy group were found. The mutants were all genetically confirmed. The frequency (expressed as mutants/locus/gamete) of enzyme-activity mutations is 2 (5.1 + 5.1-Gy group) to 10 (3.0 + 3.0-Gy group) times lower than the frequency of recessive specific-locus mutations. (Auth.)

  14. Mex3c mutation reduces adiposity partially through increasing physical activity.

    Science.gov (United States)

    Han, Changjie; Jiao, Yan; Zhao, Qingguo; Lu, Baisong

    2014-06-01

    MEX3C is an RNA-binding protein with unknown physiological function. We have recently reported that a Mex3c mutation in mice causes growth retardation and reduced adiposity, but how adiposity is reduced remains unclear. Herein, we show that homozygous Mex3c gene trap mice have increased physical activity. The Mex3c mutation consistently conferred full protection from diet-induced obesity, hyperglycemia, insulin resistance, hyperlipidemia, and hepatic steatosis. In ob/ob mice with leptin deficiency, the Mex3c mutation also increased physical activity and improved glucose and lipid profiles. Expressing cre in the neurons of Mex3c gene trap mice, an attempt to partially restoring neuronal Mex3c expression, significantly increased white adipose tissue deposition, but had no effects on body length. Our data suggest that one way in which Mex3c regulates adiposity is through controlling physical activity, and that neuronal Mex3c expression could play an important role in this process. © 2014 Society for Endocrinology.

  15. Dopamine Induces Oscillatory Activities in Human Midbrain Neurons with Parkin Mutations.

    Science.gov (United States)

    Zhong, Ping; Hu, Zhixing; Jiang, Houbo; Yan, Zhen; Feng, Jian

    2017-05-02

    Locomotor symptoms in Parkinson's disease (PD) are accompanied by widespread oscillatory neuronal activities in basal ganglia. Here, we show that activation of dopamine D1-class receptors elicits a large rhythmic bursting of spontaneous excitatory postsynaptic currents (sEPSCs) in midbrain neurons differentiated from induced pluripotent stem cells (iPSCs) of PD patients with parkin mutations, but not normal subjects. Overexpression of wild-type parkin, but not its PD-causing mutant, abolishes the oscillatory activities in patient neurons. Dopamine induces a delayed enhancement in the amplitude of spontaneous, but not miniature, EPSCs, thus increasing quantal content. The results suggest that presynaptic regulation of glutamatergic transmission by dopamine D1-class receptors is significantly potentiated by parkin mutations. The aberrant dopaminergic regulation of presynaptic glutamatergic transmission in patient-specific iPSC-derived midbrain neurons provides a mechanistic clue to PD pathophysiology, and it demonstrates the usefulness of this model system in understanding how mutations of parkin cause movement symptoms in Parkinson's disease. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations.

    Science.gov (United States)

    Osada, Takuya; Chen, Minyong; Yang, Xiao Yi; Spasojevic, Ivan; Vandeusen, Jeffrey B; Hsu, David; Clary, Bryan M; Clay, Timothy M; Chen, Wei; Morse, Michael A; Lyerly, H Kim

    2011-06-15

    Wnt/β-catenin pathway activation caused by adenomatous polyposis coli (APC) mutations occurs in approximately 80% of sporadic colorectal cancers (CRC). The antihelminth compound niclosamide downregulates components of the Wnt pathway, specifically Dishevelled-2 (Dvl2) expression, resulting in diminished downstream β-catenin signaling. In this study, we determined whether niclosamide could inhibit the Wnt/β-catenin pathway in human CRCs and whether its inhibition might elicit antitumor effects in the presence of APC mutations. We found that niclosamide inhibited Wnt/β-catenin pathway activation, downregulated Dvl2, decreased downstream β-catenin signaling, and exerted antiproliferative effects in human colon cancer cell lines and CRC cells isolated by surgical resection of metastatic disease, regardless of mutations in APC. In contrast, inhibition of NF-κB or mTOR did not exert similar antiproliferative effects in these CRC model systems. In mice implanted with human CRC xenografts, orally administered niclosamide was well tolerated, achieved plasma and tumor levels associated with biologic activity, and led to tumor control. Our findings support clinical explorations to reposition niclosamide for the treatment of CRC.

  17. Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

    Science.gov (United States)

    Zhuang, P L; Yu, L X; Tao, Y; Zhou, Y; Zhi, Q H; Lin, H C

    2016-04-11

    Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans. The point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. SrtA△N40(UA159) and the mutant enzymes, SrtA△N40(D56E) and SrtA△N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtA△N40(D56E) and SrtA△N40(R157H) remained approximately equal to the affinity of SrtA△N40(UA159), as determined by the Michaelis constant (K m ). However, the catalytic rate constant (k cat ) and catalytic efficiency (k cat /K m ) of SrtA△N40(D56E) were reduced compared with those of SrtA△N40(R157H) and SrtA△N40(UA159), whereas the k cat and k cat /K m values of SrtA△N40(R157H) were slightly lower than those of SrtA△N40(UA159). The findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.

  18. Novel Oncogenic Mutations of CBL in Human Acute Myeloid Leukemia That Activate Growth and Survival Pathways Depend on Increased Metabolism*

    Science.gov (United States)

    Fernandes, Margret S.; Reddy, Mamatha M.; Croteau, Nicole J.; Walz, Christoph; Weisbach, Henry; Podar, Klaus; Band, Hamid; Carroll, Martin; Reiter, Andreas; Larson, Richard A.; Salgia, Ravi; Griffin, James D.; Sattler, Martin

    2010-01-01

    Acute myeloid leukemia (AML) is characterized by multiple mutagenic events that affect proliferation, survival, as well as differentiation. Recently, gain-of-function mutations in the α helical structure within the linker sequence of the E3 ubiquitin ligase CBL have been associated with AML. We identified four novel CBL mutations, including a point mutation (Y371H) and a putative splice site mutation in AML specimens. Characterization of these two CBL mutants revealed that coexpression with the receptor tyrosine kinases FLT3 (Fms-like tyrosine kinase 3) or KIT-induced ligand independent growth or ligand hyperresponsiveness, respectively. Growth of cells expressing mutant CBL required expression and kinase activity of FLT3. In addition to the CBL-dependent phosphorylation of FLT3 and CBL itself, transformation was associated with activation of Akt and STAT5 and required functional expression of the small GTPases Rho, Rac, and Cdc42. Furthermore, the mutations led to constitutively elevated intracellular reactive oxygen species levels, which is commonly linked to increased glucose metabolism in cancer cells. Inhibition of hexokinase with 2-deoxyglucose blocked the transforming activity of CBL mutants and reduced activation of signaling mechanisms. Overall, our data demonstrate that mutations of CBL alter cellular biology at multiple levels and require not only the activation of receptor proximal signaling events but also an increase in cellular glucose metabolism. Pathways that are activated by CBL gain-of-function mutations can be efficiently targeted by small molecule drugs. PMID:20622007

  19. Byzantine seals from the Ras fortress

    Directory of Open Access Journals (Sweden)

    Ivanišević Vujadin

    2013-01-01

    Full Text Available In this paper, seals found at the location of the Ras fortress (Tvrđava Ras have been published. Inscriptions on these seals show that they used to belong to persons which could be identified with certain military commanders who served under Alexios I Komnenos. The seals in question are: the seals of protonobelissimos Eustathios Kamytzes, Constantine Dalassenos Doukas, protoproedros and doux Constantine Kekaumenos and a certain person called Alexios. [Projekat Ministarstva nauke Republike Srbije, br. 177021 i br. 177032

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Correlation between MEK signature and Ras gene alteration in advanced gastric cancer.

