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Sample records for cyclin t1 regulates

  1. Limited redundancy in genes regulated by Cyclin T2 and Cyclin T1

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    Yu Wendong

    2011-07-01

    Full Text Available Abstract Background The elongation phase, like other steps of transcription by RNA Polymerase II, is subject to regulation. The positive transcription elongation factor b (P-TEFb complex allows for the transition of mRNA synthesis to the productive elongation phase. P-TEFb contains Cdk9 (Cyclin-dependent kinase 9 as its catalytic subunit and is regulated by its Cyclin partners, Cyclin T1 and Cyclin T2. The HIV-1 Tat transactivator protein enhances viral gene expression by exclusively recruiting the Cdk9-Cyclin T1 P-TEFb complex to a RNA element in nascent viral transcripts called TAR. The expression patterns of Cyclin T1 and Cyclin T2 in primary monocytes and CD4+ T cells suggests that Cyclin T2 may be generally involved in expression of constitutively expressed genes in quiescent cells, while Cyclin T1 may be involved in expression of genes up-regulated during macrophage differentiation, T cell activation, and conditions of increased metabolic activity To investigate this issue, we wished to identify the sets of genes whose levels are regulated by either Cyclin T2 or Cyclin T1. Findings We used shRNA lentiviral vectors to stably deplete either Cyclin T2 or Cyclin T1 in HeLa cells. Total RNA extracted from these cells was subjected to cDNA microarray analysis. We found that 292 genes were down- regulated by depletion of Cyclin T2 and 631 genes were down-regulated by depletion of Cyclin T1 compared to cells transduced with a control lentivirus. Expression of 100 genes was commonly reduced in either knockdown. Additionally, 111 and 287 genes were up-regulated when either Cyclin T2 or Cyclin T1 was depleted, respectively, with 45 genes in common. Conclusions These results suggest that there is limited redundancy in genes regulated by Cyclin T1 or Cyclin T2.

  2. The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1;3

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    Peng Qi; Hong-Xuan Lin; You-Shun Lin; Xian-Jun Song; Jin-Bo Shen; Wei Huang; Jun-Xiang Shan; Mei-Zhen Zhu; Liwen Jiang; Ji-Ping Gao

    2012-01-01

    Increased crop yields are required to support rapid population growth worldwide.Grain weight is a key component of rice yield,but the underlying molecular mechanisms that control it remain elusive.Here,we report the cloning and characterization of a new quantitative trait locus(QTL)for the control of rice grain length,weight and yield.This locus,GL3.1,encodes a protein phosphatase kelch(PPKL)family — Ser/Thr phosphatase.GL3.1 is a member of the large grain WY3 variety,which is associated with weaker dephosphorylation activity than the small grain FAZ1 variety.GL3.1-WY3 influences protein phosphorylation in the spikelet to accelerate cell division,thereby resulting in longer grains and higher yields.Further studies have shown that GL3.1 directly dephosphorylates its substrate,Cyclin-T1;3,which has only been rarely studied in plants.The downregulation of Cyclin-T1;3 in rice resulted in a shorter grain,which indicates a novel function for Cyclin-T in cell cycle regulation.Our findings suggest a new mechanism for the regulation of grain size and yield that is driven through a novel phosphatase-mediated process that affects the phosphorylation of Cyclin-T1;3 during cell cycle progression,and thus provide new insight into the mechanisms underlying crop seed development.We bred a new variety containing the natural GL3.1 allele that demonstrated increased grain yield,which indicates that GL3.1 is a powerful tool for breeding high-yield crops.

  3. Crystal Structure of Human Cyclin K, A Positive Regulator of Cyclin-Dependent Kinase 9

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    Baek,K.; Brown, R.; Birrane, G.; Ladias, J.

    2007-01-01

    K and the closely related cyclins T1, T2a, and T2b interact with cyclin-dependent kinase 9 (CDK9) forming multiple nuclear complexes, referred to collectively as positive transcription elongation factor b (P-TEFb). Through phosphorylation of the C-terminal domain of the RNA polymerase II largest subunit, distinct P-TEFb species regulate the transcriptional elongation of specific genes that play central roles in human physiology and disease development, including cardiac hypertrophy and human immunodeficiency virus-1 pathogenesis. We have determined the crystal structure of human cyclin K (residues 11-267) at 1.5 {angstrom} resolution, which represents the first atomic structure of a P-TEFb subunit. The cyclin K fold comprises two typical cyclin boxes with two short helices preceding the N-terminal box. A prominent feature of cyclin K is an additional helix (H4a) in the first cyclin box that obstructs the binding pocket for the cell-cycle inhibitor p27{sup Kip1}. Modeling of CDK9 bound to cyclin K provides insights into the structural determinants underlying the formation and regulation of this complex. A homology model of human cyclin T1 generated using the cyclin K structure as a template reveals that the two proteins have similar structures, as expected from their high level of sequence identity. Nevertheless, their CDK9-interacting surfaces display significant structural differences, which could potentially be exploited for the design of cyclin-targeted inhibitors of the CDK9-cyclin K and CDK9-cyclin T1 complexes.

  4. Effect of berberine on Cdk9 and cyclin T1 expressions in myocardium of diabetic rats

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    Zhou Jiyin; Zhou Shiwen; Tang Jianlin; Xu Ying; Ying Yi

    2008-01-01

    Objective: To investigate the effect of berberine, one of the main alkaloids of Rhizoma coptidis, on myocardial orphology and the expressions of cyclin-dependent kinase 9 (Cdk9) and cyclin T1 protein in the myocardium of type diabetic rats. Methods: Type 2 diabetes mellitus rats were induced by an injection of 35 mg/kg streptozotocin (STZ) nd a high-carbohydrate/high-fat diet for 16 weeks. Diabetic rats were given low-, middle-, high-dose berberine (75,150, 300 mg/kg), fenofibrate (100 mg/kg) and rosiglitazone (4 mg/kg) for another 16 weeks, respectively. The myocardium structure was observed with hematoxylin & eosin (H&E) staining and Cdk9 and cyclin T1 protein expressions were detected by immunohistochemistry. Results: Middle-dose, high-dose berberine improved myocardial hypertrophy and interstitial fibrosis of diabetic rats. Cdk9 and cyclin T1 protein were significantly lower in diabetic myocardium than in control one (P<0.01), and middle-dose, high-dose berberine and fenofibrate obviously increased oth Cdk9 and cyclin T1 expression to near control level (P<0.01). Conclusion: Berberine modulates Cdk9 and cyclin I protein expression in diabetic myocardium which may contribute to ameliorate myocardium damage.

  5. Cyclin T1-dependent genes in activated CD4 T and macrophage cell lines appear enriched in HIV-1 co-factors.

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    Wendong Yu

    Full Text Available HIV-1 is dependent upon cellular co-factors to mediate its replication cycle in CD4(+ T cells and macrophages, the two major cell types infected by the virus in vivo. One critical co-factor is Cyclin T1, a subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is targeted directly by the viral Tat protein to activate proviral transcription. Cyclin T1 is up-regulated when resting CD4(+ T cells are activated and during macrophage differentiation or activation, conditions that are also necessary for high levels of HIV-1 replication. Because Cyclin T1 is a subunit of a transcription factor, the up-regulation of Cyclin T1 in these cells results in the induction of cellular genes, some of which might be HIV-1 co-factors. Using shRNA depletions of Cyclin T1 and transcriptional profiling, we identified 54 cellular mRNAs that appear to be Cyclin T1-dependent for their induction in activated CD4(+ T Jurkat T cells and during differentiation and activation of MM6 cells, a human monocytic cell line. The promoters for these Cyclin T1-dependent genes (CTDGs are over-represented in two transcription factor binding sites, SREBP1 and ARP1. Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value<0.00021. The results of siRNA depletion and dominant-negative protein experiments with two CTDGs identified here, CDK11 and Casein kinase 1 gamma 1, suggest that these genes are involved either directly or indirectly in HIV-1 replication. It is likely that the 54 CTDGs identified here include novel HIV-1 co-factors. The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors.

  6. The human I-mfa domain-containing protein, HIC, interacts with cyclin T1 and modulates P-TEFb-dependent transcription.

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    Young, Tara M; Wang, Qi; Pe'ery, Tsafi; Mathews, Michael B

    2003-09-01

    Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.

  7. Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4+ T cells

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    Rice Andrew P

    2006-10-01

    Full Text Available Abstract Background The latent reservoir of human immunodeficiency virus type 1 (HIV-1 in resting CD4+ T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART. Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4+ T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9 that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity. Results Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without a functional Tat protein, we found that prostratin stimulation of HIV-1 gene expression appears to require a functional Tat protein. Microarray analyses were performed and several genes related to HIV biology, including APOBEC3B, DEFA1, and S100 calcium-binding protein genes, were found to be regulated by prostratin. Conclusion Prostratin induces Cyclin T1 expression and P-TEFb function and this is likely to be involved in prostratin reactivation of latent HIV-1 proviruses. The large increase in association of 7SK and HEXIM1 with P-TEFb following prostratin treatment may reflect a requirement in CD4+ T cells for a precise balance between

  8. Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis.

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    Keqing Wang

    Full Text Available Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

  9. Regulation of cyclin E stability in Xenopus laevis embryos

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    Brandt-(Webb), Yekaterina

    Cyclin-Cdk complexes positively regulate cell cycle progression. Cyclins are regulatory subunits that bind to and activate cyclin-dependent kinases or Cdks. Cyclin E associates with Cdk2 to mediate G1/S phase transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how this protein is downregulated during development in order to determine if these mechanisms are disrupted during tumorigenesis, leading to its overexpression. Using Xenopus laevis embryos as a model, we have shown previously that during the first 12 embryonic cell cycles Cyclin E levels remain constant yet Cdk2 activity oscillates twice per cell cycle. Cyclin E is abruptly destabilized by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT). Based on work our work and work by others, we have hypothesized that differential phosphorylation and a change in localization result in Cyclin E degradation by the 26S proteasome at the MBT. To test this, we generated a series of point mutations in conserved threonine/serine residues implicated in degradation of human Cyclin E. Using Western blot analysis, we show that similarly to human Cyclin E, mutation of these residues to unphosphorylatable alanine stabilizes Cyclin E past the MBT when they are expressed in vivo. Cyclin E localization was studied by immunofluorescence analysis of endogenous and exogenous protein in pre-MBT, MBT, and post-MBT embryos. In addition, we developed a novel method of conjugating recombinant His6-tagged Cyclin E to fluorescent (CdSe)ZnS nanoparticles (quantum dots) capped with dihydrolipoic acid. Confocal microscopy was used to visualize His6Cyclin E-quantum dot complexes inside embryo cells in real time. We found that re-localization at the MBT from the cytoplasm to the nucleus

  10. Developmental regulators containing the I-mfa domain interact with T cyclins and Tat and modulate transcription.

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    Wang, Qi; Young, Tara M; Mathews, Michael B; Pe'ery, Tsafi

    2007-03-30

    Positive transcription elongation factor b (P-TEFb) complexes, composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1 or T2, are engaged by many cellular transcription regulators that activate or inhibit transcription from specific promoters. The related I-mfa (inhibitor of MyoD family a) and HIC (human I-mfa-domain-containing) proteins function in myogenic differentiation and embryonic development by participating in the Wnt signaling pathway. We report that I-mfa is a novel regulator of P-TEFb. Both HIC and I-mfa interact through their homologous I-mfa domains with cyclin T1 and T2 at two binding sites. One site is the regulatory histidine-rich domain that interacts with CDK9 substrates including RNA polymerase II. The second site contains a lysine and arginine-rich motif that is highly conserved between the two T cyclins. This site overlaps and includes the previously identified Tat/TAR recognition motif of cyclin T1 required for activation of human immunodeficiency virus type 1 (HIV-1) transcription. HIC and I-mfa can serve as substrates for P-TEFb. Their I-mfa domains also bind the activation domain of HIV-1 Tat and inhibit Tat- and P-TEFb-dependent transcription from the HIV-1 promoter. This transcriptional repression is cell-type specific and can operate via Tat and cyclin T1. Genomic and sequence comparisons indicate that the I-mf and HIC genes, as well as flanking genes, diverged from a duplicated chromosomal region. Our findings link I-mfa and HIC to viral replication, and suggest that P-TEFb is modulated in the Wnt signaling pathway.

  11. Tripartite Motif-Containing Protein 22 Interacts with Class II Transactivator and Orchestrates Its Recruitment in Nuclear Bodies Containing TRIM19/PML and Cyclin T1

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    Greta Forlani

    2017-05-01

    Full Text Available Among interferon (IFN inducible antiviral factors both tripartite motif-containing protein 22 (TRIM22 and class II transactivator (CIITA share the capacity of repressing human immunodeficiency virus type 1 (HIV-1 proviral transcription. TRIM22 is constitutively expressed in a subset of U937 cell clones poorly permissive to HIV-1 replication, whereas CIITA has been shown to inhibit virus multiplication in both T lymphocytic and myeloid cells, including poorly HIV-1 permissive U937 cells, by suppressing Tat-mediated transactivation of HIV-1 transcription. Therefore, we tested whether TRIM22 and CIITA could form a nuclear complex potentially endowed with HIV-1 repressive functions. Indeed, we observed that TRIM22, independent of its E3 ubiquitin ligase domain, interacts with CIITA and promotes its recruitment into nuclear bodies. Importantly, TRIM19/promyelocytic leukemia (PML protein, another repressor of HIV-1 transcription also acting before proviral integration, colocalize in these nuclear bodies upon TRIM22 expression induced by IFN-γ. Finally, tTRIM22 nuclear bodies also contained CyclinT1, a crucial elongation factor of HIV-1 primary transcripts. These findings show that TRIM22 nuclear bodies are a site of recruitment of factors crucial for the regulation of HIV-1 transcription and highlight the potential existence of a concerted action between TRIM22, CIITA, and TRIM19/PML to maintain a state of proviral latency, at least in myeloid cells.

  12. Mice transgenic for equine cyclin T1 and ELR1 are susceptible to equine infectious anemia virus infection.

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    Du, Cheng; Ma, Jian; Liu, Qiang; Li, Yun-Fei; He, Xi-Jun; Lin, Yue-Zhi; Wang, Xue-Feng; Meng, Qing-Wen; Wang, Xiaojun; Zhou, Jian-Hua

    2015-04-28

    As a member of the tumor necrosis factor receptor (TNFR) protein superfamily, equine lentivirus receptor 1 (ELR1) has been shown to be expressed in various equine cells that are permissive for equine infectious anemia virus (EIAV) replication. The EIAV Tat protein (eTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter through a unique mechanism that requires the recruitment of the equine cyclin T1 (eCT1) cofactor into the viral TAR RNA target element. In vitro studies have demonstrated that mouse fibroblast cell lines (e.g., NIH 3T3 cells) that express the EIAV receptor ELR1 and eCT1 support the productive replication of EIAV. Therefore, we constructed transgenic eCT1- and ELR1-expressing mice to examine whether they support in vivo EIAV replication. For the first time, we constructed mice transgenic for ELR1 and eCT1. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis confirmed that ELR1 and eCT1 were expressed in the transgenic mouse tissues, particularly in the intestines, spleen and lymph nodes. Consistent with the results of EIAV infection in NIH 3T3 cells expressing ELR1 and eCT1, mouse embryonic fibroblasts (MEFs) from the transgenic mice could support EIAV replication. More importantly, this virus could infect and replicate in mouse blood monocyte-derived macrophages (mMDMs). Macrophages are the principle target cell of EIAV in its natural hosts. Furthermore, after the transgenic mice were inoculated with EIAV, the virus could be detected not only in the plasma of the circulating blood but also in multiple organs, among which, the spleen and lymph nodes were the predominant sites of EIAV replication. Finally, we found that consistent with high viral replication levels, the relevant pathological changes occurred in the spleen and lymph nodes. Our results show that mice transgenic for ELR1 and eCT1 are susceptible to EIAV infection and replication. Further, EIAV infection can cause

  13. Down-regulating cyclin-dependent kinase 9 of alloreactive CD4+ T cells prolongs allograft survival

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    Zhan, Yang; Han, Yeming; Sun, Hukui; Liang, Ting; Zhang, Chao; Song, Jing; Hou, Guihua

    2016-01-01

    CDK9 (Cyclin-dependent kinase 9)/Cyclin T1/RNA polymerase II pathway has been demonstrated to promote the development of several inflammatory diseases, such as arthritis or atherosclerosis, however, its roles in allotransplantation rejection have not been addressed. Here, we found that CDK9/Cyclin T1 were apparently up-regulated in the allogeneic group, which was positively correlated with allograft damage. CDK9 was inhibited obviously in naive splenic CD4+ T cells treated 6 h with 3 μM PHA767491 (a CDK9 inhibitor), and adoptive transfer of these CD4+ T cells into allografted SCID mice resulted in prolonged survival compared with the group without PHA767491 pretreated. Decelerated rejection was correlated with enhanced IL-4 and IL-10 production and with decreased IFN-γ production by alloreactive T cells. More interestingly, we found that CDK942, not CDK955, was high expressed in allorejection group, which could be prominently dampened with PHA767491 treatment. The expression of CDK942 was consistent with its downstream molecule RNA polymerase II. Altogether, our findings revealed the crucial role of CDK9/Cyclin T1/Pol II pathway in promoting allorejection at multiple levels and may provide a new approach for transplantation tolerance induction through targeting CDK9. PMID:27102157

  14. Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo

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    Littman Dan R

    2009-01-01

    Full Text Available Abstract Background Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1. Results Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. Conclusion Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.