    Science.gov (United States)

    Ahn, Soomin; Brant, Roz; Sharpe, Alan; Dry, Jonathan R; Hodgson, Darren R; Kilgour, Elaine; Kim, Kyung; Kim, Seung Tae; Park, Se Hoon; Kang, Won Ki; Kim, Kyoung-Mee; Lee, Jeeyun

    2017-12-08

    MEK inhibitor (selumetinib) is a potent, orally active inhibitor of MAPK/ERK pathway. It is important to develop an accurate and robust method indicative of RAS pathway activity to stratify potential patients who can benefit from selumetinib treatment in gastric cancer (GC). First, we surveyed the sensitivity to selumetinib in a panel of 22 GC cell lines and correlated with MEK signature to selumetinib sensitivity. Next, we analyzed MEK signature via nanostring assay in two Asian cohorts using clinical samples ( n = 218) and then performed a correlative analysis with MEK signature status and KRAS genotype in GC. MEK signature was predictive of response of selumetinib in GC cell lines regardless of KRAS mutation status. The proportion of high MEK signature by nanostring assay was 6.9% and the proportion of high MEK signature was significantly higher in KRAS altered group in a Korean cohort. None of PIK3CA altered cases belonged to high MEK signature group. MEK high signature was more prevalent in intestinal type by Lauren classification. The correlation between MEK signature, KRAS alteration and treatment response to selumetinib should be validated in prospective clinical trials.

  2. Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase.

    Science.gov (United States)

    McDonald, Charles J; Acheff, Eric; Kennedy, Ryan; Taylor, Lynn; Curthoys, Norman P

    2015-09-01

    The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGACΔ1 plasmid. The wild type and mutated proteins were purified by Ni(+)-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K311A and K328A) and the one in the dimer:dimer interface (K396A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K320A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Mechanism for activation of mutated epidermal growth factor receptors in lung cancer.

    Science.gov (United States)

    Red Brewer, Monica; Yun, Cai-Hong; Lai, Darson; Lemmon, Mark A; Eck, Michael J; Pao, William

    2013-09-17

    The initiation of epidermal growth factor receptor (EGFR) kinase activity proceeds via an asymmetric dimerization mechanism in which a "donor" tyrosine kinase domain (TKD) contacts an "acceptor" TKD, leading to its activation. In the context of a ligand-induced dimer, identical wild-type EGFR TKDs are thought to assume the donor or acceptor roles in a random manner. Here, we present biochemical reconstitution data demonstrating that activated EGFR mutants found in lung cancer preferentially assume the acceptor role when coexpressed with WT EGFR. Mutated EGFRs show enhanced association with WT EGFR, leading to hyperphosphorylation of the WT counterpart. Mutated EGFRs also hyperphosphorylate the related erythroblastic leukemia viral oncogene (ErbB) family member, ErbB-2, in a similar manner. This directional "superacceptor activity" is particularly pronounced in the drug-resistant L834R/T766M mutant. A 4-Å crystal structure of this mutant in the active conformation reveals an asymmetric dimer interface that is essentially the same as that in WT EGFR. Asymmetric dimer formation induces an allosteric conformational change in the acceptor subunit. Thus, superacceptor activity likely arises simply from a lower energetic cost associated with this conformational change in the mutant EGFR compared with WT, rather than from any structural alteration that impairs the donor role of the mutant. Collectively, these findings define a previously unrecognized mode of mutant-specific intermolecular regulation for ErbB receptors, knowledge of which could potentially be exploited for therapeutic benefit.

  4. Deep intronic mutation and pseudo exon activation as a novel muscular hypertrophy modifier in cattle.

    Directory of Open Access Journals (Sweden)

    Claire Bouyer

    Full Text Available Myostatin is essential for proper regulation of myogenesis, and inactivation of Myostatin results in muscle hypertrophy. Here, we identified an unexpected mutation in the myostatin gene which is almost fixed in Blonde d'Aquitaine cattle. In skeletal muscle, the mutant allele was highly expressed leading to an abnormal transcript consisting of a 41-bp inclusion and premature termination codons and to residual levels of a correctly spliced transcript. This expression pattern, caused by a leaky intronic mutation with regard to spliceosome activity and its apparent stability with regard to surveillance mechanisms, could contribute to the moderate muscle hypertrophy in this cattle breed. This finding is of importance for genetic counseling for meat quantity and quality in livestock production and possibly to manipulate myostatin pre-mRNA in human muscle diseases.

  5. 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.

  6. Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway.

    Directory of Open Access Journals (Sweden)

    George Dialynas

    2015-05-01

    Full Text Available Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, we performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, we modeled these mutations in Drosophila Lamin C and expressed the mutant lamins in muscle. We found that the structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of our Drosophila model. Thus, novel

  7. Mechanism of Ras Activation by TGFBeta

    Science.gov (United States)

    2002-07-01

    cells. JBiol Chem 271, 13931-13934. Verschueren, K., Remade, J. E., Collart, C, Kraft , H., Baker, B. S., Tylza- nowski, P., Nelles, L., Wuytens, G...within specific functional domains. Penn State University Dinner and Poster Session, Penn State Univ., University Park, PA, April 2002 Tang, Q., Staub

  8. Society News: Monica Grady awarded CBE; Grubb Parsons Lecture 2012; Join the RAS; Astronomy on radio for kids; New Fellows; Peter D Hingley

    Science.gov (United States)

    2012-08-01

    RAS Fellow Prof. Monica Grady has been made a Commander of the Most Excellent Order of the British Empire (CBE), in recognition of her services to space science. The RAS sponsors the annual Grubb Parsons Lecture, which this year took place on 6 June at the University of Durham. If you are a professional astronomer, geophysicist, or similar, a student studying these disciplines, or simply someone with a serious interest in them, we urge you to apply for membership of the RAS. Outreach is an important activity for the RAS. We recently supported an astronomy series called Deep Space High on the digital radio channel Fun Kids.

  9. 1, 25 Dihydroxyvitamin D Regulation of Glucose Metabolism in Harvey-ras Transformed MCF10A Human Breast Epithelial Cells

    Science.gov (United States)

    Zheng, Wei; Tayyari, Fariba; Gowda, G. A. Nagana; Raftery, Daniel; McLamore, Eric S.; Shi, Jin; Porterfield, D. Marshall; Donkin, Shawn; Bequette, Brian; Teegarden, Dorothy

    2014-01-01

    This study was designed to investigate the impact of 1,25 dihydroxyvitamin D (1,25(OH)2D) on glucose metabolism during early cancer progression. Untransformed and ras-oncogene transfected (ras) MCF10A human breast epithelial cells were employed to model early breast cancer progression. 1,25(OH)2D modified the response of the ras cells to glucose restriction, suggesting 1,25(OH)2D may reduce the ras cell glucose addiction noted in cancer cells. To understand the 1,25(OH)2D regulation of glucose metabolism, following four-day 1,25(OH)2D treatment, metabolite fluxes at the cell membrane were measured by a nanoprobe biosensor, [13C6]glucose flux by 13C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of related enzymes assessed. Treatment with 1,25(OH)2D reduced glycolysis as flux of glucose to 3-phosphoglycerate was reduced by 15% (P = 0.017) and 32% (P MCF10A and ras cells respectively. In the ras cells, 1,25(OH)2D reduced lactate dehydrogenase activity by 15% (P MCF10A and ras cells, respectively, suggesting a reduction in tricarboxylic acid (TCA) cycle activity. The results suggest a novel mechanism involving the regulation of glucose metabolism by which 1,25(OH)2D may prevent breast cancer progression. PMID:23619337

  10. Novel somatic mutations in large granular lymphocytic leukemia affecting the STAT-pathway and T-cell activation

    International Nuclear Information System (INIS)

    Andersson, E I; Rajala, H L M; Eldfors, S; Ellonen, P; Olson, T; Jerez, A; Clemente, M J; Kallioniemi, O; Porkka, K; Heckman, C; Loughran, T P Jr; Maciejewski, J P; Mustjoki, S

    2013-01-01

    T-cell large granular lymphocytic (T-LGL) leukemia is a clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene, whereas STAT5 mutations are present in 2% of patients. In order to identify putative disease-causing genetic alterations in the remaining T-LGL patients, we performed exome sequencing from three STAT mutation-negative patients and validated the findings in 113 large granular lymphocytic (LGL) leukemia patients. On average, 11 CD8+ LGL leukemia cell-specific high-confidence nonsynonymous somatic mutations were discovered in each patient. Interestingly, all patients had at least one mutation that affects either directly the STAT3-pathway (such as PTPRT) or T-cell activation (BCL11B, SLIT2 and NRP1). In all three patients, the STAT3 pathway was activated when studied by RNA expression or pSTAT3 analysis. Screening of the remaining 113 LGL leukemia patients did not reveal additional patients with same mutations. These novel mutations are potentially biologically relevant and represent rare genetic triggers for T-LGL leukemia, and are associated with similar disease phenotype as observed in patients with mutations in the STAT3 gene

  11. Disruption of Dopamine Neuron Activity Pattern Regulation through Selective Expression of a Human KCNN3 Mutation

    Science.gov (United States)

    Soden, Marta E.; Jones, Graham L.; Sanford, Christina A.; Chung, Amanda S.; Güler, Ali D.; Chavkin, Charles; Luján, Rafael; Zweifel, Larry S.

    2013-01-01

    Summary The calcium-activated small conductance potassium channel, SK3, plays an essential role in the regulation of dopamine neuron activity patterns. Here we demonstrate that expression of a human disease-related SK3 mutation (hSK3Δ) in dopamine neurons of mice disrupts the balance between tonic and phasic dopamine neuron activity. Expression of hSK3Δ suppressed endogenous SK currents, reducing coupling between SK channels and NMDA receptors (NMDARs) and increasing permissiveness for burst firing. Consistent with enhanced excitability of dopamine neurons, hSK3Δ increased evoked calcium signals in dopamine neurons in vivo and potentiated evoked dopamine release. Specific expression of hSK3Δ led to deficits in attention and sensory gating and heightened sensitivity to a psychomimetic drug. Sensory-motor alterations and psychomimetic sensitivity were recapitulated in a mouse model of transient, reversible dopamine neuron activation. These results demonstrate the cell-autonomous effects of a human ion channel mutation on dopamine neuron physiology and the impact of activity pattern disruption on behavior. PMID:24206670

  12. Epidermal Growth Factor Receptor (EGFR) gene copy number (GCN) correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH) and chromogenic in situ hybridization (CISH) analysis.

    Science.gov (United States)

    Scartozzi, Mario; Bearzi, Italo; Mandolesi, Alessandra; Pierantoni, Chiara; Loupakis, Fotios; Zaniboni, Alberto; Negri, Francesca; Quadri, Antonello; Zorzi, Fausto; Galizia, Eva; Berardi, Rossana; Biscotti, Tommasina; Labianca, Roberto; Masi, Gianluca; Falcone, Alfredo; Cascinu, Stefano

    2009-08-27

    K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Forty-four patients were available for analysis. We observed a partial remission in 9 (60%) and 2 (9%) cases with a FISH EGFR GCN >or= 2.6 and CISH EGFR GCN >or= 2.12 and CISH EGFR GCN whereas it was 2.9 and 3.1 months in those with low FISH and CISH EGFR GCN (p = 0.04 and 0.02 respectively). FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.

  13. Epidermal Growth Factor Receptor (EGFR) gene copy number (GCN) correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH) and chromogenic in situ hybridization (CISH) analysis

    International Nuclear Information System (INIS)

    Scartozzi, Mario; Galizia, Eva; Berardi, Rossana; Biscotti, Tommasina; Labianca, Roberto; Masi, Gianluca; Falcone, Alfredo; Cascinu, Stefano; Bearzi, Italo; Mandolesi, Alessandra; Pierantoni, Chiara; Loupakis, Fotios; Zaniboni, Alberto; Negri, Francesca; Quadri, Antonello; Zorzi, Fausto

    2009-01-01

    K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Forty-four patients were available for analysis. We observed a partial remission in 9 (60%) and 2 (9%) cases with a FISH EGFR GCN ≥ 2.6 and < 2.6 respectively (p = 0.002) and in 10 (36%) and 1 (6%) cases with a CISH EGFR GCN ≥ 2.12 and < 2.12 respectively (p = 0.03). Median TTP was 7.7 and 6.4 months in patients showing increased FISH and CISH EGFR GCN whereas it was 2.9 and 3.1 months in those with low FISH and CISH EGFR GCN (p = 0.04 and 0.02 respectively). FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab

  14. Status of neutron complex of INR RAS

    International Nuclear Information System (INIS)

    Grachev, M.I.; Koptelov, E.A.; Kravchuk, L.V.; Matveev, V.A.; Perekrestenko, A.D.; Sidorkin, S.F.; Stavissky, Y.Y.

    2001-01-01

    The neutron complex of INR RAS consists of two sources of neutrons, beam stop, lead slowing down spectrometer and solid state spectrometers. The description of objects and their condition, the program of planned researches, co-operation with other institutes of the Moscow Region, progress reached for last two years are introduced in the article. (author)

  15. Is het flevolanderschaap een zeldzaam ras?

    NARCIS (Netherlands)

    Oldenbroek, J.K.

    2012-01-01

    In 2011 heeft een internationale werkgroep bepaald dat een ras inheems is als het 40 jaar en 6 generaties in een land aanwezig is. De vraag is dan hoe je om moet gaan met recent gevormde rassen die bedreigd worden in hun voortbestaan. Die vraag wordt in dit artikel, met als voorbeeld het

  16. The landscape of somatic mutations in Infant MLL rearranged acute lymphoblastic leukemias

    Science.gov (United States)

    Andersson, Anna K; Ma, Jing; Wang, Jianmin; Chen, Xiang; Gedman, Amanda Larson; Dang, Jinjun; Nakitandwe, Joy; Holmfeldt, Linda; Parker, Matthew; Easton, John; Huether, Robert; Kriwacki, Richard; Rusch, Michael; Wu, Gang; Li, Yongjin; Mulder, Heather; Raimondi, Susana; Pounds, Stanley; Kang, Guolian; Shi, Lei; Becksfort, Jared; Gupta, Pankaj; Payne-Turner, Debbie; Vadodaria, Bhavin; Boggs, Kristy; Yergeau, Donald; Manne, Jayanthi; Song, Guangchun; Edmonson, Michael; Nagahawatte, Panduka; Wei, Lei; Cheng, Cheng; Pei, Deqing; Sutton, Rosemary; Venn, Nicola C; Chetcuti, Albert; Rush, Amanda; Catchpoole, Daniel; Heldrup, Jesper; Fioretos, Thoas; Lu, Charles; Ding, Li; Pui, Ching-Hon; Shurtleff, Sheila; Mullighan, Charles G; Mardis, Elaine R; Wilson, Richard K; Gruber, Tanja A; Zhang, Jinghui; Downing, James R

    2015-01-01

    Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R) and 20 older children (MLL-R cases) with leukemia. Our data demonstrated infant MLL-R ALL to have one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite the paucity of mutations, activating mutations in kinase/PI3K/RAS signaling pathways were detected in 47%. Surprisingly, however, these mutations were often sub-clonal and frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (a mean of 6.5/case versus 1.3/case, P=7.15×10−5) and contained frequent mutations (45%) in epigenetic regulators, a category of genes that with the exception of MLL was rarely mutated in infant MLL-R ALL. PMID:25730765

  17. The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias

    DEFF Research Database (Denmark)

    Andersson, Anna K; Ma, Jing; Wang, Jianmin

    2015-01-01

    Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children...... (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase......-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10...