  15. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells.

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    Bhattacharya, Sujoy; Ray, Ramesh M; Johnson, Leonard R

    2014-03-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.

  16. Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

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    Guo, Zheng [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Dadao Bei, Guangzhou 510515 (China); Zhou, Yuning [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Evers, B. Mark [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Department of Surgery, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Wang, Qingding, E-mail: qingding.wang@uky.edu [Markey Cancer Center, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States); Department of Surgery, The University of Kentucky, 800 Rose Street, Lexington, KY 40536 (United States)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer Rictor associates with FBXW7 to form an E3 complex. Black-Right-Pointing-Pointer Knockdown of rictor decreases ubiquitination of c-Myc and cylin E. Black-Right-Pointing-Pointer Knockdown of rictor increases protein levels of c-Myc and cylin E. Black-Right-Pointing-Pointer Overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Black-Right-Pointing-Pointer Rictor regulation of c-Myc and cyclin E requires FBXW7. -- Abstract: Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.

  17. CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain.

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    Yan, Junpeng; Hao, Caili; DeLucia, Maria; Swanson, Selene; Florens, Laurence; Washburn, Michael P; Ahn, Jinwoo; Skowronski, Jacek

    2015-05-22

    SAMHD1 is a nuclear deoxyribonucleoside triphosphate triphosphohydrolase that contributes to the control of cellular deoxyribonucleoside triphosphate (dNTP) pool sizes through dNTP hydrolysis and modulates the innate immune response to viruses. CyclinA2-CDK1/2 phosphorylates SAMHD1 at Thr-592, but how this modification controls SAMHD1 functions in proliferating cells is not known. Here, we show that SAMHD1 levels remain relatively unchanged during the cell division cycle in primary human T lymphocytes and in monocytic cell lines. Inactivation of the bipartite cyclinA2-CDK-binding site in the SAMHD1 C terminus described herein abolished SAMHD1 phosphorylation on Thr-592 during S and G2 phases thus interfering with DNA replication and progression of cells through S phase. The effects exerted by Thr-592 phosphorylation-defective SAMHD1 mutants were associated with activation of DNA damage checkpoint and depletion of dNTP concentrations to levels lower than those seen upon expression of wild type SAMHD1 protein. These disruptive effects were relieved by either mutation of the catalytic residues of the SAMHD1 phosphohydrolase domain or by a Thr-592 phosphomimetic mutation, thus linking the Thr-592 phosphorylation state to the control of SAMHD1 dNTPase activity. Our findings support a model in which phosphorylation of Thr-592 by cyclinA2-CDK down-modulates, but does not inactivate, SAMHD1 dNTPase in S phase, thereby fine-tuning SAMHD1 control of dNTP levels during DNA replication. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain*

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    Yan, Junpeng; Hao, Caili; DeLucia, Maria; Swanson, Selene; Florens, Laurence; Washburn, Michael P.; Ahn, Jinwoo; Skowronski, Jacek

    2015-01-01

    SAMHD1 is a nuclear deoxyribonucleoside triphosphate triphosphohydrolase that contributes to the control of cellular deoxyribonucleoside triphosphate (dNTP) pool sizes through dNTP hydrolysis and modulates the innate immune response to viruses. CyclinA2-CDK1/2 phosphorylates SAMHD1 at Thr-592, but how this modification controls SAMHD1 functions in proliferating cells is not known. Here, we show that SAMHD1 levels remain relatively unchanged during the cell division cycle in primary human T lymphocytes and in monocytic cell lines. Inactivation of the bipartite cyclinA2-CDK-binding site in the SAMHD1 C terminus described herein abolished SAMHD1 phosphorylation on Thr-592 during S and G2 phases thus interfering with DNA replication and progression of cells through S phase. The effects exerted by Thr-592 phosphorylation-defective SAMHD1 mutants were associated with activation of DNA damage checkpoint and depletion of dNTP concentrations to levels lower than those seen upon expression of wild type SAMHD1 protein. These disruptive effects were relieved by either mutation of the catalytic residues of the SAMHD1 phosphohydrolase domain or by a Thr-592 phosphomimetic mutation, thus linking the Thr-592 phosphorylation state to the control of SAMHD1 dNTPase activity. Our findings support a model in which phosphorylation of Thr-592 by cyclinA2-CDK down-modulates, but does not inactivate, SAMHD1 dNTPase in S phase, thereby fine-tuning SAMHD1 control of dNTP levels during DNA replication. PMID:25847232

  19. Mutation analysis of the negative regulator cyclin G2 in gastric cancer

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... Key words: Cyclin G2, gastric cancer, negative regulator, mutation screen. ... has been reported in thyroid cancer, breast cancer, oral cancer and acute ..... transformation of papillary carcinoma of the thyroid. Anticancer. Res.

  20. Involvement of cyclin K posttranscriptional regulation in the formation of Artemia diapause cysts.

    Directory of Open Access Journals (Sweden)

    Yang Zhao

    Full Text Available BACKGROUND: Artemia eggs tend to develop ovoviviparously to yield nauplius larvae in good rearing conditions; while under adverse situations, they tend to develop oviparously and encysted diapause embryos are formed instead. However, the intrinsic mechanisms regulating this process are not well understood. PRINCIPAL FINDING: This study has characterized the function of cyclin K, a regulatory subunit of the positive transcription elongation factor b (P-TEFb in the two different developmental pathways of Artemia. In the diapause-destined embryo, Western blots showed that the cyclin K protein was down-regulated as the embryo entered dormancy and reverted to relatively high levels of expression once development resumed, consistent with the fluctuations in phosphorylation of position 2 serines (Ser2 in the C-terminal domain (CTD of the largest subunit (Rpb1 of RNA polymerase II (RNAP II. Interestingly, the cyclin K transcript levels remained constant during this process. In vitro translation data indicated that the template activity of cyclin K mRNA stored in the postdiapause cyst was repressed. In addition, in vivo knockdown of cyclin K in developing embryos by RNA interference eliminated phosphorylation of the CTD Ser2 of RNAP II and induced apoptosis by inhibiting the extracellular signal-regulated kinase (ERK survival signaling pathway. CONCLUSIONS/SIGNIFICANCE: Taken together, these findings reveal a role for cyclin K in regulating RNAP II activity during diapause embryo development, which involves the post-transcriptional regulation of cyclin K. In addition, a further role was identified for cyclin K in regulating the control of cell survival during embryogenesis through ERK signaling pathways.

  1. Cyclin D1 in the Liver: Role of Noncanonical Signaling in Liver Steatosis and Hormone Regulation

    Science.gov (United States)

    Núñez, Kelley G.; Gonzalez-Rosario, Janet; Thevenot, Paul T.; Cohen, Ari J.

    2017-01-01

    Background: Cyclin D1 is an important protein for cell cycle progression; however, functions independent of the cell cycle have been described in the liver. Cyclin D1 is also involved in DNA repair, is overexpressed in many cancers, and functions as a proto-oncogene. The lesser-known roles of Cyclin D1, specifically in hepatocytes, impact liver steatosis and hormone regulation in the liver. Methods: A comprehensive search of PubMed was conducted using the keywords Cyclin D1, steatosis, lipogenesis, and liver transplantation. In this article, we review the results from this literature search, with a focus on the role of Cyclin D1 in hepatic lipogenesis and gluconeogenesis, as well as the impact and function of this protein in hepatic steatosis. Results: Cyclin D1 represses carbohydrate response element binding protein (ChREBP) and results in a decrease in transcription of fatty acid synthase (FAS) and acetyl-coenzyme A carboxylase (ACC). Cyclin D1 also inhibits peroxisome proliferator-activated receptor gamma (PPARγ) which is involved in hepatic lipogenesis. Cyclin D1 inhibits both hepatocyte nuclear factor 4 alpha (HNF4α) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) and represses transcription of lipogenic genes FAS and liver-type pyruvate kinase (Pklr), along with the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Conclusion: Cyclin D1 represses multiple proteins involved in both lipogenesis and gluconeogenesis in the liver. Targeting Cyclin D1 to decrease hepatic steatosis in patients with nonalcoholic fatty liver disease or alcoholic fatty liver disease may help improve patient health and the quality of the donor liver pool.

  2. Flowcytometric evaluation of cell cycle regulators (cyclins and cyclin dependent kinase inhibitors expressed on bone marrow cells of patients with chronic myelogenous leukemia and multiple myeloma

    Directory of Open Access Journals (Sweden)

    Selami Koçak Toprak

    2012-03-01

    Full Text Available OBJECTIVE: Etiopathology of malignancy can be demonstrated by the comparison of the quantified changes in the different phases of the cycle about cyclins and cyclin dependent kinase inhibitors (CDKI in healthy and malignant proliferated cells. The aim of this study is to analyze flow cytometric expression of cell cycle regulating elements in the malignant diseases with low and high proliferative signature. METHODS: The levels of cyclin D, E, A, B and CDKI's p16, p21 were studied by flowcytometry in patients with chronic myeloid leukemia (CML (n=16, multiple myeloma (MM (n=13 and control subjects (n=15. RESULTS: The distributions of the cell cycle S phase were 10, 63%, 6, 72% and 3, 59%; for CML, MM and control subjects, respectively. Among all the cyclins expressed during the S phase, cyclin D expression was the lowest, in CML patients. While the distribution of cyclins and CDKI’s was similar between MM and control groups in G2/M phase; cyclins expressions were parallel in all three phases in MM and chronic myeloid leukemia groups. CONCLUSION: CML and MM are diseases presenting with variable degrees of proliferation. The increase of cyclins in cell cycle phases in patient group was not associated with the augmentation of the expression of CDKI’s. This finding may contribute the mechanisms effective in the etiopathogenesis of hematological malignancy.

  3. 小檗碱对链脲菌素和高糖高脂饲料诱导糖尿病大鼠骨骼肌CDK9和cyclin T1表达的影响%Effect of berberine on CDK9 and cyclin T1 expression in skeletal muscle of diabeticrats induced by streptozotocin and a high-carbohydrate/high-fat diet

    Institute of Scientific and Technical Information of China (English)

    周吉银; 周世文

    2008-01-01

    目的 观察小檗碱对2型糖尿病大鼠骨骼肌病理结构改变及CDK9和cyclin T1蛋白表达的影响.方法 腹腔注射链脲菌素(35mg·kg-1)加高糖高脂饲料喂养16 wk建立2型糖尿病大鼠模型,随后16 wk每天分别拌食给予低中高剂量小檗碱(75、150、300mg·kg-1)、非诺贝特(100 mg·kg-1)和罗格列酮(4 mg·kg-1),用HE染色检查骨骼肌结构病变和免疫组化检测CDK9和cyclin T1的表达.结果 骨骼肌纤维在各组大鼠中仍正常分布,中高剂量小檗碱部分地改善糖尿病肌纤维的萎缩;中高剂量小檗碱和罗格列酮都能明显恢复糖尿病大鼠骨骼肌中表达降低的CDK9和cyclin T1至正常水平(P<0.01).结论 小檗碱调控骨骼肌CDK9和cyclin T1蛋白的表达可能是其改善糖尿病骨骼肌纤维萎缩的机制之一.%AIM To investigate the effect of berberine on damaged skeletal muscle and on the cyclin-dependent kinase 9(CDK9)and cyclin T1 expression in type 2 diabetic rats.METHODS Type 2 diabetes mellitus rats were in duced by an injection of 35 mg·kg-1 streptozotocin and a high-carbohydrate/high-fat diet for 16 wk.From wk 17 to wk 32,diabetic rats were given low-,middle-,high-dose berberine(75,150,300 mg·kg-1),fenofibrate(100 mg·kg-1) and rosiglitazone(4 mg·kg-1)by oral administration,respectively.The skeletal muscle structure was observed with hematoxylin-eosin(HE)staining and the CDK9 and cyclin T1 expression Were detected by immunohistochemistry.RESULTS Fibre distribution remained normal in skeletal muscles of all the groups.Middle-,high-dose berberine partly im proved diabetic fibre atrophy.Middle-,high-dose berberine and rosiglitazone significantly increased the expressions for both of CDK9 and cyclin T1(P<0.01=.CONCLUSION Berberine modulates the CDK9 and cyclin T1 expressions in diabetic skeletal muscle and ameliorates the fibre damage.

  4. Cyclin E-Mediated Human Proopiomelanocortin Regulation as a Therapeutic Target for Cushing Disease

    Science.gov (United States)

    Liu, Ning-Ai; Araki, Takako; Cuevas-Ramos, Daniel; Hong, Jiang; Ben-Shlomo, Anat; Tone, Yukiko; Tone, Masahide

    2015-01-01

    Context: Cushing disease, due to pituitary corticotroph tumor ACTH hypersecretion, drives excess adrenal cortisol production with adverse morbidity and mortality. Loss of glucocorticoid negative feedback on the hypothalamic-pituitary-adrenal axis leads to autonomous transcription of the corticotroph precursor hormone proopiomelanocortin (POMC), consequent ACTH overproduction, and adrenal hypercortisolism. We previously reported that R-roscovitine (CYC202, seliciclib), a 2,6,9-trisubstituted purine analog, suppresses cyclin-dependent-kinase 2/cyclin E and inhibits ACTH in mice and zebrafish. We hypothesized that intrapituitary cyclin E signaling regulates corticotroph tumor POMC transcription independently of cell cycle progression. The aim was to investigate whether R-roscovitine inhibits human ACTH in corticotroph tumors by targeting the cyclin-dependent kinase 2/cyclin E signaling pathway. Methods: Primary cell cultures of surgically resected human corticotroph tumors were treated with or without R-roscovitine, ACTH measured by RIA and quantitative PCR, and/or Western blot analysis performed to investigate ACTH and lineage-specific transcription factors. Cyclin E and E2F transcription factor 1 (E2F1) small interfering RNA (siRNA) transfection was performed in murine corticotroph tumor AtT20 cells to elucidate mechanisms for drug action. POMC gene promoter activity in response to R-roscovitine treatment was analyzed using luciferase reporter and chromatin immunoprecipitation assays. Results: R-roscovitine inhibits human corticotroph tumor POMC and Tpit/Tbx19 transcription with decreased ACTH expression. Cyclin E and E2F1 exhibit reciprocal positive regulation in corticotroph tumors. R-roscovitine disrupts E2F1 binding to the POMC gene promoter and suppresses Tpit/Tbx19 and other lineage-specific POMC transcription cofactors via E2F1-dependent and -independent pathways. Conclusion: R-roscovitine inhibits human pituitary corticotroph tumor ACTH by targeting the

  5. Determining the Functions of HIV-1 Tat and a Second Magnesium Ion in the CDK9/Cyclin T1 Complex: A Molecular Dynamics Simulation Study.