  18. Effect of Vandetanib on Lung Tumorigenesis in Transgenic Mice Carrying an Activating Egfr Gene Mutation.

    Science.gov (United States)

    Osawa, Masahiro; Ohashi, Kadoaki; Kubo, Toshio; Ichihara, Eiki; Takata, Saburo; Takigawa, Nagio; Takata, Minoru; Tanimoto, Mitsune; Kiura, Katsuyuki

    2016-08-01

    Vandetanib (ZactimaTM) is a novel, orally available inhibitor of both vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) tyrosine kinase. In the present study, a line of transgenic mice with a mouse Egfr gene mutation (delE748-A752) corresponding to a human EGFR mutation (delE746-A750) was established. The transgenic mice developed atypical adenomatous hyperplasia to adenocarcinoma of the lung at around 5 weeks of age and died of lung tumors at approximately 17 weeks of age. In the mice treated with vandetanib (6mg/kg/day), these lung tumors disappeared and the phosphorylations of EGFR and VEGFR-2 were reduced in lung tissues to levels comparable to those of non-transgenic control mice. The median overall survival time of the transgenic mice was 28 weeks in the vandetanib-treated group and 17 weeks in the vehicle-treated group. Vandetanib significantly prolonged the survival of the transgenic mice (log-rank test, p< 0.01); resistance to vandetanib occurred at 20 weeks of age and the animals died from their lung tumors at about 28 weeks of age. These data suggest that vandetanib could suppress the progression of tumors harboring an activating EGFR mutation.

  19. Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway

    OpenAIRE

    Wu, C-H; Chen, Y-F; Wang, J-Y; Hsieh, M-C; Yeh, C-S; Lian, S-T; Shin, S-J; Lin, S-R

    2002-01-01

    The result of our previous study has shown that the K-ras mutant (pK568MRSV) transfected human adrenocortical cells can significantly increase cortisol production and independently cause cell transformation. The aim of this study is to investigate the effect of the active K-ras oncogene on the cortisol production in normal human adrenocortical cells. First we used isopropyl thiogalactoside to induce the inducible mutant K-ras expression plasmid, pK568MRSV, in the stable transfected human adre...

  20. Second RAS Symposium Brings Together World’s Leading RAS Scientists | Poster

    Science.gov (United States)

    From December 6–8, the Advanced Technology Research Facility of the Frederick National Laboratory for Cancer Research was abuzz with conversation and collaboration as nearly 450 scientists, academics, and industry partners gathered for the Second RAS Initiative Symposium. Attendees hailed from 14 nations, dozens of institutions, and diverse scientific backgrounds, but they shared a common purpose: to present and discuss their research on RAS genes and cancer.

  1. Activating mutations in ERBB2 and their impact on diagnostics and treatment

    Directory of Open Access Journals (Sweden)

    Grit Sophie Herter-Sprie

    2013-04-01

    Full Text Available Despite the ongoing ‘war on cancer’, cancer remains one of the major causes of human morbidity and mortality. A new paradigm of targeted therapies holds the most promise for the future, making identification of tumor-specific therapeutic targets of prime importance. ERBB2, best known for its role in breast cancer tumorigenesis, can be targeted by two types of pharmacological manipulation: antibody therapy against the extracellular receptor domain and small molecule compounds against the intracellular tyrosine kinase domain. Aberrant activation of ERBB2 by gene amplification has been shown to participate in the pathophysiology of breast, ovarian, gastric, colorectal, lung, brain and head and neck tumors. However, the advent of next-generation sequencing technologies has enabled efficient identification of activating molecular alterations of ERBB2. In this review, we will focus on the functional role of these somatic mutations that cause ERBB2 receptor activation. We will additionally discuss the current preclinical and clinical therapeutic strategies for targeting mutationally activated ERBB2.

  2. Potential late-onset Alzheimer's disease-associated mutations in the ADAM10 gene attenuate {alpha}-secretase activity.

    Science.gov (United States)

    Kim, Minji; Suh, Jaehong; Romano, Donna; Truong, Mimy H; Mullin, Kristina; Hooli, Basavaraj; Norton, David; Tesco, Giuseppina; Elliott, Kathy; Wagner, Steven L; Moir, Robert D; Becker, K David; Tanzi, Rudolph E

    2009-10-15

    ADAM10, a member of a disintegrin and metalloprotease family, is an alpha-secretase capable of anti-amyloidogenic proteolysis of the amyloid precursor protein. Here, we present evidence for genetic association of ADAM10 with Alzheimer's disease (AD) as well as two rare potentially disease-associated non-synonymous mutations, Q170H and R181G, in the ADAM10 prodomain. These mutations were found in 11 of 16 affected individuals (average onset age 69.5 years) from seven late-onset AD families. Each mutation was also found in one unaffected subject implying incomplete penetrance. Functionally, both mutations significantly attenuated alpha-secretase activity of ADAM10 (>70% decrease), and elevated Abeta levels (1.5-3.5-fold) in cell-based studies. In summary, we provide the first evidence of ADAM10 as a candidate AD susceptibility gene, and report two potentially pathogenic mutations with incomplete penetrance for late-onset familial AD.

  3. The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.

    Science.gov (United States)

    Ando, Maya; Fiesel, Fabienne C; Hudec, Roman; Caulfield, Thomas R; Ogaki, Kotaro; Górka-Skoczylas, Paulina; Koziorowski, Dariusz; Friedman, Andrzej; Chen, Li; Dawson, Valina L; Dawson, Ted M; Bu, Guojun; Ross, Owen A; Wszolek, Zbigniew K; Springer, Wolfdieter

    2017-04-24

    Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson's disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients' fibroblasts and in cell-based, biochemical assays. Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon

  4. Optimization ofRAS/BRAFMutational Analysis Confirms Improvement in Patient Selection for Clinical Benefit to Anti-EGFR Treatment in Metastatic Colorectal Cancer.

    Science.gov (United States)

    Santos, Cristina; Azuara, Daniel; Garcia-Carbonero, Rocio; Alfonso, Pilar Garcia; Carrato, Alfredo; Elez, Mª Elena; Gomez, Auxiliadora; Losa, Ferran; Montagut, Clara; Massuti, Bartomeu; Navarro, Valenti; Varela, Mar; Lopez-Doriga, Adriana; Moreno, Victor; Valladares, Manuel; Manzano, Jose Luis; Vieitez, Jose Maria; Aranda, Enrique; Sanjuan, Xavier; Tabernero, Josep; Capella, Gabriel; Salazar, Ramon

    2017-09-01

    In metastatic colorectal cancer (mCRC), recent studies have shown the importance to accurately quantify low-abundance mutations of the RAS pathway because anti-EGFR therapy may depend on certain mutation thresholds. We aimed to evaluate the added predictive value of an extended RAS panel testing using two commercial assays and a highly sensitive and quantitative digital PCR (dPCR). Tumor samples from 583 mCRC patients treated with anti-EGFR- ( n = 255) or bevacizumab- ( n = 328) based therapies from several clinical trials and retrospective series from the TTD/RTICC Spanish network were analyzed by cobas, therascreen , and dPCR. We evaluated concordance between techniques using the Cohen kappa index. Response rate, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutant allele fraction (MAF). Concordance between techniques was high when analyzing RAS and BRAF (Cohen kappa index around 0.75). We observed an inverse correlation between MAF and response in the anti-EGFR cohort ( P < 0.001). Likelihood ratio analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value. PFS and OS were significantly longer in RAS / BRAF wild-type patients, independently of the technique. However, the predictability of both PFS and OS were higher when we considered a threshold of 1% in the RAS scenario (HR = 1.53; CI 95%, 1.12-2.09 for PFS, and HR = 1.9; CI 95%, 1.33-2.72 for OS). Although the rate of mutations observed among techniques is different, RAS and BRAF mutational analysis improved prediction of response to anti-EGFR therapy. Additionally, dPCR with a threshold of 1% outperformed the other platforms. Mol Cancer Ther; 16(9); 1999-2007. ©2017 AACR . ©2017 American Association for Cancer Research.

  5. Ras-dva, a member of novel family of small GTPases, is required for the anterior ectoderm patterning in the Xenopus laevis embryo.