    Directory of Open Access Journals (Sweden)

    Hai-Xiao Jin

    Full Text Available The current paradigm of cyclin-dependent kinase (CDK regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1 Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg2+ ion at site 1 (Mg12+. The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg12+ ion, coupled with the Mg22+ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg12+ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg12+ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg12+ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.

  6. DDX3 regulates cell growth through translational control of cyclin E1.

    Science.gov (United States)

    Lai, Ming-Chih; Chang, Wen-Cheng; Shieh, Sheau-Yann; Tarn, Woan-Yuh

    2010-11-01

    DDX3 belongs to the DEAD box family of RNA helicases, but the details of its biological function remain largely unclear. Here we show that knockdown of DDX3 expression impedes G(1)/S-phase transition of the cell cycle. To know how DDX3 may act in cell cycle control, we screened for cellular mRNA targets of DDX3. Many of the identified DDX3 targets encoded cell cycle regulators, including G(1)/S-specific cyclin E1. DDX3 depletion specifically downregulates translation of cyclin E1 mRNA. Moreover, our data suggest that DDX3 participates in translation initiation of targeted mRNAs as well as in cell growth control via its RNA helicase activity. Consistent with these findings, we show that in the temperature-sensitive DDX3 mutant hamster cell line tsET24, cyclin E1 expression is downregulated at a nonpermissive temperature that inactivates mutant DDX3. Taken together, our results indicate that DDX3 is critical for translation of cyclin E1 mRNA, which provides an alternative mechanism for regulating cyclin E1 expression during the cell cycle.

  7. Cdc2/cyclin B1 regulates centrosomal Nlp proteolysis and subcellular localization.

    Science.gov (United States)

    Zhao, Xuelian; Jin, Shunqian; Song, Yongmei; Zhan, Qimin

    2010-11-01

    The formation of proper mitotic spindles is required for appropriate chromosome segregation during cell division. Aberrant spindle formation often causes aneuploidy and results in tumorigenesis. However, the underlying mechanism of regulating spindle formation and chromosome separation remains to be further defined. Centrosomal Nlp (ninein-like protein) is a recently characterized BRCA1-regulated centrosomal protein and plays an important role in centrosome maturation and spindle formation. In this study, we show that Nlp can be phosphorylated by cell cycle protein kinase Cdc2/cyclin B1. The phosphorylation sites of Nlp are mapped at Ser185 and Ser589. Interestingly, the Cdc2/cyclin B1 phosphorylation site Ser185 of Nlp is required for its recognition by PLK1, which enable Nlp depart from centrosomes to allow the establishment of a mitotic scaffold at the onset of mitosis . PLK1 fails to dissociate the Nlp mutant lacking Ser185 from centrosome, suggesting that Cdc2/cyclin B1 might serve as a primary kinase of PLK1 in regulating Nlp subcellular localization. However, the phosphorylation at the site Ser589 by Cdc2/cyclin B1 plays an important role in Nlp protein stability probably due to its effect on protein degradation. Furthermore, we show that deregulated expression or subcellular localization of Nlp lead to multinuclei in cells, indicating that scheduled levels of Nlp and proper subcellular localization of Nlp are critical for successful completion of normal cell mitosis, These findings demonstrate that Cdc2/cyclin B1 is a key regulator in maintaining appropriate degradation and subcellular localization of Nlp, providing novel insights into understanding on the role of Cdc2/cyclin B1 in mitotic progression.

  8. Glucose Regulates Cyclin D2 Expression in Quiescent and Replicating Pancreatic β-Cells Through Glycolysis and Calcium Channels

    Science.gov (United States)

    Salpeter, Seth J.; Klochendler, Agnes; Weinberg-Corem, Noa; Porat, Shay; Granot, Zvi; Shapiro, A. M. James; Magnuson, Mark A.; Eden, Amir; Grimsby, Joseph; Glaser, Benjamin

    2011-01-01

    Understanding the molecular triggers of pancreatic β-cell proliferation may facilitate the development of regenerative therapies for diabetes. Genetic studies have demonstrated an important role for cyclin D2 in β-cell proliferation and mass homeostasis, but its specific function in β-cell division and mechanism of regulation remain unclear. Here, we report that cyclin D2 is present at high levels in the nucleus of quiescent β-cells in vivo. The major regulator of cyclin D2 expression is glucose, acting via glycolysis and calcium channels in the β-cell to control cyclin D2 mRNA levels. Furthermore, cyclin D2 mRNA is down-regulated during S-G2-M phases of each β-cell division, via a mechanism that is also affected by glucose metabolism. Thus, glucose metabolism maintains high levels of nuclear cyclin D2 in quiescent β-cells and modulates the down-regulation of cyclin D2 in replicating β-cells. These data challenge the standard model for regulation of cyclin D2 during the cell division cycle and suggest cyclin D2 as a molecular link between glucose levels and β-cell replication. PMID:21521747

  9. Cyclin K dependent regulation of Aurora B affects apoptosis and proliferation by induction of mitotic catastrophe in prostate cancer.

    Science.gov (United States)

    Schecher, Sabrina; Walter, Britta; Falkenstein, Michael; Macher-Goeppinger, Stephan; Stenzel, Philipp; Krümpelmann, Kristina; Hadaschik, Boris; Perner, Sven; Kristiansen, Glen; Duensing, Stefan; Roth, Wilfried; Tagscherer, Katrin E

    2017-10-15

    Cyclin K plays a critical role in transcriptional regulation as well as cell development. However, the role of Cyclin K in prostate cancer is unknown. Here, we describe the impact of Cyclin K on prostate cancer cells and examine the clinical relevance of Cyclin K as a biomarker for patients with prostate cancer. We show that Cyclin K depletion in prostate cancer cells induces apoptosis and inhibits proliferation accompanied by an accumulation of cells in the G2/M phase. Moreover, knockdown of Cyclin K causes mitotic catastrophe displayed by multinucleation and spindle multipolarity. Furthermore, we demonstrate a Cyclin K dependent regulation of the mitotic kinase Aurora B and provide evidence for an Aurora B dependent induction of mitotic catastrophe. In addition, we show that Cyclin K expression is associated with poor biochemical recurrence-free survival in patients with prostate cancer treated with an adjuvant therapy. In conclusion, targeting Cyclin K represents a novel, promising anti-cancer strategy to induce cell cycle arrest and apoptotic cell death through induction of mitotic catastrophe in prostate cancer cells. Moreover, our results indicate that Cyclin K is a putative predictive biomarker for clinical outcome and therapy response for patients with prostate cancer. © 2017 UICC.

  10. Regulation of cell proliferation and cell density by the inorganic phosphate transporter PiT1

    Directory of Open Access Journals (Sweden)

    Byskov Kristina

    2012-03-01

    Full Text Available Abstact Background The inorganic phosphate (Pi transporter, PiT1 (SLC20A1, is ubiquitously expressed in mammalian cells. It has previously been shown that down-regulation of PiT1 severely impaired the proliferation of two transformed human cells lines, HepG2 and HeLa, and the tumorigenicity of HeLa cells in nude mice. Moreover, PiT1 knock-out mice do not survive past E12.5 and from E10.5, the embryos were found to be growth-retarded and showed reduced proliferation of liver cells. Isolated mouse embryonic fibroblasts with knocked out as well as reduced PiT1 expression levels also exhibited impaired proliferation. Together these results suggest that a certain level of PiT1 is important for proliferation. We have here investigated the role of PiT1 in regulation of cell proliferation using two strictly density-inhibited cells lines, the murine MC3T3-E1 and NIH3T3 cells. Results We found that knock-down of PiT1 in MC3T3-E1 cells led to impaired proliferation supporting that at least a certain level of PiT1 is important for wildtype level of proliferation. We, however, also observed that MC3T3-E1 and NIH3T3 cells themselves regulate their endogenous PiT1 mRNA levels with lower levels in general correlating with decreased proliferation/increased cell density. Moreover, over-expression of human PiT1 led to increased proliferation of both MC3T3-E1 and NIH3T3 cultures and resulted in higher cell densities in cultures of these two strictly density-inhibited cell lines. In addition, when we transformed NIH3T3 cells by cultivation in fetal bovine serum, cells over-expressing human PiT1 formed more colonies in soft agar than control cells. Conclusions We conclude that not only is a certain level of PiT1 necessary for normal cell division as suggested by previously published studies, rather the cellular PiT1 level is involved in regulating cell proliferation and cell density and an increased PiT1 expression can indeed make NIH3T3 cells more sensitive to

  11. The regulation of SIRT2 function by cyclin-dependent kinases affects cell motility.

    NARCIS (Netherlands)

    Pandithage, R.; Lilischkis, R.; Harting, K.; Wolf, A.; Jedamzik, B.; Luscher-Firzlaff, J.; Vervoorts, J.; Lasonder, E.; Kremmer, E.; Knoll, B.; Luscher, B.

    2008-01-01

    Cyclin-dependent kinases (Cdks) fulfill key functions in many cellular processes, including cell cycle progression and cytoskeletal dynamics. A limited number of Cdk substrates have been identified with few demonstrated to be regulated by Cdk-dependent phosphorylation. We identify on protein express

  12. DDX3 Regulates Cell Growth through Translational Control of Cyclin E1▿ †

    OpenAIRE

    2010-01-01

    DDX3 belongs to the DEAD box family of RNA helicases, but the details of its biological function remain largely unclear. Here we show that knockdown of DDX3 expression impedes G1/S-phase transition of the cell cycle. To know how DDX3 may act in cell cycle control, we screened for cellular mRNA targets of DDX3. Many of the identified DDX3 targets encoded cell cycle regulators, including G1/S-specific cyclin E1. DDX3 depletion specifically downregulates translation of cyclin E1 mRNA. Moreover, ...

  13. NFAT1 transcription factor regulates cell cycle progression and cyclin E expression in B lymphocytes.

    Science.gov (United States)

    Teixeira, Leonardo K; Carrossini, Nina; Sécca, Cristiane; Kroll, José E; DaCunha, Déborah C; Faget, Douglas V; Carvalho, Lilian D S; de Souza, Sandro J; Viola, João P B

    2016-09-01

    The NFAT family of transcription factors has been primarily related to T cell development, activation, and differentiation. Further studies have shown that these ubiquitous proteins are observed in many cell types inside and outside the immune system, and are involved in several biological processes, including tumor growth, angiogenesis, and invasiveness. However, the specific role of the NFAT1 family member in naive B cell proliferation remains elusive. Here, we demonstrate that NFAT1 transcription factor controls Cyclin E expression, cell proliferation, and tumor growth in vivo. Specifically, we show that inducible expression of NFAT1 inhibits cell cycle progression, reduces colony formation, and controls tumor growth in nude mice. We also demonstrate that NFAT1-deficient naive B lymphocytes show a hyperproliferative phenotype and high levels of Cyclin E1 and E2 upon BCR stimulation when compared to wild-type B lymphocytes. NFAT1 transcription factor directly regulates Cyclin E expression in B cells, inhibiting the G1/S cell cycle phase transition. Bioinformatics analysis indicates that low levels of NFAT1 correlate with high expression of Cyclin E1 in different human cancers, including Diffuse Large B-cell Lymphomas (DLBCL). Together, our results demonstrate a repressor role for NFAT1 in cell cycle progression and Cyclin E expression in B lymphocytes, and suggest a potential function for NFAT1 protein in B cell malignancies.

  14. Cannabinoids Regulate Bcl-2 and Cyclin D2 Expression in Pancreatic β Cells.

    Directory of Open Access Journals (Sweden)

    Jihye Kim

    Full Text Available Recent reports have shown that cannabinoid 1 receptors (CB1Rs are expressed in pancreatic β cells, where they induce cell death and cell cycle arrest by directly inhibiting insulin receptor activation. Here, we report that CB1Rs regulate the expression of the anti-apoptotic protein Bcl-2 and cell cycle regulator cyclin D2 in pancreatic β cells. Treatment of MIN6 and βTC6 cells with a synthetic CB1R agonist, WIN55,212-2, led to a decrease in the expression of Bcl-2 and cyclin D2, in turn inducing cell cycle arrest in G0/G1 phase and caspase-3-dependent apoptosis. Additionally, genetic deletion and pharmacological blockade of CB1Rs after injury in mice led to increased levels of Bcl-2 and cyclin D2 in pancreatic β cells. These findings provide evidence for the involvement of Bcl-2 and cyclin D2 mediated by CB1Rs in the regulation of β-cell survival and growth, and will serve as a basis for developing new therapeutic interventions to enhance β-cell function and growth in diabetes.

  15. Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site

    DEFF Research Database (Denmark)

    Schulze, A; Zerfass, K; Spitkovsky, D

    1995-01-01

    Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation...

  16. Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells.

    Science.gov (United States)

    Battisti, Pier-Luigi; Daher, Aïcha; Bannwarth, Sylvie; Voortman, Johannes; Peden, Keith W C; Hiscott, John; Mouland, Andrew J; Benarous, Richard; Gatignol, Anne

    2003-09-01

    Tat-mediated trans-activation of the HIV-1 long terminal repeat (LTR) occurs through the phosphorylation of the carboxy-terminal domain of the RNA polymerase II. The kinase complex, pTEFb, composed of cyclin T1 (CycT1) and CDK9, mediates this process. The trans-activation response (TAR) RNA-binding protein 2 (TRBP2) increases HIV-1 LTR expression through TAR and protein kinase R (PKR) binding, but not through interactions with the Tat-CycT1-CDK9 complex. TRBP2 and the Tat-CycT1-CDK9 complex have overlapping binding sites on TAR RNA. TRBP2 and CycT1 increased Tat trans-activation in NIH 3T3 cells with additive effects. Upon transfection of HIV-1 pLAI, pNL4-3, pMAL, and pAD molecular clones, reverse transcriptase (RT) activity and p24 concentration were decreased 200- to 900-fold in NIH 3T3 cells compared with HeLa cells in both cells and supernatants. In murine cells, cotransfection of the HIV clones with CycT1 or TRBP2 increased modestly the expression of RT activity in cell extracts. The analysis of Gag expression in murine cells transfected with CycT1 compared with human cells showed a 20-fold decrease in expression and a strong processing defect. The expression of both CycT1 and TRBP2 had a more than additive activity on RT function in cell extracts and on viral particle production in supernatant of murine cells. These results suggest an activity of CycT1 and TRBP2 at different steps in HIV-1 expression and indicate the requirement for another posttranscriptional factor in murine cells for full HIV replication.

  17. Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells

    OpenAIRE

    Wauson, Eric M.; Guerra, Marcy L.; Dyachok, Julia; McGlynn, Kathleen; Giles, Jennifer; Ross, Elliott M.; Cobb, Melanie H.

    2015-01-01

    The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic β-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic β-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We ...