    Science.gov (United States)

    Tereshina, Maria B; Zaraisky, Andrey G; Novoselov, Vladimir V

    2006-02-01

    Ras-like small GTPases are involved in the regulation of many processes essential for the specification of the vertebrate body plan. Recently, we identified the gene of novel small GTPase Ras-dva, which is specifically expressed at the anterior margin of the neural plate of the Xenopus laevis embryo. Now, we demonstrate that Ras-dva and its homologs in other species constitute a novel protein family, distinct from the previously known families of small GTPases. We show that the expression of Ras-dva begins during gastrulation throughout the anterior ectoderm and is activated by the homeodomain transcription factor Otx2; however, later on, Ras-dva expression is inhibited in the anterior neural plate by another homeodomain factor Xanf1. Downregulation of Ras-dva functioning by the dominant-negative mutant or by the antisense morpholino oligonucleotides results in severe malformations of the forebrain and derivatives of the cranial placodes. Importantly, although the observed abnormalities can be rescued by co-injection of the Ras-dva mRNA, they cannot be rescued by the mRNA of the closest Ras-dva homolog from another family of small GTPases, Ras. This fact indicates functional specificity of the Ras-dva signaling pathway. At the molecular level, downregulation of Ras-dva inhibits the expression of several regulators of the anterior neural plate and folds patterning, such as Otx2, BF-1 (also known as Foxg1), Xag2, Pax6, Slug and Sox9, and interferes with FGF8 signaling within the anterior ectoderm. By contrast, expression of the epidermal regulator BMP4 and its target genes, Vent1, Vent2b and Msx1, is upregulated. Together, the data obtained indicate that Ras-dva is an essential component of the signaling network that patterns the early anterior neural plate and the adjacent ectoderm in the Xenopus laevis embryos.

  6. 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.

  7. Utilizing ras signaling pathway to direct selective replication of herpes simplex virus-1.

    Science.gov (United States)

    Pan, Weihong; Bodempudi, Vidya; Esfandyari, Tuba; Farassati, Faris

    2009-08-04

    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.

  8. Characterization of two MODY2 mutations with different susceptibility to activation

    Energy Technology Data Exchange (ETDEWEB)

    Langer, Sara; Platz, Christian; Waterstradt, Rica; Baltrusch, Simone, E-mail: simone.baltrusch@med.uni-rostock.de

    2015-09-04

    Glucokinase plays a key role in glucose sensing in pancreatic beta cells and in liver metabolism. Heterozygous inactivating glucokinase mutations cause the autosomal dominantly inherited MODY2 subtype of maturity-onset diabetes of the young. The goal of this study was to elucidate the pathogenicity of the recently described glucokinase mutants L304P and L315H, located in an alpha-helix and connecting region, respectively, at the outer region of the large domain of glucokinase. Both mutants showed wild-type-like cytosolic localization, but faster protein degradation in insulin-secreting MIN6 cells. However, strongly reduced nuclear/cytoplasmic localization of the mutants was observed in primary hepatocytes suggesting reduced interaction with the liver specific glucokinase regulatory protein. Both mutants displayed a significantly lowered glucokinase activity compared to the wild-type protein. Even though the L315H protein showed the lowest enzymatic activity, this mutant was very sensitive to allosteric activation. The endogenous activator fructose-2,6-bisphosphatase evoked an increase in glucokinase activity for both mutants, but much stronger for L315H compared to L304P. The synthetic activator RO281675 was ineffective against the L304P mutant. Expression of the mutant proteins evoked loss of glucose-induced insulin secretion in MIN6 cells. Administration of RO281675 increased insulin secretion, however, only for the L315H mutant. Thus, a glucokinase activator drug therapy may help MODY2 patients not in general, but seems to be a useful strategy for carriers of the L315H glucokinase mutation. - Highlights: • The GK mutants L304P and L315H display a highly reduced enzymatic activity. • In hepatocytes both mutations lower the nuclear/cytoplasmic localization ratio of GK. • Both mutants inhibit stimulus-secretion coupling in insulin-producing cells. • Activation by fructose-2,6-bisphosphatase and by RO281675 is stronger for L315H. • RO281675 stimulates

  9. 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.

  10. MEK and PI3K-AKT inhibitors synergistically block activated IL7 receptor signaling in T-cell acute lymphoblastic leukemia

    NARCIS (Netherlands)

    K. Canté-Barrett (Kirsten); J.A.P. Spijkers-Hagelstein (J A P); J.G.C.A.M. Buijs-Gladdines (Jessica); J.C.M. Uitdehaag (Joost C. M.); W.K. Smits; J. van der Zwet; R.C. Buijsman; G.J.R. Zaman; R. Pieters (Rob); J.P.P. Meijerink (Jules)

    2016-01-01

    textabstractWe identified mutations in the IL7Ra gene or in genes encoding the downstream signaling molecules JAK1, JAK3, STAT5B, N-RAS, K-RAS, NF1, AKT and PTEN in 49% of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Strikingly, these mutations (except RAS/NF1) were mutually

  11. Mutational analysis of primary and metastatic colorectal cancer samples underlying the resistance to cetuximab-based therapy

    Directory of Open Access Journals (Sweden)

    Nemecek R

    2016-07-01

    . Finally, new occurrences of activating KRAS mutations were identified in a group of patients initially determined as wild-type RAS by routinely used qPCR-based RAS mutational tests. All newly detected activating KRAS mutations most likely led to cetuximab treatment failure. Conclusion: The results of the present study suggest a need of careful consideration of previously published results of anti-EGFR-targeted therapy with regard to potentially inaccurate diagnostic tools used in the past. Based on our findings, we recommend more extensive use of next-generation sequencing testing in daily clinical practice, as it brings a significant added value in terms of validity of the diagnostic procedure. Keywords: colorectal cancer, cetuximab, resistance to anti-EGFR therapy, next-generation sequencing, FBXW7, KRAS

  12. Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

    DEFF Research Database (Denmark)

    Klein, H H; Müller, R; Vestergaard, H

    1999-01-01

    We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ...... activation of the receptor kinase in skeletal muscle was reduced about 70%. Selection of only those receptors that bound to anti-phosphotyrosine antibody showed that these receptors had normal kinase activity and that the reduction in overall kinase activity was due to the inability of about 70......% of the receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother...

  13. Hotspot mutations in KIT receptor differentially modulate its allosterically coupled conformational dynamics: impact on activation and drug sensitivity.

    Directory of Open Access Journals (Sweden)

    Isaure Chauvot de Beauchêne

    2014-07-01

    Full Text Available Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D localized in crucial regulatory segments, the juxtamembrane region (JMR and the activation (A- loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts.

  14. Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth.

    Science.gov (United States)

    Gaglio, Daniela; Metallo, Christian M; Gameiro, Paulo A; Hiller, Karsten; Danna, Lara Sala; Balestrieri, Chiara; Alberghina, Lilia; Stephanopoulos, Gregory; Chiaradonna, Ferdinando

    2011-08-16

    Oncogenes such as K-ras mediate cellular and metabolic transformation during tumorigenesis. To analyze K-Ras-dependent metabolic alterations, we employed ¹³C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD) of ¹⁵N-labeled glutamine, and transcriptomic profiling in mouse fibroblast and human carcinoma cell lines. Stable isotope-labeled glucose and glutamine tracers and computational determination of intracellular fluxes indicated that cells expressing oncogenic K-Ras exhibited enhanced glycolytic activity, decreased oxidative flux through the tricarboxylic acid (TCA) cycle, and increased utilization of glutamine for anabolic synthesis. Surprisingly, a non-canonical labeling of TCA cycle-associated metabolites was detected in both transformed cell lines. Transcriptional profiling detected elevated expression of several genes associated with glycolysis, glutamine metabolism, and nucleotide biosynthesis upon transformation with oncogenic K-Ras. Chemical perturbation of enzymes along these pathways further supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle. These results provide evidence for a role of oncogenic K-Ras in the metabolic reprogramming of cancer cells.