  18. STAR syndrome-associated CDK10/Cyclin M regulates actin network architecture and ciliogenesis.

    Science.gov (United States)

    Guen, Vincent J; Gamble, Carly; Perez, Dahlia E; Bourassa, Sylvie; Zappel, Hildegard; Gärtner, Jutta; Lees, Jacqueline A; Colas, Pierre

    2016-01-01

    CDK10/CycM is a protein kinase deficient in STAR (toe Syndactyly, Telecanthus and Anogenital and Renal malformations) syndrome, which results from mutations in the X-linked FAM58A gene encoding Cyclin M. The biological functions of CDK10/CycM and etiology of STAR syndrome are poorly understood. Here, we report that deficiency of CDK10/Cyclin M promotes assembly and elongation of primary cilia. We establish that this reflects a key role for CDK10/Cyclin M in regulation of actin network organization, which is known to govern ciliogenesis. In an unbiased screen, we identified the RhoA-associated kinase PKN2 as a CDK10/CycM phosphorylation substrate. We establish that PKN2 is a bone fide regulator of ciliogenesis, acting in a similar manner to CDK10/CycM. We discovered that CDK10/Cyclin M binds and phosphorylates PKN2 on threonines 121 and 124, within PKN2's core RhoA-binding domain. Furthermore, we demonstrate that deficiencies in CDK10/CycM or PKN2, or expression of a non-phosphorylatable version of PKN2, destabilize both the RhoA protein and the actin network architecture. Importantly, we established that ectopic expression of RhoA is sufficient to override the induction of ciliogenesis resulting from CDK10/CycM knockdown, indicating that RhoA regulation is critical for CDK10/CycM's negative effect on ciliogenesis. Finally, we show that kidney sections from a STAR patient display dilated renal tubules and abnormal, elongated cilia. Altogether, these results reveal CDK10/CycM as a key regulator of actin dynamics and a suppressor of ciliogenesis through phosphorylation of PKN2 and promotion of RhoA signaling. Moreover, they suggest that STAR syndrome is a ciliopathy.

  19. miR-379 regulates cyclin B1 expression and is decreased in breast cancer.

    Directory of Open Access Journals (Sweden)

    Sonja Khan

    Full Text Available MicroRNAs are small non-coding RNA molecules that control gene expression post-transcriptionally, and are known to be altered in many diseases including breast cancer. The aim of this study was to determine the relevance of miR-379 in breast cancer. miR-379 expression was quantified in clinical samples including tissues from breast cancer patients (n=103, healthy controls (n=30 and patients with benign breast disease (n=35. The level of miR-379 and its putative target Cyclin B1 were investigated on all breast tissue specimens by RQ-PCR. Potential relationships with gene expression and patient clinicopathological details were also determined. The effect of miR-379 on Cyclin B1 protein expression and function was investigated using western blot, immunohistochemistry and proliferation assays respectively. Finally, the levels of circulating miR-379 were determined in whole blood from patients with breast cancer (n=40 and healthy controls (n=34. The level of miR-379 expression was significantly decreased in breast cancer (Mean(SEM 1.9 (0.09 Log10 Relative Quantity (RQ compared to normal breast tissues (2.6 (0.16 Log10 RQ, p<0.01. miR-379 was also found to decrease significantly with increasing tumour stage. A significant negative correlation was determined between miR-379 and Cyclin B1 (r=-0.31, p<0.001. Functional assays revealed reduced proliferation (p<0.05 and decreased Cyclin B1 protein levels following transfection of breast cancer cells with miR-379. Circulating miR-379 was not significantly dysregulated in patients with breast cancer compared to healthy controls (p=0.42. This data presents miR-379 as a novel regulator of Cyclin B1 expression, with significant loss of the miRNA observed in breast tumours.

  20. MicroRNA-16 Modulates HuR Regulation of Cyclin E1 in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xun Guo

    2015-03-01

    Full Text Available RNA binding protein (RBPs and microRNAs (miRNAs or miRs are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC. Despite this, miR-16 and HuR do not affect the other’s expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA.

  1. Morphogenesis signaling components influence cell cycle regulation by cyclin dependent kinase

    Directory of Open Access Journals (Sweden)

    Bevis Brooke J

    2009-07-01

    Full Text Available Abstract Background The yeast cell cycle is largely controlled by the cyclin-dependent kinase (CDK Cdc28. Recent evidence suggests that both CDK complex stability as well as function during mitosis is determined by precise regulation of Swe1, a CDK inhibitory kinase and cyclin binding partner. A model of mitotic progression has been provided by study of filamentous yeast. When facing nutrient-limited conditions, Ras2-mediated PKA and MAPK signaling cascades induce a switch from round to filamentous morphology resulting in delayed mitotic progression. Results To delineate how the dimorphic switch contributes to cell cycle regulation, temperature sensitive cdc28 mutants exhibiting constitutive filamentation were subjected to epistasis analyses with RAS2 signaling effectors. It was found that Swe1-mediated inhibitory tyrosine phosphorylation of Cdc28 during filamentous growth is in part mediated by Ras2 activation of PKA, but not Kss1-MAPK, signaling. This pathway is further influenced by Cks1, a conserved CDK-binding partner of elusive function with multiple proposed roles in CDK activation, transcriptional regulation and ubiquitin-mediated proteasome degradation. Conclusion The dynamic balance between Cks1- and Swe1-dependent regulation of Cdc28 and, thereby, the timing of mitosis during yeast dimorphism is regulated in part by Ras2/cAMP-mediated PKA signaling, a key pathway controlling filamentous growth.

  2. Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization

    DEFF Research Database (Denmark)

    Petersen, B O; Lukas, J; Sørensen, Claus Storgaard

    1999-01-01

    by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo...

  3. Inhibitor of CDK interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

    DEFF Research Database (Denmark)

    Baumer, Nicole; Tickenbrock, Lara; Tschanter, Petra

    2011-01-01

    The cell cycle is driven by the kinase activity of cyclin/CDK complexes which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as interaction partner and substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin binding site...... in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inihibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, while it was induced by cell cycle arrest. We established a deletional mouse model that showed increased...... CDK2 activity in spleen with altered spleen architecture in Inca1-/- mice. Inca1-/- embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from ALL and AML patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control...

  4. The Hematopoietic Transcription Factor AML1 (RUNX1) Is Negatively Regulated by the Cell Cycle Protein Cyclin D3

    Science.gov (United States)

    Peterson, Luke F.; Boyapati, Anita; Ranganathan, Velvizhi; Iwama, Atsushi; Tenen, Daniel G.; Tsai, Schickwann; Zhang, Dong-Er

    2005-01-01

    The family of cyclin D proteins plays a crucial role in the early events of the mammalian cell cycle. Recent studies have revealed the involvement of AML1 transactivation activity in promoting cell cycle progression through the induction of cyclin D proteins. This information in combination with our previous observation that a region in AML1 between amino acids 213 and 289 is important for its function led us to investigate prospective proteins associating with this region. We identified cyclin D3 by a yeast two-hybrid screen and detected AML1 interaction with the cyclin D family by both in vitro pull-down and in vivo coimmunoprecipitation assays. Furthermore, we demonstrate that cyclin D3 negatively regulates the transactivation activity of AML1 in a dose-dependent manner by competing with CBFβ for AML1 association, leading to a decreased binding affinity of AML1 for its target DNA sequence. AML1 and its fusion protein AML1-ETO have been shown to shorten and prolong the mammalian cell cycle, respectively. In addition, AML1 promotes myeloid cell differentiation. Thus, our observations suggest that the direct association of cyclin D3 with AML1 functions as a putative feedback mechanism to regulate cell cycle progression and differentiation. PMID:16287839

  5. Methionine Regulates mTORC1 via the T1R1/T1R3-PLCβ-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

    Directory of Open Access Journals (Sweden)

    Yuanfei Zhou

    2016-10-01

    Full Text Available The mammalian target of rapamycin complex 1 (mTORC1 integrates amino acid (AA availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2 activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and T1R3 subunits were expressed in C2C12 myotubes, and l-AA sensing was accomplished by T1R1/T1R3 to activate mTORC1. In response to l-AAs, such as serine (Ser, arginine (Arg, threonine (Thr, alanine (Ala, methionine (Met, glutamine (Gln, and glycine (Gly, Met induced mTORC1 activation and promoted protein synthesis. Met also regulated mTORC1 via T1R1/T1R3-PLCβ-Ca2+-ERK1/2 signal transduction. Results revealed a new role for Met-regulated mTORC1 via an AA receptor. Further studies should be performed to determine the role of T1R1/T1R3 in mediating extracellular AA to regulate mTOR signaling and to reveal its mechanism.

  6. Methionine Regulates mTORC1 via the T1R1/T1R3-PLCβ-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

    Science.gov (United States)

    Zhou, Yuanfei; Ren, Jiao; Song, Tongxing; Peng, Jian; Wei, Hongkui

    2016-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) integrates amino acid (AA) availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R) is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2) activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and T1R3 subunits were expressed in C2C12 myotubes, and l-AA sensing was accomplished by T1R1/T1R3 to activate mTORC1. In response to l-AAs, such as serine (Ser), arginine (Arg), threonine (Thr), alanine (Ala), methionine (Met), glutamine (Gln), and glycine (Gly), Met induced mTORC1 activation and promoted protein synthesis. Met also regulated mTORC1 via T1R1/T1R3-PLCβ-Ca2+-ERK1/2 signal transduction. Results revealed a new role for Met-regulated mTORC1 via an AA receptor. Further studies should be performed to determine the role of T1R1/T1R3 in mediating extracellular AA to regulate mTOR signaling and to reveal its mechanism. PMID:27727170

  7. Cyclin Y Is Involved in the Regulation of Adipogenesis and Lipid Production.

    Directory of Open Access Journals (Sweden)

    Weiwei An

    Full Text Available A new member of the cyclin family cyclin Y (CCNY is involved in the regulation of various physiological processes. In this study, the role of CCNY in energy metabolism was characterized. We found that compared with wild-type (WT mice, Ccny knockout (KO mice had both lower body weight and lower fat content. The Ccny KO mice also had a higher metabolic rate, resisted the stress of a high-fat diet, and were sensitive to calorie restriction. The expression levels of UCP1 and PGC1α were significantly higher in the brown adipose tissue (BAT of the Ccny KO mice than that of the WT littermate controls, whereas there was no significant difference in BAT weight between the WT and the Ccny KO mice. In addition, the down-regulation of Ccny resulted in suppression of white adipocyte differentiation both in vivo and in vitro, while the expression of Ccny was up-regulated by C/EBPα. Furthermore, both hepatocytes and HepG2 cells that were depleted of Ccny were insensitive to insulin stimulation, consistent with the significant inhibition of insulin sensitivity in the liver of the Ccny KO mice, but no significant changes in WAT and muscle, indicating that CCNY is involved in regulating the hepatic insulin signaling pathway. The hepatic insulin resistance generated by Ccny depletion resulted in down-regulation of the sterol-regulatory element-binding protein (SREBP1 and fatty acid synthase (FASN. Together, these results provide a new link between CCNY and lipid metabolism in mice, and suggest that inhibition of CCNY may offer a therapeutic approach to obesity and diabetes.

  8. Zebrafish cyclin Dx is required for development of motor neuron progenitors, and its expression is regulated by hypoxia-inducible factor 2α.

    Science.gov (United States)

    Lien, Huang-Wei; Yuan, Rey-Yue; Chou, Chih-Ming; Chen, Yi-Chung; Hung, Chin-Chun; Hu, Chin-Hwa; Hwang, Sheng-Ping L; Hwang, Pung-Pung; Shen, Chia-Ning; Chen, Chih-Lung; Cheng, Chia-Hsiung; Huang, Chang-Jen

    2016-06-21

    Cyclins play a central role in cell-cycle regulation; in mammals, the D family of cyclins consists of cyclin D1, D2, and D3. In Xenopus, only homologs of cyclins D1 and D2 have been reported, while a novel cyclin, cyclin Dx (ccndx), was found to be required for the maintenance of motor neuron progenitors during embryogenesis. It remains unknown whether zebrafish possess cyclin D3 or cyclin Dx. In this study, we identified a zebrafish ccndx gene encoding a protein which can form a complex with Cdk4. Through whole-mount in situ hybridization, we observed that zccndx mRNA is expressed in the motor neurons of hindbrain and spinal cord during development. Analysis of a 4-kb promoter sequence of the zccndx gene revealed the presence of HRE sites, which can be regulated by HIF2α. Morpholino knockdown of zebrafish Hif2α and cyclin Dx resulted in the abolishment of isl1 and oligo2 expression in the precursors of motor neurons, and also disrupted axon growth. Overexpression of cyclin Dx mRNA in Hif2α morphants partially rescued zccndx expression. Taken together, our data indicate that zebrafish cyclin Dx plays a role in maintaining the precursors of motor neurons.

  9. Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells.

    Science.gov (United States)

    Wauson, Eric M; Guerra, Marcy L; Dyachok, Julia; McGlynn, Kathleen; Giles, Jennifer; Ross, Elliott M; Cobb, Melanie H

    2015-08-01

    The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic β-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic β-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that G(I) is not central to either pathway. Although glucagon-like peptide 1, an agonist for a G(s-)coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca(2+) entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective G(q) inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for G(q). Inhibition of G(12/13) by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways.

  10. Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number.

    Science.gov (United States)

    Sankaran, Vijay G; Ludwig, Leif S; Sicinska, Ewa; Xu, Jian; Bauer, Daniel E; Eng, Jennifer C; Patterson, Heide Christine; Metcalf, Ryan A; Natkunam, Yasodha; Orkin, Stuart H; Sicinski, Piotr; Lander, Eric S; Lodish, Harvey F

    2012-09-15

    Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components-a finding resulting from the biological follow-up of unbiased human genetic studies.

  11. Cdc6 and Cyclin E2 Are PTEN-Regulated Genes Associated with Human Prostate Cancer Metastasis

    Directory of Open Access Journals (Sweden)

    Zhong Wu

    2009-01-01

    Full Text Available Phosphatase and tensin homolog deleted on chromosome 10 (PTEN is frequently inactivated in metastatic prostate cancer, yet the molecular consequences of this and their association with the metastatic phenotype are incompletely understood. We performed transcriptomic analysis and identified genes altered by conditional PTEN reexpression in C4-2, a human metastatic prostate cancer cell line with inactive PTEN. PTEN-regulated genes were disproportionately represented among genes altered in human prostate cancer progression and metastasis but not among those associated with tumorigenesis. From the former set, we identified two novel putative PTEN targets, cdc6 and cyclin E2, which were overexpressed in metastatic human prostate cancer and up-regulated as a function of PTEN depletion in poorly metastatic DU145 human prostate cancer cells harboring a wild type PTEN. Inhibition of cdc6 and cyclin E2 levels as a consequence of PTEN expression was associated with cell cycle G1 arrest, whereas use of PTEN activity mutants revealed that regulation of these genes was dependent on PTEN lipid phosphatase activity. Computational and promoter-reporter evaluations implicated the E2F transcription factor in PTEN regulation of cdc6 and cyclin E2 expression. Our results suggest a hypothetical model whereby PTEN loss upregulates cell cycle genes such as cdc6 and cyclin E2 that in turn promote metastatic colonization at distant sites.

  12. Cyclin-dependent kinase 5 regulates PSD-95 ubiquitination in neurons.

    Science.gov (United States)

    Bianchetta, Michael J; Lam, TuKiet T; Jones, Stephen N; Morabito, Maria A

    2011-08-17

    Cyclin-dependent kinase 5 (Cdk5) and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer's, learning and memory, and synapse maturation and plasticity. However, the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the ubiquitin E3 ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a nonproteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiquitination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis.

  13. Regulation of μ and δ opioid receptor functions: involvement of cyclin-dependent kinase 5

    Science.gov (United States)

    Beaudry, H; Mercier-Blais, A-A; Delaygue, C; Lavoie, C; Parent, J-L; Neugebauer, W; Gendron, L

    2015-01-01

    Background and Purpose Phosphorylation of δ opioid receptors (DOP receptors) by cyclin-dependent kinase 5 (CDK5) was shown to regulate the trafficking of this receptor. Therefore, we aimed to determine the role of CDK5 in regulating DOP receptors in rats treated with morphine or with complete Freund's adjuvant (CFA). As μ (MOP) and DOP receptors are known to be co-regulated, we also sought to determine if CDK5-mediated regulation of DOP receptors also affects MOP receptor functions. Experimental Approach The role of CDK5 in regulating opioid receptors in CFA- and morphine-treated rats was studied using roscovitine as a CDK inhibitor and a cell-penetrant peptide mimicking the second intracellular loop of DOP receptors (C11-DOPri2). Opioid receptor functions were assessed in vivo in a series of behavioural experiments and correlated by measuring ERK1/2 activity in dorsal root ganglia homogenates. Key Results Chronic roscovitine treatment reduced the antinociceptive and antihyperalgesic effects of deltorphin II (Dlt II) in morphine- and CFA-treated rats respectively. Repeated administrations of C11-DOPri2 also robustly decreased Dlt II-induced analgesia. Interestingly, DAMGO-induced analgesia was significantly increased by roscovitine and C11-DOPri2. Concomitantly, in roscovitine-treated rats the Dlt II-induced ERK1/2 activation was decreased, whereas the DAMGO-induced ERK1/2 activation was increased. An acute roscovitine treatment had no effect on Dlt II- or DAMGO-induced analgesia. Conclusions and Implications Together, our results demonstrate that CDK5 is a key player in the regulation of DOP receptors in morphine- and CFA-treated rats and that the regulation of DOP receptors by CDK5 is sufficient to modulate MOP receptor functions through an indirect process. PMID:25598508

  14. CYCD3 D-type cyclins regulate cambial cell proliferation and secondary growth in Arabidopsis.

    Science.gov (United States)

    Collins, Carl; Maruthi, N M; Jahn, Courtney E

    2015-08-01

    A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-of-function approach was used here to reveal a rate-limiting role for the Arabidopsis CYCLIN D3 (CYCD3) subgroup of cell-cycle genes in the control of cambial cell proliferation and secondary growth, providing conclusive evidence of a direct link between the cell cycle and vascular development. It is shown that all three CYCD3 genes are specifically expressed in the cambium throughout vascular development. Analysis of a triple loss-of-function CYCD3 mutant revealed a requirement for CYCD3 in promoting the cambial cell cycle since mutant stems and hypocotyls showed a marked reduction in diameter linked to reduced mitotic activity in the cambium. Conversely, loss of CYCD3 provoked an increase in xylem cell size and the expression of differentiation markers, showing that CYCD3 is required to restrain the differentiation of xylem precursor cells. Together, our data show that tight control of cambial cell division through developmental- and cell type-specific regulation of CYCD3 is required for normal vascular development, constituting part of a novel mechanism controlling organ growth in higher plants.

  15. Regulation of the retinoblastoma protein-related p107 by G1 cyclin complexes

    NARCIS (Netherlands)

    Beijersbergen, R.L.; Carlée, L.; Kerkhoven, R.M.; Bernards, R.A.

    1995-01-01

    The orderly progression through the cell cycle is mediated by the sequential activation of several cyclin/cyclin-dependent kinase (cdk) complexes. These kinases phosphorylate a number of cellular substrates, among which is the product of the retinoblastoma gene, pRb. Phosphorylation of pRb in late G

  16. A Limited Role for the Cell Cycle Regulator Cyclin A1 in Murine Leukemogenesis.

    Directory of Open Access Journals (Sweden)

    Nicole Bäumer

    Full Text Available The quest for novel therapeutic targets in acute myeloid leukemia (AML is still ongoing. One of such targets, cyclin A1, was shown to be overexpressed in AML including AML stem cells. However, the function of cyclin A1 in AML is largely unknown, and the data on its impact on patients' survival remain controversial. Therefore, we developed a transgenic mouse model of stem cell-directed inducible cyclin A1 overexpression and crossed these mice with PML-RARα-knockin mice, which develop an AML M3-like phenotype. To observe the effects of cyclin A1 loss-of-function, we also crossed PML-RARα-knockin mice to cyclin A1-knockout mice. Neither overexpression nor loss of cyclin A1 significantly altered leukemogenesis in PML-RARα-knockin mice. These findings imply that upregulation of cyclin A1 is not essential for leukemogenesis. Our data suggest that cyclin A1 does not represent a suitable target for AML therapy.

  17. Differential Regulation of Cyclin E by Yorkie-Scalloped Signaling in Organ Development

    Directory of Open Access Journals (Sweden)

    Zhiqiang Shu

    2017-03-01

    Full Text Available Tissue integrity and homeostasis are accomplished through strict spatial and temporal regulation of cell growth and proliferation during development. Various signaling pathways have emerged as major growth regulators across metazoans; yet, how differential growth within a tissue is spatiotemporally coordinated remains largely unclear. Here, we report a role of a growth modulator Yorkie (Yki, the Drosophila homolog of Yes-associated protein (YAP, that differentially regulates its targets in Drosophila wing imaginal discs; whereby Yki interacts with its transcriptional partner, Scalloped (Sd, the homolog of the TEAD/TEF family transcription factor in mammals, to control an essential cell cycle regulator Cyclin E (CycE. Interestingly, when Yki was coexpressed with Fizzy-related (Fzr, a Drosophila endocycle inducer and homolog of Cdh1 in mammals, surrounding hinge cells displayed larger nuclear size than distal pouch cells. The observed size difference is attributable to differential regulation of CycE, a target of Yki and Sd, the latter of which can directly bind to CycE regulatory sequences, and is expressed only in the pouch region of the wing disc starting from the late second-instar larval stage. During earlier stages of larval development, when Sd expression was not detected in the wing disc, coexpression of Fzr and Yki did not cause size differences between cells along the proximal–distal axis of the disc. We show that ectopic CycE promoted cell proliferation and apoptosis, and inhibited transcriptional activity of Yki targets. These findings suggest that spatiotemporal expression of transcription factor Sd induces differential growth regulation by Yki during wing disc development, highlighting coordination between Yki and CycE to control growth and maintain homeostasis.

  18. The Rho GTPase Effector ROCK Regulates Cyclin A, Cyclin D1, and p27Kip1 Levels by Distinct Mechanisms

    OpenAIRE

    Croft, Daniel R; Olson, Michael F.

    2006-01-01

    The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. H...

  19. The ubiquitin ligase STUB1 regulates stability and activity of RUNX1 and RUNX1-RUNX1T1.

    Science.gov (United States)

    Yonezawa, Taishi; Takahashi, Hirotaka; Shikata, Shiori; Liu, Xiaoxiao; Tamura, Moe; Asada, Shuhei; Fukushima, Tsuyoshi; Fukuyama, Tomofusa; Tanaka, Yosuke; Sawasaki, Tatsuya; Kitamura, Toshio; Goyama, Susumu

    2017-07-28

    RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Chromosomal translocations involving the RUNX1 gene are associated with several types of leukemia, including acute myeloid leukemia driven by a leukemogenic fusion protein RUNX1-RUNX1T1. Previous studies have shown that RUNX1 is an unstable protein and is subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. However, the precise mechanisms of RUNX1 ubiquitination have not been fully understood. Furthermore, much less is known about the mechanisms to regulate the stability of RUNX1-RUNX1T1. In this study, we identified several RUNX1-interacting E3 ubiquitin ligases using a novel high-throughput binding assay. Among them, we found that STUB1 bound to RUNX1 and induced its ubiquitination and degradation mainly in the nucleus. Immunofluorescence analyses revealed that the STUB1-induced ubiquitination also promoted nuclear export of RUNX1, which probably contributes to the reduced transcriptional activity of RUNX1 in STUB1-overexpressing cells. STUB1 also induced ubiquitination of RUNX1-RUNX1T1 and down-regulated its expression. Importantly, STUB1 overexpression showed a substantial growth-inhibitory effect in myeloid leukemia cells that harbor RUNX1-RUNX1T1, whereas it showed only a marginal effect in other non-RUNX1-RUNX1T1 leukemia cells and normal human cord blood cells. Taken together, these data suggest that the E3 ubiquitin ligase STUB1 is a negative regulator of both RUNX1 and RUNX1-RUNX1T1. Activation of STUB1 could be a promising therapeutic strategy for RUNX1-RUNX1T1 leukemia. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Expression of cell cycle regulator p57kip2, cyclinE protein and proliferating cell nuclear antigen in human pancreatic cancer: An immunohistochemical study

    Institute of Scientific and Technical Information of China (English)

    Hui Yue; Hui-Yong Jiang

    2005-01-01

    AIM: To investigate the effects of p57kip2, cyclinE protein and proliferating cell nuclear antigen (PCNA) on occurrence and progression of human pancreatic cancer.METHODS: The expression of p57kip2, cyclinE protein and PCNA in tumor tissues and adjacent tissues from 32patients with pancreatic cancer was detected by SP immunohistochemical technique.RESULTS: The positive expression rate of p57kip2 protein in tumor tissues was 46.9%, which was lower than that in adjacent pancreatic tissues (x2 = 5.317, P<0.05). P57kip2protein positive expression remarkably correlated with tumor cell differentiation (P<0.05), but not with lymph node metastasis (P>0.05). The positive expression rate of cyclinE protein in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues (x2 = 4.063,P<0.05). CyclinE protein positive expression significantly correlated with tumor cell differentiation and lymph node metastasis (P<0.05). The positive expression rate of PCNA in the tumor tissues was 71.9%, which was higher than that in adjacent pancreatic tissues (x2 = 5.189, P<0.05).PCNA positive expression remarkably correlated with tumor cell differentiation and lymph node metastasis (P<0.05).CONCLUSION: The decreased expression of p57kip2 and/or overexpression of cyclinE protein and PCNA may contribute to the occurrence and progression of pancreatic cancer.p57kip2, cyclinE protein, and PCNA play an important role in occurrence and progression of pancreatic cancer.

  1. Regulation of a Myb transcription factor by cyclin-dependent kinase 2 in Giardia lamblia.

    Science.gov (United States)

    Cho, Chao-Cheng; Su, Li-Hsin; Huang, Yu-Chang; Pan, Yu-Jiao; Sun, Chin-Hung

    2012-02-03

    The protozoan Giardia lamblia parasitizes the human small intestine to cause diseases. It undergoes differentiation into infectious cysts by responding to intestinal stimulation. How the activated signal transduction pathways relate to encystation stimulation remain largely unknown. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately up-regulated by a Myb2 transcription factor. Because cell differentiation is linked to cell cycle regulation, we tried to understand the role of cell cycle regulators, cyclin-dependent kinases (Cdks), in encystation. We found that the recombinant Myb2 was phosphorylated by Cdk-associated complexes and the levels of phosphorylation increased significantly during encystation. We have identified a putative cdk gene (cdk2) by searching the Giardia genome database. Cdk2 was found to localize in the cytoplasm with higher expression during encystation. Interestingly, overexpression of Cdk2 resulted in a significant increase of the levels of cwp gene expression and cyst formation. In addition, the Cdk2-associated complexes can phosphorylate Myb2 and the levels of phosphorylation increased significantly during encystation. Mutations of important catalytic residues of Cdk2 resulted in a significant decrease of kinase activity and ability of inducing cyst formation. Addition of a Cdk inhibitor, purvalanol A, significantly decreased the Cdk2 kinase activity and the levels of cwp gene expression and cyst formation. Our results suggest that the Cdk2 pathway may be involved in phosphorylation of Myb2, leading to activation of the Myb2 function and up-regulation of cwp genes during encystation. The results provide insights into the use of Cdk inhibitory drugs in disruption of Giardia differentiation into cysts.

  2. The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

    Directory of Open Access Journals (Sweden)

    Laura Graf

    2013-12-01

    Full Text Available The human cytomegalovirus (HCMV-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.

  3. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Lan [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Wang, Yongsheng [Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2013-05-31

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

  4. Effects of Cyclin D1 Antisense Oligodeoxyneucleotides on the Growth and Expression of G1 Phase Regulators in Gastric Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    帅晓明; 韩高雄; 王国斌

    2003-01-01

    To investigate the effects of Cyclin D1 antisense oligodeoxyneucleotides (ASODN) on the growth, cell cycle progression and expression of G1 phase regulators in human gastric carcinoma cell lines SGC7901 and HS746T, phosphorothioate-modified Cyclin D1 ASODN were encapsulated by LipofectAMINE2000 and transfected into gastric carcinoma cells. Dose-dependent inhibitory effects were induced by Cyclin D1 ASODN in two gastric carcinoma cell lines. Treatment of gastric carcinoma cells with 0.2 μmol/L Cyclin D1 ASODN for 24 h could significantly inhibit their growth in vitro and in vivo, reduce expression of Cyclin DlmRNA to 26.3 % (SGC7901) and 17.3 %(HS746T) respectively. The percentage of cells in G0/G1 phase was increased as revealed by flow cytometry. Immunohistochemical staining showed that the expression of p21 was increased and the expression of Cyclin D1 and pRb was decreased in the two cell lines; the expression of p27 was increased in HS746T, but unchanged in SGC7901. Cyclin D1 ASODN could inhibit the growth and the expression of Cyclin D1 mRNA in gastric carcinoma cells, influence the cell cycle and expression of its regulators.

  5. Cyclin C regulates adipogenesis by stimulating transcriptional activity of CCAAT/enhancer binding protein alpha.

    Science.gov (United States)

    Song, Ziyi; Xiaoli, Alus M; Zhang, Quanwei; Zhang, Yi; Yang, Ellen S T; Wang, Sven; Chang, Rui; Zhang, Zhengdong D; Yang, Gongshe; Strich, Randy; Pessin, Jeffrey E; Yang, Fajun

    2017-03-28

    Brown adipose tissue (BAT) is important for maintaining energy homeostasis and adaptive thermogenesis in rodents and humans. As disorders arising from dysregulated energy metabolism, such as obesity and metabolic diseases, have increased, so has interest in the molecular mechanisms in adipocyte biology. Using a functional screen, we identified cyclin C (CycC), a conserved subunit of the Mediator complex, as a novel regulator for brown adipocyte formation. siRNA-mediated CycC knockdown (KD) in brown preadipocytes impaired the early transcriptional program of differentiation, and genetic knockout (KO) of CycC completely blocked the differentiation process. RNA-seq analyses of CycC-KD revealed a critical role of CycC in activating genes co-regulated by peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα). Overexpression of PPARγ2 or addition of the PPARγ ligand rosiglitazone rescued the defects in CycC-KO brown preadipocytes, and efficiently activated the PPARγ-responsive promoters in both wild-type (WT) and CycC-KO cells, suggesting that CycC is not essential for PPARγ transcriptional activity. In contrast, CycC-KO significantly reduced C/EBPα-dependent gene expression. Unlike for PPARγ, overexpression of C/EBPα could not induce C/EBPα target gene expression in CycC-KO cells or rescue the CycC-KO defects in brown adipogenesis, suggesting that CycC is essential for C/EBPα-mediated gene activation. CycC physically interacted with C/EBPα and this interaction was required for C/EBPα transactivation domain activity. Consistent with the role of C/EBPα in white adipogenesis, CycC-KD also inhibited differentiation of 3T3-L1 cells into white adipocytes. Together, these data indicate that CycC activates adipogenesis by stimulating the transcriptional activity of C/EBPα.

  6. Role of cyclin B1/Cdc2 up-regulation in the development of mitotic prometaphase arrest in human breast cancer cells treated with nocodazole.

    Directory of Open Access Journals (Sweden)

    Hye Joung Choi

    Full Text Available BACKGROUND: During a normal cell cycle, the transition from G₂ phase to mitotic phase is triggered by the activation of the cyclin B1-dependent Cdc2 kinase. Here we report our finding that treatment of MCF-7 human breast cancer cells with nocodazole, a prototypic microtubule inhibitor, results in strong up-regulation of cyclin B1 and Cdc2 levels, and their increases are required for the development of mitotic prometaphase arrest and characteristic phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: It was observed that there was a time-dependent early increase in cyclin B1 and Cdc2 protein levels (peaking between 12 and 24 h post treatment, and their levels started to decline after the initial increase. This early up-regulation of cyclin B1 and Cdc2 closely matched in timing the nocodazole-induced mitotic prometaphase arrest. Selective knockdown of cyclin B1or Cdc2 each abrogated nocodazole-induced accumulation of prometaphase cells. The nocodazole-induced prometaphase arrest was also abrogated by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or with cycloheximide, a protein synthesis inhibitor that was found to suppress cyclin B1 and Cdc2 up-regulation. In addition, we found that MAD2 knockdown abrogated nocodazole-induced accumulation of cyclin B1 and Cdc2 proteins, which was accompanied by an attenuation of nocodazole-induced prometaphase arrest. CONCLUSIONS/SIGNIFICANCE: These observations demonstrate that the strong early up-regulation of cyclin B1 and Cdc2 contributes critically to the rapid and selective accumulation of prometaphase-arrested cells, a phenomenon associated with exposure to microtubule inhibitors.