  15. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Monika B Dolinska

    Full Text Available Tyrosinase (TYR catalyzes the rate-limiting, first step in melanin production and its gene (TYR is mutated in many cases of oculocutaneous albinism (OCA1, an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.The intra-melanosomal domain of human tyrosinase (residues 19-469 and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  16. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    Science.gov (United States)

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  17. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Science.gov (United States)

    Dolinska, Monika B; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

    2014-01-01

    Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  18. Consistent absence of BRAF mutations in salivary gland carcinomas

    Directory of Open Access Journals (Sweden)

    Nooshin Mohtasham

    2017-06-01

    Full Text Available Introduction: Malignant salivary gland tumors are rare entities. Despite advances in surgery, radiation therapy and chemotherapy, the rate of the mortality and five-year survival has not been improved markedly over the last few decades. The activation of EGFR- RAS-RAF signaling pathway contributes to the initiation and progression of many human cancers, promising a key pathway for therapeutic molecules. Thus, the objective of this study was to evaluate BRAF mutations in salivary gland carcinomas. Methods: We designed PCR- RFLP (Polymerase Chain Reaction -Restriction Fragment Length Polymorphism and screened 50 salivary gland carcinomas (SGCs including mucoepidermoid carcinoma (MEC, adenoid cystic carcinoma (AdCC and polymorphous low grade adenocarcinoma (PLGA for the BRAF V600E mutation. Results: PCR-RFLP analyses demonstrated no mutation in BRAF exon 15 for SGC samples at position V600, which is the most commonly mutated site for BRAF in human cancer. Conclusions: According to our results SGCs didn’t acquire BRAF mutations that result in a constitutive activation of the signaling cascade downstream of EGFR, hence SGCs can be a good candidate for anti EGFR therapies.

  19. Dynamics of Ras Complexes Observed in Living Cells

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    Xiangyong Li

    2012-07-01

    Full Text Available K-Ras works as a switch in many important intracellular signaling pathways and plays important roles in cell growth, proliferation, differentiation and carcinogenesis. For signal transduction from K-Ras to Raf1, the best-characterized effector of K-Ras, the general view is that Ras recruits Raf1 from the cytoplasm to the cell membrane. To elucidate this process, we constructed a series of fusion proteins (including Raf1 and K-Ras fused with either fluorescent proteins or fluorescent protein fragments to compare subcellular localizations of these proteins. Bimolecular fluorescence complementation (BiFC and a co-transfection system were used. In the BiFC system, the K-Ras/Raf1 complexes were mainly located in the cell membrane, while the Raf1 control was uniformly distributed in the cytoplasm. However, the complexes of Raf1 and K-RasC185S, a K-Ras mutant which loses membrane-localization, were also able to accumulate in the cell membrane. In contrast, an apparent cytosolic distribution pattern was observed in cells co-transfected with mcerulean-Raf1 and EGFP-K-RasC185S, suggesting that the membrane localization of K-Ras/Raf1 complexes is not entirely dependent on K-Ras, and that other factors, such as the irreversible conformation formed between K-Ras and Raf1 may play a role. This study sheds light on the interaction between K-Ras and Raf1 and provides a practical method to elucidate the mechanism underlying K-Ras and Raf1 binding to the cell membrane.

  20. Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

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    Miriam Kaltenbach

    2016-10-01

    Full Text Available The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1 from phosphotriesterase to arylesterase, and (2 from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak

  1. Transcriptionally Silenced Transgenes in Maize Are Activated by Three Mutations Defective in Paramutation

    Science.gov (United States)

    McGinnis, Karen M.; Springer, Catherine; Lin, Yan; Carey, Charles C.; Chandler, Vicki

    2006-01-01

    Plants with mutations in one of three maize genes, mop1, rmr1, and rmr2, are defective in paramutation, an allele-specific interaction that leads to meiotically heritable chromatin changes. Experiments reported here demonstrate that these genes are required to maintain the transcriptional silencing of two different transgenes, suggesting that paramutation and transcriptional silencing of transgenes share mechanisms. We hypothesize that the transgenes are silenced through an RNA-directed chromatin mechanism, because mop1 encodes an RNA-dependent RNA polymerase. In all the mutants, DNA methylation was reduced in the active transgenes relative to the silent transgenes at all of the CNG sites monitored within the transgene promoter. However, asymmetrical methylation persisted at one site within the reactivated transgene in the rmr1-1 mutant. With that one mutant, rmr1-1, the transgene was efficiently resilenced upon outcrossing to reintroduce the wild-type protein. In contrast, with the mop1-1 and rmr2-1 mutants, the transgene remained active in a subset of progeny even after the wild-type proteins were reintroduced by outcrossing. Interestingly, this immunity to silencing increased as the generations progressed, consistent with a heritable chromatin state being formed at the transgene in plants carrying the mop1-1 and rmr2-1 mutations that becomes more resistant to silencing in subsequent generations. PMID:16702420

  2. Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria.

    Science.gov (United States)

    Zhang, Can; Wang, Yuanchao; Sun, Huzhi; Ren, Huiying

    2015-10-01

    The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7 (Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a pET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated (L99A, M102E) to construct pET28a-Bp7Δe, with pET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.

  3. Germline activating MTOR mutation arising through gonadal mosaicism in two brothers with megalencephaly and neurodevelopmental abnormalities.

    Science.gov (United States)

    Mroske, Cameron; Rasmussen, Kristen; Shinde, Deepali N; Huether, Robert; Powis, Zoe; Lu, Hsiao-Mei; Baxter, Ruth M; McPherson, Elizabeth; Tang, Sha

    2015-11-05

    In humans, Mammalian Target of Rapamycin (MTOR) encodes a 300 kDa serine/ threonine protein kinase that is ubiquitously expressed, particularly at high levels in brain. MTOR functions as an integrator of multiple cellular processes, and in so doing either directly or indirectly regulates the phosphorylation of at least 800 proteins. While somatic MTOR mutations have been recognized in tumors for many years, and more recently in hemimegalencephaly, germline MTOR mutations have rarely been described. We report the successful application of family-trio Diagnostic Exome Sequencing (DES) to identify the underlying molecular etiology in two brothers with multiple neurological and developmental lesions, and for whom previous testing was non-diagnostic. The affected brothers, who were 6 and 23 years of age at the time of DES, presented symptoms including but not limited to mild Autism Spectrum Disorder (ASD), megalencephaly, gross motor skill delay, cryptorchidism and bilateral iris coloboma. Importantly, we determined that each affected brother harbored the MTOR missense alteration p.E1799K (c.5395G>A). This exact variant has been previously identified in multiple independent human somatic cancer samples and has been shown to result in increased MTOR activation. Further, recent independent reports describe two unrelated families in whom p.E1799K co-segregated with megalencephaly and intellectual disability (ID); in both cases, p.E1799K was shown to have originated due to germline mosaicism. In the case of the family reported herein, the absence of p.E1799K in genomic DNA extracted from the blood of either parent suggests that this alteration most likely arose due to gonadal mosaicism. Further, the p.E1799K variant exerts its effect by a gain-of-function (GOF), autosomal dominant mechanism. Herein, we describe the use of DES to uncover an activating MTOR missense alteration of gonadal mosaic origin that is likely to be the causative mutation in two brothers who present

  4. Physiochemical and Thermodynamic Characterization of Highly Active Mutated Aspergillus niger β-glucosidase for Lignocellulose Hydrolysis.

    Science.gov (United States)

    Javed, Muhammad Rizwan; Rashid, Muhammad Hamid; Riaz, Muhammad; Nadeem, Habibullah; Qasim, Muhammad; Ashiq, Nourin

    2018-01-30

    The extracellular β-glucosidases (BGL) from Aspergillus niger NIBGE (P) and its 2-Deoxy-D-glucose resistant mutant derivative NIBGE-06 (M-6), produced under submerged growth conditions were purified to homogeneity level by ammonium sulfate precipitation and FPLC system. The BGLs from both strains were dimeric in nature, with subunit and native molecular masses of 130 kDa and 252 kDa, respectively. The comparative analysis of nucleotides of bgl genes revealed 8 point mutations within the bgl gene of mutant strain as compared to parental strain. Out of these 8 mutations 2 were true substitutions, 2 were semi-conserved and 3 were conserved substitutions, while one was silent mutation. The enzyme belongs to family 3 of glycoside hydrolases with retaining catalytic mechanism. Significant improvement was observed in the kinetic properties of the mutant BGL relative to the wild type enzyme. Arrhenius plot for energy of activation (Ea) showed a biphasic trend and ES-complex formation required Ea of 50 and 42 kJ mol-1 by BGL from parent and mutant, respectively. The pKa1 and pKa2 of the active site residues were 3.4 & 5.5 and 3.2 & 5.6, respectively. The heat of ionization for the acidic limb (ΔHI-AL) and the basic limb (ΔHI-BL) of BGL from both strains were equal to 56 & 41 and 71 & 45 kJ mol-1, respectively. Kinetic constants of cellobiose hydrolysis for BGL from both strains were determined as follows: kcat = 2,589 and 4,135 s-1, Km = 0.24 and 0.26 mM cellobiose, kcat/Km = 10,872 and 15,712 s-1 mM-1 cellobiose, respectively. Thermodynamic parameters for cellobiose hydrolysis also suggested that mutant BGL is more efficient compared to the parent enzyme. Comparative analysis of Ea(d), ΔH* and ΔG* for irreversible thermostability indicated that the thermostabilization of mutant enzyme was due to higher functional energy (free energy), which enabled the enzyme to resist against unfolding of its transition state. Hence, directed mutation resulted in the engineering of