  7. Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

    Science.gov (United States)

    Ferrero, Macarena; Ferragud, Juan; Orlando, Leonardo; Valero, Luz; Sánchez del Pino, Manuel; Farràs, Rosa; Font de Mora, Jaime

    2011-01-01

    Background Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. Methodology/Principal Findings Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. Conclusions Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the

  8. Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene

    OpenAIRE

    El Messaoudi, Selma; Fabbrizio, Eric; Rodriguez, Carmen; Chuchana, Paul; Fauquier, Lucas; Cheng, Donghang; Theillet, Charles; Vandel, Laurence; Bedford, Mark T.; Sardet, Claude

    2006-01-01

    The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation/methylation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 an...

  9. Phosphorylation of SAMHD1 by Cyclin A2/CDK1 Regulates Its Restriction Activity toward HIV-1

    Directory of Open Access Journals (Sweden)

    Alexandra Cribier

    2013-04-01

    Full Text Available SAMHD1 restricts HIV-1 replication in myeloid and quiescent CD4+ T cells. Here, we show that SAMHD1 restriction activity is regulated by phosphorylation. SAMHD1 interacts with cyclin A2/cdk1 only in cycling cells. Cyclin A2/CDK1 phosphorylates SAMHD1 at the Threonine 592 residue both in vitro and in vivo. Phosphorylation of SAMHD1 Thr592 correlates with loss of its ability to restrict HIV-1. Indeed, while PMA treatment of proliferating THP1 cells results in reduced Thr592 phosphorylation, activation of resting peripheral blood mononuclear cells (PBMCs and purified quiescent CD4+ T cells results in increased phosphorylation of SAMHD1 Thr592. Interestingly, we found that treatment of cells by type 1 interferon reduced Thr592 phosphorylation, reinforcing the link between the phosphorylation of SAMHD1 and its antiviral activity. Unlike wild-type SAMHD1, a phosphorylation-defective mutant was able to restrict HIV-1 replication in both PMA-treated and untreated cells. Our results uncover the phosphorylation of SAMHD1 at Thr592 by cyclin A2/CDK1 as a key regulatory mechanism of its antiviral activity.

  10. Upregulation of ATF-3 is correlated with prognosis and proliferation of laryngeal cancer by regulating Cyclin D1 expression.

    Science.gov (United States)

    Feng, Jiapeng; Sun, Qingfeng; Wu, Tianyi; Lu, Jianguang; Qu, Lingmei; Sun, Yanan; Tian, Linli; Zhang, Binghui; Li, Dandan; Liu, Ming

    2013-01-01

    This study aimed to investigate the expression and significance of ATF-3 in laryngeal squamous cell carcinoma (LSCC). Expression of ATF-3 was examined using immunohistochemistry methods in samples from 83 cases of LSCC carcinoma. MTT assay was used to detect proliferation of Hep-2 cells after ATF-3 knocked down by siRNA lentivirus. A mouse model was used to investigate the inhibitive role of ATF-3 siRNA in LSCC xenografts. Realtime RCR was used to detect Cyclin D1 expression after ATF-3 downregulation in Hep-2 cells. The expression of ATF-3 was positively detected in all the 83 cases of LSCC cancer tissues while Only 4 cases of adjacent non-neoplastic tissues were detected with positive ATF-3 expression. The ATF-3 expression was statistically related with T stage, neck nodal metastasis, clinical stage and prognosis of LSCC. Both cell proliferation in vitro and tumor growth in vivo were suppressed after ATF-3 knockdown. Furthermore, the expression of Cyclin D1 was decreased after ATF-3 downregulation in Hep-2 cells. ATF-3 is involved in the progress of LSCC, and may provide clinical information for evaluation of prognosis of LSCC. The oncologic role of ATF-3 may be correlated with Cyclin D1 regulation.

  11. D-type Cyclins are important downstream effectors of cytokine signaling that regulate the proliferation of normal and neoplastic mammary epithelial cells.

    Science.gov (United States)

    Zhang, Qian; Sakamoto, Kazuhito; Wagner, Kay-Uwe

    2014-01-25

    In response to the ligand-mediated activation of cytokine receptors, cells decide whether to proliferate or to undergo differentiation. D-type Cyclins (Cyclin D1, D2, or D3) and their associated Cyclin-dependent kinases (CDK4, CDK6) connect signals from cytokines to the cell cycle machinery, and they propel cells through the G1 restriction point and into the S phase, after which growth factor stimulation is no longer essential to complete cell division. D-type Cyclins are upregulated in many human malignancies including breast cancer to promote an uncontrolled proliferation of cancer cells. After summarizing important aspects of the cytokine-mediated transcriptional regulation and the posttranslational modification of D-type Cyclins, this review will highlight the physiological significance of these cell cycle regulators during normal mammary gland development as well as the initiation and promotion of breast cancer. Although the vast majority of published reports focus almost exclusively on the role of Cyclin D1 in breast cancer, we summarize here previous and recent findings that demonstrate an important contribution of the remaining two members of this Cyclin family, in particular Cyclin D3, for the growth of ErbB2-associated breast cancer cells in humans and in mouse models. New data from genetically engineered models as well as the pharmacological inhibition of CDK4/6 suggest that targeting the combined functions of D-type Cyclins could be a suitable strategy for the treatment of ErbB2-positive and potentially other types of breast cancer.

  12. DNA-PKcs Negatively Regulates Cyclin B1 Protein Stability through Facilitating Its Ubiquitination Mediated by Cdh1-APC/C Pathway.

    Science.gov (United States)

    Shang, Zeng-Fu; Tan, Wei; Liu, Xiao-Dan; Yu, Lan; Li, Bing; Li, Ming; Song, Man; Wang, Yu; Xiao, Bei-Bei; Zhong, Cai-Gao; Guan, Hua; Zhou, Ping-Kun

    2015-01-01

    The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair. DNA-PKcs has also been shown recently functioning in mitotic regulation. Here, we report that DNA-PKcs negatively regulates the stability of Cyclin B1 protein through facilitating its ubiquitination mediated by Cdh1 / E 3 ubiquitin ligase APC/C pathway. Loss of DNA-PKcs causes abnormal accumulation of Cyclin B1 protein. Cyclin B1 degradation is delayed in DNA-PKcs-deficient cells as result of attenuated ubiquitination. The impact of DNA-PKcs on Cyclin B1 stability relies on its kinase activity. Our study further reveals that DNA-PKcs interacts with APC/C core component APC2 and its co-activator Cdh1. The destruction of Cdh1 is accelerated in the absence of DNA-PKcs. Moreover, overexpression of exogenous Cdh1 can reverse the increase of Cyclin B1 protein in DNA-PKcs-deficient cells. Thus, DNA-PKcs, in addition to its direct role in DNA damage repair, functions in mitotic progression at least partially through regulating the stability of Cyclin B1 protein.

  13. Hormonal regulation of dipeptide transporter (PepT1) in Caco-2 cells with normal and anoxia/reoxygenation management

    Institute of Scientific and Technical Information of China (English)

    Bing-Wei Sun; Xiao-Chen Zhao; Guang-Ji Wang; Ning Li; Jie-Shou Li

    2003-01-01

    AIM: To determine the regulation effects of recombinant human growth hormone (rhGH) on dipeptide transporter (PepT1) in Caco-2 cells with normal culture and anoxia/reoxygenation injury.METHODS: A human intestinal cell monolayer (Caco-2) was used as the in vitro model of human small intestine and cephalexin as the model substrate for dipeptide transporter (PepT1). Caco-2 cells grown on Transwell membrane filters were preincubated in the presence of rhGH in the culture medium for 4 d, serum was withdrawn from monolayers for 24 h before each experiment. The transport experiments of cephalexin across apical membromes were then conducted;Caco-2 cells grown on multiple well dishes (24 pore) with normal culture or anoxia/reoxygenation injury were preincubated with rhGH as above and uptake of cephalexin was then measured.RESULTS: The transport and uptake of cephelaxin across apical membranes of Caco-2 cells after preincubation with rhGH were significantly increased compared with controls (P=0.045, 0.0223). Also, addition of rhGH at physiological concentration (34 nM) to incubation medium greatly stimulates cephalexin uptake by anoxia/reoxygenation injuried Caco-2 cells (P=0.0116), while the biological functions of PepT1 in injured Caco-2 cells without rhGH were markedly downregulated. Northem blot analysis showed that the level of PepT1 mRNA of rhGH-treated injured Caco-2cells was greatly increased compared to controls.CONCLUSION: The present results of rhGH stimulating the uptake and transport of cephalexin indicated that rhGH greatly upregulates the physiological effects of dipeptide transporters of Caco-2 cells. The alteration in the gene expression may be a mechanism of regulation of PepT1. In addition, Caco-2 cells take up cephalexin by the Proton-dependent dipeptide transporters that closely resembles the transporters present in the intestine. Caco-2 cells represent an ideal cellular model for future studies of the dipeptide transporter.

  14. The Regulation of Human Cyclin E Protein Levels by the Ubiquitin Proteolytic Pathway

    Science.gov (United States)

    2000-09-01

    7-11 Paper published in Molecular Cell ................................................. 8-11 4 INTRODUCTION Cyclins have been...the CDK2 inhibitor p21. These data were published this year in Molecular Cell in February (see appended copy of paper). KEY RESEARCH ACCOMPLISHMENTS...during the second year of support appeared in the February issue of Molecular Cell . I am applying for a faculty position with the data reported here as

  15. Effects of metformin alone or in combination with compound C on proliferation and cyclinD1 expression in mouse breast cancer 4T1 cells%二甲双胍单用或联合compound C对小鼠4T1乳腺癌细胞增殖和细胞周期蛋白D1表达的影响

    Institute of Scientific and Technical Information of China (English)

    杜洪泉; 黄丽莉; 贾爱华; 蒋群龙; 张光珍; 白洁

    2014-01-01

    目的:研究二甲双胍及单磷酸腺苷活化蛋白激酶( AMPK)抑制剂( compound C )对体外培养的小鼠乳腺癌4T1细胞株增殖抑制及细胞周期蛋白D1(cyclin D1)表达的影响,进一步探索AMPK通路在二甲双胍抗肿瘤的可能作用。方法体外培养小鼠乳腺癌4 T1细胞株至对数生长期,分别以2.5、5、10、20、40 mmol/L二甲双胍及20μmol/L compound C 对其进行单一或10 mmol/L二甲双胍联合20μmol/L compound C干预24、48、72 h。应用CCK-8方法检测不同浓度的二甲双胍、20μmol/L compound C、20μmol/L compound C+10 mmol/L二甲双胍对该细胞株增殖的抑制情况;Western blot法检测各组细胞内cyclin D1的表达情况。多组比较采用单因素方差分析,两两比较采用q检验,治疗前后用重复测量数据资料的方差分析。结果 CCK-8法结果显示在体外细胞培养实验中2.5 mmol/L二甲双胍组作用于该细胞株24、48 h后分别和正常对照组比较,细胞存活率差异无统计学意义(24 h: F=2.87, P=0.129;48 h:F=1.63, P=0.290),作用72 h后,差异有统计学意义( F=6.40, P=0.000)。5、10、20、40 mmol/L二甲双胍组作用于该细胞株24、48、72 h后,该细胞株的增殖被显著抑制(均P=0.000),且抑制程度随着药物浓度的增大( F=332.89、363.54、352.11, P均=0.000)及作用时间的延长而逐渐增强, compound C单独处理组及10 mmol/L二甲双胍联合20μmol/L compound C处理细胞24、48、72 h后,细胞存活率与正常对照组相比较,差异无统计学意义( F=1.08,P=2.283; F=1.92, P=0.050)。 Western blot结果显示随着二甲双胍浓度的增加,各组细胞 cyclin D1表达水平逐渐降低( F=54.41、61.69、75.84, P均=0.000),10 mmol/L二甲双胍联合20μmol/L compound C 作用时,与正常对照组比较, cyclin D1表达水平无明显变化(F=1.87, P=0.190

  16. Id2 regulates the proliferation of squamous cell carcinoma in vitro via the NF-κB/Cyclin D1 pathway

    Institute of Scientific and Technical Information of China (English)

    Chuan Wang; Qiang Chen; Yuki Hamajima; Wei Sun; Yi-Qing Zheng; Xiao-Hua Hu; Frank G.Ondrey; Ji-Zhen Lin

    2012-01-01

    Squamous cell carcinoma (SCC) is a significant cause of cancer morbidity and mortality worldwide,with an incidence of up to 166 cases per 100 000 population.It arises in the skin,upper aerodigestive tract,lung,and cervix and affects more than 200 000 Americans each year.We report here that a microarray experiment comparing 41 SCC and 13 normal tissue specimens showed that Id2,a gene that controls the cell cycle,was significantly up-regulated in SCC.Enforced expression of Id2 in vitro stimulated the proliferation of SCC cells and up-regulated the transcription of nuclear factor kappa B (NF-κB) and cyclin D1.Enhancement of the NF-κB activity with p65 significantly increased the cell proliferation and the transcription of cyclin D1,whereas inhibition of the NF-κB activity with I kappa B alpha mutant (IκBα M) and pyrroline dithiocarbamate (PDTC) abrogated cell proliferation and transcription of cyclin D1.Furthermore,a mutated NF-κB binding site in the cyclin D1 promoter fully abrogated the Id2- induced transcription of cyclin D1.Taken together,these data indicate that Id2 induces SCC tumor growth and proliferation through the NF-κB/cyclin D1 pathway.

  17. The regulators of yeast PHO system participate in the transcriptional regulation of G1 cyclin under alkaline stress conditions.

    Science.gov (United States)

    Nishizawa, Masafumi

    2015-03-01

    The yeast Pho85 kinase oversees whether environmental conditions are favourable for cell growth and enables yeast cells to express only genes that are appropriate for the conditions. Alkaline stress perturbs transport of molecules across the plasma membrane that is vital for cell survival. Progression through the cell cycle is halted until the cells can adapt to the stress conditions. I found that Pho85 is required for CLN2 expression and that overproduction of the transcription factors Pho4, Rim101 and Crz1, all targets of Pho85, inhibited CLN2 expression. CLN2 expression in the absence of Pho85 could be recovered only when all the three transcription factors were deleted. Whi5, a functional homologue of the mammalian Rb protein, represses CLN2 expression and is inactivated when phosphorylated by either of the CDK-cyclin complexes, Cdc28-Cln3 or Pho85-Pcl9. Under alkaline conditions, the absence of Whi5 caused an increase in CLN2 expression but failed to do so when Pho85 was also absent, or when Pho4 was overproduced. The expression level of CLN2 in a Δpho85 Δpho4 Δrim101 Δcrz1 quadruple mutant was stimulated when the Whi5 activity was repressed by overproduction of Pho85-Pcl9. These results indicate that Whi5 is also under control of alkaline stress. The inhibitory function of Whi5 on CLN2 is dependent on Rpd3 HDAC, and the absence of Rpd3 could also suppress the inhibitory effect of Pho4 overproduction. Based on these findings, a model is presented in which Pho85 and Pho4 functions in CLN2 regulation under alkaline conditions.

  18. SWAP-70 controls formation of the splenic marginal zone through regulating T1B-cell differentiation.

    Science.gov (United States)

    Chopin, Michaël; Quemeneur, Laurence; Ripich, Tatsiana; Jessberger, Rolf

    2010-12-01

    T1 and T2 transitional B cells are precursors for marginal zone B cells (MZB), which surround splenic follicles. MZB are essential for marginal zone formation, are central to the innate immune response, and contribute to adaptive immunity. Differentiation, migration, and homing of MZB and their precursors remain to be fully understood. We show that SWAP-70, a RhoGTPase-interacting and F-actin-binding protein with functions in cell polarization, migration, and adhesion regulates MZB development and marginal zone formation. The percentage of MZB in spleen of Swap70(-/-) mice was reduced to about one-third of that found in WT mice. Swap70(-/-) T1 cells accumulated in integrin ligand(high) regions of the splenic red pulp and failed to efficiently develop into T2 cells. Adoptive transfer and mixed BM chimera experiments demonstrated this to be a B-cell intrinsic phenotype. T-cell-independent antibody production was not impaired, however, and thus suggests that this process does not require correct homing of MZB precursors. B-cell adhesion through α(L)β(2) and α(4)β(1) integrins was hyper-activated in vitro and on tissue sections, and S1P-stimulated chemokinesis of MZB was reduced in the absence of SWAP-70. Thus, SWAP-70 acts as a regulator of the adhesion process, particularly important for differentiation control of B-cell precursors and their contribution to splenic tissue formation.

  19. Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity.

    Directory of Open Access Journals (Sweden)

    Indra Tumurbaatar

    Full Text Available Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.

  20. Polycomb protein SCML2 regulates the cell cycle by binding and modulating CDK/CYCLIN/p21 complexes.

    Science.gov (United States)

    Lecona, Emilio; Rojas, Luis Alejandro; Bonasio, Roberto; Johnston, Andrew; Fernández-Capetillo, Oscar; Reinberg, Danny

    2013-12-01

    Polycomb group (PcG) proteins are transcriptional repressors of genes involved in development and differentiation, and also maintain repression of key genes involved in the cell cycle, indirectly regulating cell proliferation. The human SCML2 gene, a mammalian homologue of the Drosophila PcG protein SCM, encodes two protein isoforms: SCML2A that is bound to chromatin and SCML2B that is predominantly nucleoplasmic. Here, we purified SCML2B and found that it forms a stable complex with CDK/CYCLIN/p21 and p27, enhancing the inhibitory effect of p21/p27. SCML2B participates in the G1/S checkpoint by stabilizing p21 and favoring its interaction with CDK2/CYCE, resulting in decreased kinase activity and inhibited progression through G1. In turn, CDK/CYCLIN complexes phosphorylate SCML2, and the interaction of SCML2B with CDK2 is regulated through the cell cycle. These findings highlight a direct crosstalk between the Polycomb system of cellular memory and the cell-cycle machinery in mammals.

  1. Mxi1 inhibits the proliferation of U87 glioma cells through down-regulation of cyclin B1 gene expression

    Science.gov (United States)

    Manni, I; Tunici, P; Cirenei, N; Albarosa, R; Colombo, B M; Roz, L; Sacchi, A; Piaggio, G; Finocchiaro, G

    2002-01-01

    Mxi1 is a Mad family member that plays a role in cell proliferation and differentiation. To test the role of Mxi1 on tumorigenesis of glioma cells we transfected a CMV-driven MXI1 cDNA in U87 human glioblastoma cells. Two clones were isolated expressing MXI1 levels 18- and 3.5-fold higher than wild-type U87 cells (clone U87.Mxi1.14 and U87.Mxi1.22, respectively). In vivo, U87.Mxi1.14 cells were not tumorigenic in nude mice and delayed development of tumours was observed with U87.Mxi1.22 cells. In vitro, the proliferation rate was partially and strongly inhibited in U87.Mxi1.22 and U87.Mxi1.14 cells respectively. The cell cycle analysis revealed a relevant accumulation of U87.Mxi1.14 cells in the G2/M phase. Interestingly, the expression of cyclin B1 was inhibited to about 60% in U87.Mxi1.14 cells. This inhibition occurs at the transcriptional level and depends, at least in part, on the E-box present on the cyclin B1 promoter. Consistent with this, the endogenous Mxi1 binds this E-box in vitro. Thus, our findings indicate that Mxi1 can act as a tumour suppressor in human glioblastomas through a molecular mechanism involving the transcriptional down-regulation of cyclin B1 gene expression. British Journal of Cancer (2002) 86, 477–484. DOI: 10.1038/sj/bjc/6600065 www.bjcancer.com © 2002 The Cancer Research Campaign PMID:11875718

  2. Changes of biological functions of dipeptide transporter (PepT1)and hormonal regulation in severe scald rats

    Institute of Scientific and Technical Information of China (English)

    Bing-Wei Sun; Xiao-Chen Zhao; Guang-Ji Wang; Ning Li; Jie-Shou Li

    2003-01-01

    AIM: To determine the regulatory effects of recombinant human growth hormone (rhGH) on dipeptide transport (PepT1) in normal and severe scald rats.METHODS: Male Sprague-Dawley rats with 30 % total body surface area (TBSA) Ⅲ degree scald were employed as the model. In this study rhGH was used at the dose of 2 IU.kg-1d-1. An everted sleeve of intestine 4 cm long obtained from mid-jejunum was securely incubated in Kreb's solution with radioactive dipeptide (3H-glycylsarcosine, 3H-Gly-Sar,10 μCi/ml) at 37 ℃ for 15 min to measure the effects of uptake and transport of PepT1 of small intestinal epithelial cells in normal and severe scald rats.RESULTS: Abundant blood supply to intestine and mesentery was observed in normal and scald rats administered rhGH,while less supply of blood to intestine and mesentery was observed in rats without rhGH. Compared with controls, the transport of dipeptide in normal rats with injection of rhGH was not significantly increased (P=0.1926), while the uptake was significantly increased (P=0.0253). The effects of transport and uptake of PepT1 in scald rats with injection of rhGH were significantly increased (P=0.0082, 0.0391).CONCLUSION: Blood supply to intestine and mesentery of rats was increased following injection of rhGH. The effects of uptake and transport of dipeptide transporters in small intestinal epithelial cells of rats with severe scald were markedly up-regulated by rhGH.

  3. AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins.

    Science.gov (United States)

    Randall, Ricardo S; Miyashima, Shunsuke; Blomster, Tiina; Zhang, Jing; Elo, Annakaisa; Karlberg, Anna; Immanen, Juha; Nieminen, Kaisa; Lee, Ji-Young; Kakimoto, Tatsuo; Blajecka, Karolina; Melnyk, Charles W; Alcasabas, Annette; Forzani, Celine; Matsumoto-Kitano, Miho; Mähönen, Ari Pekka; Bhalerao, Rishikesh; Dewitte, Walter; Helariutta, Ykä; Murray, James A H

    2015-09-04

    Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism. © 2015. Published by The Company of Biologists Ltd.

  4. AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins

    Directory of Open Access Journals (Sweden)

    Ricardo S. Randall

    2015-10-01

    Full Text Available Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism.

  5. AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins

    Science.gov (United States)

    Randall, Ricardo S.; Miyashima, Shunsuke; Blomster, Tiina; Zhang, Jing; Elo, Annakaisa; Karlberg, Anna; Immanen, Juha; Nieminen, Kaisa; Lee, Ji-Young; Kakimoto, Tatsuo; Blajecka, Karolina; Melnyk, Charles W.; Alcasabas, Annette; Forzani, Celine; Matsumoto-Kitano, Miho; Mähönen, Ari Pekka; Bhalerao, Rishikesh; Dewitte, Walter; Helariutta, Ykä; Murray, James A. H.

    2015-01-01

    ABSTRACT Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism. PMID:26340943

  6. Cyclin D3 expression in non-Hodgkin lymphoma. Correlation with other cell cycle regulators and clinical features

    DEFF Research Database (Denmark)

    Møller, Michael Boe; Nielsen, O; Pedersen, Niels Tinggaard

    2001-01-01

    analyzed immunohistochemically for cyclin D3 expression. In 43 lymphomas (21.7%), cyclin D3 was overexpressed. T-cell lymphomas more frequently overexpressed cyclin D3 than B-cell lymphomas. Furthermore, cyclin D3-overexpressing indolent lymphomas were associated with higher proliferation rate, higher p21......Waf1 expression, lower p27Kip1 expression, and altered p53. Cyclin D3 overexpression identified a subgroup of patients with indolent B-cell lymphoma with adverse clinical features: patients were older, more frequently had "B" symptoms and extranodal involvement, and were more frequently in the high...

  7. p27Kip1 and p21Cip1 collaborate in the regulation of transcription by recruiting cyclin-Cdk complexes on the promoters of target genes.

    Science.gov (United States)

    Orlando, Serena; Gallastegui, Edurne; Besson, Arnaud; Abril, Gabriel; Aligué, Rosa; Pujol, Maria Jesus; Bachs, Oriol

    2015-08-18

    Transcriptional repressor complexes containing p130 and E2F4 regulate the expression of genes involved in DNA replication. During the G1 phase of the cell cycle, sequential phosphorylation of p130 by cyclin-dependent kinases (Cdks) disrupts these complexes allowing gene expression. The Cdk inhibitor and tumor suppressor p27(Kip1) associates with p130 and E2F4 by its carboxyl domain on the promoters of target genes but its role in the regulation of transcription remains unclear. We report here that p27(Kip1) recruits cyclin D2/D3-Cdk4 complexes on the promoters by its amino terminal domain in early and mid G1. In cells lacking p27(Kip1), cyclin D2/D3-Cdk4 did not associate to the promoters and phosphorylation of p130 and transcription of target genes was increased. In late G1, these complexes were substituted by p21(Cip1)-cyclin D1-Cdk2. In p21(Cip1) null cells cyclin D1-Cdk2 were not found on the promoters and transcription was elevated. In p21/p27 double null cells transcription was higher than in control cells and single knock out cells. Thus, our results clarify the role of p27(Kip1) and p21(Cip1) in transcriptional regulation of genes repressed by p130/E2F4 complexes in which p27(Kip1) and p21(Cip1) play a sequential role by recruiting and regulating the activity of specific cyclin-Cdk complexes on the promoters.

  8. Regulation of Wnt signaling activity for growth suppression induced by quercetin in 4T1 murine mammary cancer cells.

    Science.gov (United States)

    Kim, Haesung; Seo, Eun-Min; Sharma, Ashish R; Ganbold, Bilguun; Park, Jongbong; Sharma, Garima; Kang, Young-Hee; Song, Dong-Keun; Lee, Sang-Soo; Nam, Ju-Suk

    2013-10-01

    Quercetin is a promising chemopreventive agent against cancer that inhibits tumor progression by inducing cell cycle arrest and promoting apoptotic cell death. Recently, the Wnt/β-catenin signaling pathway has been implicated in mammary tumorigenesis, where its abnormal activation is associated with the development of breast cancer. Thus, the objective of this study was to examine the biological activities of quercetin against mammary cancer cells, and to determine whether quercetin could regulate the Wnt/β-catenin signaling pathway. Quercetin showed dose-dependent inhibition of cell growth and induced apoptosis in 4T1 cells. Treatment of 20 µM quercetin suppressed ~50% of basal TopFlash luciferase activity. Moreover, the inhibitory effect of quercetin on the Wnt/β-catenin signaling pathway was confirmed by the reduced stabilization of the β-catenin protein. Among various antagonists screened for the Wnt/β-catenin signaling pathway, the expression of DKK1, 2 and 3 was induced after treatment with 20 µM of quercetin. Stimulation with recombinant DKK1 protein, showed suppressive cell growth of mammary cancer cells instead of quercetin. When 4T1 cells were treated with recombinant Wnt3a or LiCl along with quercetin, both stimulators for the Wnt/β-catenin signaling pathway were able to restore the suppressed cell viability by quercetin. Thus, our data suggest that quercetin exerts its anticancer activity through the downregulation of Wnt/β-catenin signaling activity. These results indicate for the first time that quercetin decreases cell viability and induces apoptosis in murine mammary cancer cells, which is possibly mediated by DKK-dependent inhibition of the Wnt/β-catenin signaling pathway. In conclusion, our findings suggest that quercetin has great potential value as chemotherapeutic agent for cancer treatment, especially in breast cancer controlled by Wnt/β-catenin signaling activity.

  9. GRK2 negatively regulates IGF-1R signaling pathway and cyclins' expression in HepG2 cells.

    Science.gov (United States)

    Wei, Zhengyu; Hurtt, Reginald; Gu, Tina; Bodzin, Adam S; Koch, Walter J; Doria, Cataldo

    2013-09-01

    G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin-like growth factor 1 receptor (IGF-1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF-1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF-1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF-1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF-1R signaling pathway and cyclins' expression.

  10. Cyclin-dependent kinase inhibitor p21 controls adult neural stem cell expansion by regulating Sox2 gene expression.

    Science.gov (United States)

    Marqués-Torrejón, M Ángeles; Porlan, Eva; Banito, Ana; Gómez-Ibarlucea, Esther; Lopez-Contreras, Andrés J; Fernández-Capetillo, Oscar; Vidal, Anxo; Gil, Jesús; Torres, Josema; Fariñas, Isabel

    2013-01-01

    In the adult brain, continual neurogenesis of olfactory neurons is sustained by the existence of neural stem cells (NSCs) in the subependymal niche. Elimination of the cyclin-dependent kinase inhibitor 1A (p21) leads to premature exhaustion of the subependymal NSC pool, suggesting a relationship between cell cycle control and long-term self-renewal, but the molecular mechanisms underlying NSC maintenance by p21 remain unexplored. Here we identify a function of p21 in the direct regulation of the expression of pluripotency factor Sox2, a key regulator of the specification and maintenance of neural progenitors. We observe that p21 directly binds a Sox2 enhancer and negatively regulates Sox2 expression in NSCs. Augmented levels of Sox2 in p21 null cells induce replicative stress and a DNA damage response that leads to cell growth arrest mediated by increased levels of p19(Arf) and p53. Our results show a regulation of NSC expansion driven by a p21/Sox2/p53 axis.

  11. Metronomic Ceramide Analogs Inhibit Angiogenesis in Pancreatic Cancer through Up-regulation of Caveolin-1 and Thrombospondin-1 and Down-regulation of Cyclin D1

    Directory of Open Access Journals (Sweden)

    Guido Bocci

    2012-09-01

    Full Text Available AIMS: To evaluate the antitumor and antiangiogenic activity of metronomic ceramide analogs and their relevant molecular mechanisms. METHODS: Human endothelial cells [human dermal microvascular endothelial cells and human umbilical vascular endothelial cell (HUVEC] and pancreatic cancer cells (Capan-1 and MIA PaCa-2 were treated with the ceramide analogs (C2, AL6, C6, and C8, at low concentrations for 144 hours to evaluate any antiproliferative and proapoptotic effects and inhibition of migration and to measure the expression of caveolin-1 (CAV-1 and thrombospondin-1 (TSP-1 mRNAs by real-time reverse transcription-polymerase chain reaction. Assessment of extracellular signal-regulated kinases 1 and 2 (ERK1/2 and Akt phosphorylation and of CAV-1 and cyclin D1 protein expression was performed by ELISA. Maximum tolerated dose (MTD gemcitabine was compared against metronomic doses of the ceramide analogs by evaluating the inhibition of MIA PaCa-2 subcutaneous tumor growth in nude mice. RESULTS: Metronomic ceramide analogs preferentially inhibited cell proliferation and enhanced apoptosis in endothelial cells. Low concentrations of AL6 and C2 caused a significant inhibition of HUVEC migration. ERK1/2 and Akt phosphorylation were significantly decreased after metronomic ceramide analog treatment. Such treatment caused the overexpression of CAV-1 and TSP-1 mRNAs and proteins in endothelial cells, whereas cyclin D1 protein levels were reduced. The antiangiogenic and antitumor impact in vivo of metronomic C2 and AL6 regimens was similar to that caused by MTD gemcitabine. CONCLUSIONS: Metronomic C2 and AL6 analogs have antitumor and antiangiogenic activity, determining the up-regulation of CAV-1 and TSP-1 and the suppression of cyclin D1.

  12. ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells.

    Science.gov (United States)

    Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O

    2016-08-02

    ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.

  13. The Cln3 cyclin is down-regulated by translational repression and degradation during the G1 arrest caused by nitrogen deprivation in budding yeast.