  5. Impact of antibiotic treatments on the expression of the R plasmid tra genes and on the host innate immune activity during pRAS1 bearing Aeromonas hydrophila infection in zebrafish (Danio rerio

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    Cantas Leon

    2012-03-01

    Full Text Available Abstract Background The transfer of R plasmids between bacteria has been well studied under laboratory conditions and the transfer frequency has been found to vary between plasmids and under various physical conditions. For the first time, we here study the expression of the selected plasmid mobility genes traD, virB11 and virD4 in the 45 kb IncU plasmid, pRAS1, conferring resistance to tetracycline, trimethoprim and sulphonamide, using an in vivo zebrafish infection- treatment model. Results Three days after oral infection of adult zebrafish with Aeromonas hydrophila harboring pRAS1, elevated expression of pro-inflammatory cytokine (TNF α, IL-1β and IL-8 and complement C3 genes in the intestine coincided with disease symptoms. Tetracycline, trimethoprim and an ineffective concentration of flumequine given 48 h prior to sampling, strongly increased expression of plasmid mobility genes, whereas an effective dosage of flumequine resulted in lower levels of mRNA copies of these genes relative to placebo treatment. Following effective treatment with flumequine, and ineffective treatments with a low concentration of flumequine, with trimethoprim or with sulphonamide, the intestinal expression of immune genes was strongly induced compared to placebo treated control fish. Conclusions Treatment of zebrafish infected with an antibiotic resistant (TcR, TmR, SuR A. hydrophila with ineffective concentrations of flumequine or the ineffective antimicrobials tetracycline and trimethoprim strongly induced expression of genes mediating conjugative transfer of the R-plasmid pRAS1. Simultaneously, there was a strong induction of selected inflammatory and immune response genes, which was again evident in fish subjected to ineffective treatment protocols. Our findings point to the essential role of therapeutic practices in escalation or control of antibiotic resistance transfer, and suggest that antibiotic substances, even in sub-inhibitory concentrations, may

  6. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    Science.gov (United States)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  7. Ras signalling regulates differentiation and UCP1 expression in models of brown adipogenesis

    DEFF Research Database (Denmark)

    Murholm, Maria; Dixen, Karen; Hansen, Jacob B

    2010-01-01

    BACKGROUND: The Ras/Raf/MEK/ERK pathway has been recognised as an important signalling module in adipogenesis and adipocyte function, but whether it promotes or inhibits the formation of fat cells has not been reconciled. METHODS: Here we investigate the significance of Ras signalling intensity...... of differentiation was inefficiently rescued by forced expression of the adipogenic transcription factors C/EBPalpha and PPARgamma. However, the defective differentiation was alleviated by MEK inhibitors, suggesting that the obstruction of differentiation was dependent on activation of ERK. A dominant interfering H...

  8. The association of clinicopathological features and survival in colorectal cancer patients with kras mutation status.

    Science.gov (United States)

    Akman, Tulay; Oztop, Ilhan; Baskin, Yasemin; Unek, Ilkay Tugba; Demir, Necla; Ellidokuz, Hulya; Yilmaz, Ahmet Ugur

    2016-01-01

    KRAS mutations have a significant role in the consecutive activation of RAS.RAF.MEK.ERK pathway in colorectal cancer.Approximately 30.35% of sporadic colorectal cancers have KRAS mutation. While the predictive role of KRAS is commonly accepted at the present time, its prognostic role and association with different clinical and histopathological properties are currently unclear and inconsistent. The intent of this study, has been to evaluate the associations between KRAS gene mutations and clinicopathological features and survival times in Turkish colorectal cancer patients. In this study, the file records of 115 metastatic colorectal cancer patients who applied to the Department of Medical Oncology between 2000 and 2011 were monitored; data on clinicopathological features and survival times were collected. DNA.sequencing method with PCR amplification from archival paraffin blocks were used for KRAS mutation status analysis. The associations between KRAS mutation status and clinicopathological features and survival times were compared statistically. While a significant association hadbeen determined between KRAS mutation status and tumor localization, there was no determined significant association with other clinicopathological properties. Similarly, there was no association between KRAS mutation status and survival parameters. As a result, the effect of KRAS mutation status on clinicopathological features, survival time and prognosis is unclear.

  9. Mutation in the Plasmodium falciparum CRT Protein Determines the Stereospecific Activity of Antimalarial Cinchona Alkaloids

    Science.gov (United States)

    Griffin, Carol E.; Hoke, Jonathan M.; Samarakoon, Upeka; Duan, Junhui; Mu, Jianbing; Ferdig, Michael T.; Warhurst, David C.

    2012-01-01

    The Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (−)-quinine and (+)-quinidine. The potency of (+)-isomers is greater than the (−)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility of eight clonal lines of P. falciparum derived from the 106/1 strain, each containing a unique pfcrt allele, to four Cinchona stereoisomer pairs: quinine and quinidine; cinchonidine and cinchonine; hydroquinine and hydroquinidine; 9-epiquinine and 9-epiquinidine. Stereospecific potency of the Cinchona alkaloids was associated with changes in charge and hydrophobicity of mutable PfCRT amino acids. In isogenic chloroquine-resistant lines, the IC50 ratio of (−)/(+) CA pairs correlated with side chain hydrophobicity of the position 76 residue. Second-site PfCRT mutations negated the K76I stereospecific effects: charge-change mutations C72R or Q352K/R restored potency patterns similar to the parent K76 line, while V369F increased susceptibility to the alkaloids and nullified stereospecific differences between alkaloid pairs. Interactions between key residues of the PfCRT channel/transporter with (−) and (+) alkaloids are stereospecifically determined, suggesting that PfCRT binding plays an important role in the antimalarial activity of quinine and other Cinchona alkaloids. PMID:22869567

  10. Plant mutation reports. Vol. 1, No. 3, December 2007

    International Nuclear Information System (INIS)

    2007-12-01

    This issue of the Plant Mutation Reports emphasises the recently concluded IAEA-RCA project on Mutant Multi-location Trial and Mutational Enhancement of genetic Diversity (RAS/5/040) which has developed 33 mutant varieties and dozens of promising mutant breeding lines with high yield potantial and/or improved agronomic characters, resistance and end user quality traits

  11. High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation

    International Nuclear Information System (INIS)

    Yamakawa, Nobuhiro; Takahashi, Akihisa; Mori, Eiichiro; Imai, Yuichiro; Ohnishi, Ken; Kirita, Tadaaki; Ohnishi, Takeo; Furusawa, Yoshiya

    2008-01-01

    Although mutations in the p53 gene can lead to resistance to radiotherapy, chemotherapy and thermotherapy, high linear energy transfer (LET) radiation induces apoptosis regardless of p53 gene status in cancer cells. The aim of this study was to clarify the mechanisms involved in high LET radiation-induced apoptosis. Human gingival cancer cells (Ca9-22 cells) containing a mutated p53 (mp53) gene were irradiated with X-rays, C-ion (13-100 KeV/μm), or Fe-ion beams (200 KeV/μm). Cellular sensitivities were determined using colony forming assays. Apoptosis was detected and quantified with Hoechst 33342 staining. The activity of Caspase-3 was analyzed with Western blotting and flow cytometry. Cells irradiated with high LET radiation showed a high sensitivity with a high frequency of apoptosis induction. The relative biological effectiveness (RBE) values for the surviving fraction and apoptosis induction increased in a LET-dependent manner. Both RBE curves reached a peak at 100 KeV/μm, and then decreased at values over 100 KeV/μm. When cells were irradiated with high LET radiation, Caspase-3 was cleaved and activated, leading to poly (ADP-ribose) polymerase (PARP) cleavage. In addition, Caspase-9 inhibitor suppressed Caspase-3 activation and apoptosis induction resulting from high LET radiation to a greater extent than Caspase-8 inhibitor. These results suggest that high LET radiation enhances apoptosis by activation of Caspase-3 through Caspase-9, even in the presence of mp53. (author)

  12. Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1.

    Science.gov (United States)

    Farassati, F; Yang, A D; Lee, P W

    2001-08-01

    The importance of herpes simplex viruses (HSV) as human pathogens and the emerging prospect of using mutant derivatives of HSV-1 as potential anti-cancer therapeutics have necessitated a thorough investigation into the molecular basis of host-cell permissiveness to HSV. Here we show that NIH-3T3 cells transformed with the oncogenes v-erbB, activated sos or activated ras become significantly more permissive to HSV-1. Inhibitors of the Ras signalling pathway, such as farnesyl transferase inhibitor 1 and PD98059, effectively suppressed HSV-1 infection of ras-transformed cells. Enhanced permissiveness of the transformed cells was linked to the inhibition of virus-induced activation (phosphorylation) of the double-stranded RNA-activated protein kinase (PKR), thereby allowing viral transcripts to be translated in these cells. An HSV-1-derived oncolytic mutant, R3616, was also found to infect preferentially both transformed cells and PKR-/- (but not PKR+/+) mouse embryo fibroblasts. These observations suggest that HSV-1 specifically targets cells with an activated Ras signalling pathway, and have important ramifications in the use of engineered HSV in cancer therapy, the development of strategies against HSV infections, and the controversial role of HSV in human cancers.

  13. A strong loss-of-function mutation in RAN1 results in constitution activation of the ethylene response pathway as well as a rosette-lethal phenotype

    Science.gov (United States)

    Keith Woeste; Joseph J. Kieber

    2000-01-01

    A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resuited in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a...

  14. 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......Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane...

  15. Constitutive activation of the thyroid-stimulating hormone receptor (TSHR by mutating Ile691 in the cytoplasmic tail segment.

    Directory of Open Access Journals (Sweden)

    Zheng Liu

    Full Text Available BACKGROUND: Autosomal dominant non-autoimmune hyperthyroidism (ADNAH is a rare genetic disorder of the endocrine system. Molecular genetic studies in ADNAH have revealed heterozygous germline mutations in the TSHR. To data, mutations leading to an increase in the constitutive activation of the TSHR have been described in the transmembrane segments, exoloops and cytoplasmic loop of TSHR. These mutations result in constitutive activation of the G(αs/cAMP or G(αq/11/inositol phosphate (IP pathways, which stimulate thyroid hormone production and thyroid proliferation. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study, we reported a new TSHR mutation located in the C-terminal domain of TSHR, which results in a substitution of the conserved Ile(691 for Phe. In this study, to address the question of whether the I691F mutated receptor could be responsible for G(αs/cAMP or G(αq/11/IP constitutive activity, wild-type and TSHR mutants were expressed in COS-7 cells to determine cAMP constitutive activity and IP formation. Compared to the cell surface with expression of the A623V mutated receptor as positive control, the I691F mutated receptor showed a slight increase of cAMP accumulation. Furthermore, I691F resulted in constitutive activation of the G(αq/11/IP signaling pathway. CONCLUSIONS/SIGNIFICANCE: Our results indicate that Ile(691 not only contributes to keeping TSHR inactive in the G(αs/cAMP pathways but also in the G(αq/11/IP cascade.

  16. Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline

    DEFF Research Database (Denmark)

    Giannoulatou, Eleni; McVean, Gilean; Taylor, Indira B

    2013-01-01

    The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause...

  17. Detection of an activating c-kit mutation by real-time PCR in patients with anaphylaxis.

    Science.gov (United States)

    Lawley, Wendy; Hird, Heather; Mallinder, Philip; McKenna, Sue; Hargadon, Beverley; Murray, Alistair; Bradding, Peter

    2005-05-02

    A well-characterised gain-of-function point mutation within exon 17 of the c-kit proto-oncogene known as Asp816Val is present in patients with mastocytosis. Activation of mast cells through this receptor primes them for IgE-dependent activation, and patients with mastocytosis are at increased risk of anaphylaxis. We hypothesised that the Asp816Val mutation is associated with a history of anaphylaxis in the general population. A mismatch amplification real-time PCR assay was developed and validated to test for the Asp816Val mutation. Subjects were recruited to four subject groups: normal non-atopics, atopics without anaphylaxis, food-induced anaphylactics and non-food anaphylactics. Blood samples collected from forty subjects were tested for the presence of Asp816Val. Thirteen subjects were found to carry the mutation; normals (2/9), atopics (2/10), food anaphylactics (5/11) and non-food anaphylactics (4/10). Statistical analysis of the data determined that there was no significant difference between the numbers of subjects found to carry the Asp816Val mutation in each of the groups although a trend towards an increased occurrence in anaphylactics was observed. In summary, the hypothesis that the presence of the Asp816Val mutation is linked to the occurrence of anaphylaxis was not supported, but interestingly, we have shown for the first time Asp816Val may occur more frequently than previously reported within the general population.

  18. MTHFR variants reduce the risk of G:C->A:T transition mutations within the p53 tumor suppressor gene in colon tumors.

    Science.gov (United States)

    Ulrich, C M; Curtin, K; Samowitz, W; Bigler, J; Potter, J D; Caan, B; Slattery, M L

    2005-10-01

    5,10-Methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in folate-mediated 1-carbon metabolism. Reduced MTHFR activity has been associated with genomic DNA hypomethylation. Methylated cytosines at CpG sites are easily mutated and have been implicated in G:C-->A:T transitions in the p53 tumor suppressor gene. We investigated 2 polymorphisms in the MTHFR gene (C677T and A1298C) and their associations with colon tumor characteristics, including acquired mutations in Ki-ras and p53 genes and microsatellite instability (MSI). The study population comprised 1248 colon cancer cases and 1972 controls, who participated in a population-based case-control study and had been analyzed previously for MSI, acquired mutations in Ki-ras, p53, and germline MTHFR polymorphisms. Multivariable-adjusted odds ratios are presented. Overall, MTHFR genotypes were not associated with MSI status or the presence of any p53 or Ki-ras mutation. Individuals with homozygous variant MTHFR genotypes had a significantly reduced risk of G:C-->A:T transition mutations within the p53 gene, yet, as hypothesized, only at CpG-associated sites [677TT vs. 677CC (referent group) OR = 0.4 (95% CI: 0.1-0.8) for CpG-associated sites; OR = 1.5 (0.7-3.6) for non-CpG associated sites]. Genotypes conferring reduced MTHFR activity were associated with a decreased risk of acquired G:C-->A:T mutations within the p53 gene occurring at CpG sites. Consistent with evidence on the phenotypic effect of the MTHFR C677T variant, we hypothesize that this relation may be explained by modestly reduced genomic DNA methylation, resulting in a lower probability of spontaneous deamination of methylated cytosine to thymidine. These results suggest a novel mechanism by which MTHFR polymorphisms can affect the risk of colon cancer.

  19. Ras conformational switching: simulating nucleotide-dependent conformational transitions with accelerated molecular dynamics.

    Directory of Open Access Journals (Sweden)

    Barry J Grant

    2009-03-01

    Full Text Available Ras mediates signaling pathways controlling cell proliferation and development by cycling between GTP- and GDP-bound active and inactive conformational states. Understanding the complete reaction path of this conformational change and its intermediary structures is critical to understanding