    Science.gov (United States)

    Gallego, C; Garí, E; Colomina, N; Herrero, E; Aldea, M

    1997-12-01

    Nutrients are among the most important trophic factors in all organisms. When deprived of essential nutrients, yeast cells use accumulated reserves to complete the current cycle and arrest in the following G1 phase. We show here that the Cln3 cyclin, which has a key role in the timely activation of SBF (Swi4-Swi6)- and MBF (Mbp1-Swi6)-dependent promoters in late G1, is down-regulated rapidly at a post-transcriptional level in cells deprived of the nitrogen source. In addition to the fact that Cln3 is degraded faster by ubiquitin-dependent mechanisms, we have found that translation of the CLN3 mRNA is repressed approximately 8-fold under nitrogen deprivation conditions. As a consequence, both SBF- and MBF-dependent expression is strongly down-regulated. Mainly because of their transcriptional dependence on SBF, and perhaps with the contribution of similar post-transcriptional mechanisms to those found for Cln3, the G1 cyclins Cln1 and 2 become undetectable in starved cells. The complete loss of Cln cyclins and the sustained presence of the Clb-cyclin kinase inhibitor Sic1 in starved cells may provide the molecular basis for the G1 arrest caused by nitrogen deprivation.

  14. The RNA-binding protein Spo5 promotes meiosis II by regulating cyclin Cdc13 in fission yeast.

    Science.gov (United States)

    Arata, Mayumi; Sato, Masamitsu; Yamashita, Akira; Yamamoto, Masayuki

    2014-03-01

    Meiosis comprises two consecutive nuclear divisions, meiosis I and II. Despite this unique progression through the cell cycle, little is known about the mechanisms controlling the sequential divisions. In this study, we carried out a genetic screen to identify factors that regulate the initiation of meiosis II in the fission yeast Schizosaccharomyces pombe. We identified mutants deficient in meiosis II progression and repeatedly isolated mutants defective in spo5, which encodes an RNA-binding protein. Using fluorescence microscopy to visualize YFP-tagged protein, we found that spo5 mutant cells precociously lost Cdc13, the major B-type cyclin in fission yeast, before meiosis II. Importantly, the defect in meiosis II was rescued by increasing CDK activity. In wild-type cells, cdc13 transcripts increased during meiosis II, but this increase in cdc13 expression was weaker in spo5 mutants. Thus, Spo5 is a novel regulator of meiosis II that controls the level of cdc13 expression and promotes de novo synthesis of Cdc13. We previously reported that inhibition of Cdc13 degradation is necessary to initiate meiosis II; together with the previous information, the current findings indicate that the dual control of Cdc13 by de novo synthesis and suppression of proteolysis ensures the progression of meiosis II. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  15. Cotton GalT1 encoding a putative glycosyltransferase is involved in regulation of cell wall pectin biosynthesis during plant development.

    Science.gov (United States)

    Qin, Li-Xia; Rao, Yue; Li, Long; Huang, Jun-Feng; Xu, Wen-Liang; Li, Xue-Bao

    2013-01-01

    Arabinogalactan proteins (AGPs), are a group of highly glycosylated proteins that are found throughout the plant kingdom. To date, glycosyltransferases that glycosylate AGP backbone have remained largely unknown. In this study, a gene (GhGalT1) encoding a putative β-1,3-galactosyltransferase (GalT) was identified in cotton. GhGalT1, belonging to CAZy GT31 family, is the type II membrane protein that contains an N-terminal transmembrane domain and a C-terminal galactosyltransferase functional domain. A subcellular localization assay demonstrated that GhGalT1 was localized in the Golgi apparatus. RT-PCR analysis revealed that GhGalT1 was expressed at relatively high levels in hypocotyls, roots, fibers and ovules. Overexpression of GhGalT1 in Arabidopsis promoted plant growth and metabolism. The transgenic seedlings had much longer primary roots, higher chlorophyll content, higher photosynthetic efficiency, the increased biomass, and the enhanced tolerance to exogenous D-arabinose and D-galactose. In addition, gas chromatography (GC) analysis of monosaccharide composition of cell wall fractions showed that pectin was changed in the transgenic plants, compared with that of wild type. Three genes (GAUT8, GAUT9 and xgd1) involved in pectin biosynthesis were dramatically up-regulated in the transgenic lines. These data suggested that GhGalT1 may be involved in regulation of pectin biosynthesis required for plant development.

  16. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  17. CYCD3 D-type cyclins regulate cambial cell proliferation and secondary growth in Arabidopsis

    OpenAIRE

    Collins, Carl; Maruthi, M.N.; Courtney E. Jahn

    2015-01-01

    A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-o...

  18. DACH1 regulates cell cycle progression of myeloid cells through the control of cyclin D, Cdk 4/6 and p21{sup Cip1}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Woong; Kim, Hyeng-Soo; Kim, Seonggon; Hwang, Junmo; Kim, Young Hun; Lim, Ga Young [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Sohn, Wern-Joo [Department of Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu 700-412 (Korea, Republic of); Yoon, Suk-Ran [Cell Therapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Kim, Jae-Young [Department of Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu 700-412 (Korea, Republic of); Park, Tae Sung [Department of Laboratory Medicine, Kyung Hee University School of Medicine, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-702 (Korea, Republic of); Park, Kwon Moo [Department of Anatomy, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of); Ryoo, Zae Young [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Lee, Sanggyu, E-mail: slee@knu.ac.kr [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer DACH1 increases cyclin D, F and Cdk 1, 4, 6 in mouse myeloid progenitor cells. Black-Right-Pointing-Pointer The knockdown of DACH1 blocked the cell cycle progression of HL-60 cells. Black-Right-Pointing-Pointer The novel effect of DACH1 related with cell cycle regulation and leukemogenesis. -- Abstract: The cell-fate determination factor Dachshund, a component of the Retinal Determination Gene Network (RDGN), has a role in breast tumor proliferation through the repression of cyclin D1 and several key regulators of embryonic stem cell function, such as Nanog and Sox2. However, little is known about the role of DACH1 in a myeloid lineage as a cell cycle regulator. Here, we identified the differential expression levels of extensive cell cycle regulators controlled by DACH1 in myeloid progenitor cells. The forced expression of DACH1 induced p27{sup Kip1} and repressed p21{sup Cip1}, which is a pivotal characteristic of the myeloid progenitor. Furthermore, DACH1 significantly increased the expression of cyclin D1, D3, F, and Cdk 1, 4, and 6 in myeloid progenitor cells. The knockdown of DACH1 blocked the cell cycle progression of HL-60 promyeloblastic cells through the decrease of cyclin D1, D3, F, and Cdk 1, 4, and 6 and increase in p21{sup Cip1}, which in turn decreased the phosphorylation of the Rb protein. The expression of Sox2, Oct4, and Klf4 was significantly up-regulated by the forced expression of DACH1 in mouse myeloid progenitor cells.

  19. Transcription factor CgMTF-1 regulates CgZnT1 and CgMT expression in Pacific oyster (Crassostrea gigas) under zinc stress

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Jie; Zhang, Linlin [Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong (China); Li, Li, E-mail: lili@qdio.ac.cn [Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong (China); Li, Chunyan; Wang, Ting [Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong (China); University of Chinese Academy of Sciences, Beijing 100039 (China); Zhang, Guofan, E-mail: gfzhang@qdio.ac.cn [Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, Shandong (China)

    2015-08-15

    Highlights: • CgMTF-1 and CgZnT1 were first identified in oysters. • CgMTF-1 localized in cell nucleus under unstressed conditions. • CgMTF-1 proteins could bind with the typical MRE motif. • CgMTF-1 activated CgZnT1, CgMT1 and CgMT4 promoters and regulated their expressions under zinc exposure. - Abstract: Oysters accumulate zinc at high tissue concentrations, and the metal response element (MRE)-binding transcription factor (MTF) functions as the cellular zinc sensor that coordinates the expression of genes involved in zinc efflux and storage, as well as those that protect against metal toxicity. In this study, we cloned MTF-1 in oysters and examined its regulation mechanism for its classic target genes, including MTs and ZnT1 under zinc exposure conditions. We cloned CgMTF-1 and determined the subcellular locations of its protein product in HEK293 cells. CgMTF-1 has a 2826 bp open reading frame that encodes a predicted polypeptide with 707 amino acid residues, showing six well-conserved zinc finger domains that are required for metal binding. In HEK293 cell lines, CgMTF-1 primarily localizes in the cell nucleus under unstressed conditions and nuclear translocation was not critical for the activation of this gene. We searched for CgMTF-1-regulated genes in oysters using RNA interference. Decreased expression levels of CgMT1, CgMT4, and CgZnT1 were observed after CgMTF-1 interference (>70% inhibition) under zinc exposure, indicating the critical role of CgMTF-1 in the regulation of these genes. We searched for a direct regulation mechanism involving CgMTF-1 for CgMT1, CgMT4, and CgZnT1 in vitro. EMSA experiments indicated that CgMTF-1 can bind with the MREs found in the CgZnT1, CgMT1 and CgMT4 promoter regions. Additionally, luciferase reporter gene experiments indicated that CgMTF-1 could activate the CgMT1, CgMT4, and CgZnT1 promoters. Overall, our results suggest that CgMTF-1 directly coordinates the regulation of CgMTs and CgZnT1 expression and plays

  20. Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase

    DEFF Research Database (Denmark)

    Beck, Halfdan; Nähse, Viola; Larsen, Marie Sofie Yoo;

    2010-01-01

    are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC...

  1. Genome-wide association study follow-up identifies cyclin A2 as a regulator of the transition through cytokinesis during terminal erythropoiesis.

    Science.gov (United States)

    Ludwig, Leif S; Cho, Hyunjii; Wakabayashi, Aoi; Eng, Jennifer C; Ulirsch, Jacob C; Fleming, Mark D; Lodish, Harvey F; Sankaran, Vijay G

    2015-05-01

    Genome-wide association studies (GWAS) hold tremendous promise to improve our understanding of human biology. Recent GWAS have revealed over 75 loci associated with erythroid traits, including the 4q27 locus that is associated with red blood cell size (mean corpuscular volume). The close linkage disequilibrium block at this locus harbors the CCNA2 gene that encodes cyclin A2. CCNA2 mRNA is highly expressed in human and murine erythroid progenitor cells and regulated by the essential erythroid transcription factor GATA1. To understand the role of cyclin A2 in erythropoiesis, we have reduced expression of this gene using short hairpin RNAs in a primary murine erythroid culture system. We demonstrate that cyclin A2 levels affect erythroid cell size by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. Our study provides new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into hematopoiesis.

  2. Genome-wide association study follow-up identifies cyclin A2 as a regulator of the transition through cytokinesis during terminal erythropoiesis

    Science.gov (United States)

    Ludwig, Leif S.; Cho, Hyunjii; Wakabayashi, Aoi; Eng, Jennifer C.; Ulirsch, Jacob C.; Fleming, Mark D.; Lodish, Harvey F.; Sankaran, Vijay G.

    2015-01-01

    Genome-wide association studies (GWAS) hold tremendous promise to improve our understanding of human biology. Recent GWAS have revealed over 75 loci associated with erythroid traits, including the 4q27 locus that is associated with red blood cell size (mean corpuscular volume, MCV). The close linkage disequilibrium block at this locus harbors the CCNA2 gene that encodes cyclin A2. CCNA2 mRNA is highly expressed in human and murine erythroid progenitor cells and regulated by the essential erythroid transcription factor GATA1. To understand the role of cyclin A2 in erythropoiesis, we have reduced expression of this gene using short hairpin RNAs in a primary murine erythroid culture system. We demonstrate that cyclin A2 levels affect erythroid cell size by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. Our study provides new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into hematopoiesis. PMID:25615569

  3. BAFF induces spleen CD4{sup +} T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Fang; Chen, Rongjing [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Liu, Baojun [Laboratory of Lung, Inflammation and Cancers, Huashan Hospital, Fudan University, Shanghai (China); Zhang, Xiaoping [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Han, Junli; Wang, Haining [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Shen, Gang [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Tao, Jiang, E-mail: taojiang2012@yahoo.cn [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4{sup +} T cells. Black-Right-Pointing-Pointer Carrying out siRNA technology to study FOXO3A protein function. Black-Right-Pointing-Pointer Helpful to understand the T cell especially CD4{sup +} T cell's role in immunological reaction. -- Abstract: The TNF ligand family member 'B cell-activating factor belonging to the TNF family' (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4{sup +} spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4{sup +} T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4{sup +} spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4{sup +} T cell proliferation.

  4. Multifaceted interactions and regulation between antizyme and its interacting proteins cyclin D1, ornithine decarboxylase and antizyme inhibitor.

    Science.gov (United States)

    Liu, Yen-Chin; Lee, Chien-Yun; Lin, Chi-Li; Chen, Hui-Yi; Liu, Guang-Yaw; Hung, Hui-Chih

    2015-09-15

    Ornithine decarboxylase (ODC), cyclin D1 (CCND1) and antizyme inhibitor (AZI) promote cell growth. ODC and CCND1 can be degraded through antizyme (AZ)-mediated 26S proteasomal degradation. This paper describes a mechanistic study of the molecular interactions between AZ and its interacting proteins. The dissociation constant (Kd) of the binary AZ-CCND1 complex and the respective binding sites of AZ and CCND1 were determined. Our data indicate that CCND1 has a 4-fold lower binding affinity for AZ than does ODC and an approximately 40-fold lower binding affinity for AZ than does AZI. The Kd values of AZ-CCND1, AZ-ODC and AZ-AZI were 0.81, 0.21 and 0.02 μM, respectively. Furthermore, the Kd values for CCND1 binding to the AZ N-terminal peptide (AZ34-124) and AZ C-terminal peptide (AZ100-228) were 0.92 and 8.97 μM, respectively, indicating that the binding site of CCND1 may reside at the N-terminus of AZ, rather than the C-terminus. Our data also show that the ODC-AZ-CCND1 ternary complex may exist in equilibrium. The Kd values of the [AZ-CCND1]-ODC and [AZ-ODC]-CCND1 complexes were 1.26 and 4.93 μM, respectively. This is the first paper to report the reciprocal regulation of CCND1 and ODC through AZ-dependent 26S proteasomal degradation.

  5. SGK1-Sensitive Regulation of Cyclin-Dependent Kinase Inhibitor 1B (p27 in Cardiomyocyte Hypertrophy

    Directory of Open Access Journals (Sweden)

    Jakob Voelkl

    2015-09-01

    Full Text Available Background/Aims: The serum- and glucocorticoid-inducible kinase SGK1 participates in the orchestration of cardiac hypertrophy and remodeling. Signaling linking SGK1 activity to cardiac remodeling is, however, incompletely understood. SGK1 phosphorylation targets include cyclin-dependent kinase inhibitor 1B (p27, a protein which suppresses cardiac hypertrophy. The present study explored how effects of SGK1 on nuclear p27 localization might modulate the hypertrophic response in cardiomyocytes. Methods: Experiments were performed in HL-1 cardiomyocytes and in SGK1-deficient (sgk1-/- and corresponding wild-type (sgk1+/+ mice following pressure overload by transverse aortic constriction (TAC. Transcript levels were quantified by RT-PCR, protein abundance by Western blotting and protein localization by confocal microscopy. Results: In HL-1 cardiomyocytes, overexpression of constitutively active SGK1 (SGK1S422D but not of inactive SGK1 (SGK1K127N increased significantly the cell size and transcript levels encoding Acta1, a molecular marker of hypertrophy. Those effects were paralleled by almost complete relocation of p27 in the cytoplasm. Treatment of HL-1 cardiomyocytes with isoproterenol was followed by up-regulation of SGK1 expression. Moreover, isoproterenol treatment stimulated the hypertrophic response and was followed by disappearance of p27 from the nuclei, effects prevented by the SGK1 inhibitor EMD638683. The effect of SGK1S422D overexpression on Acta1 mRNA levels was disrupted by overexpression of p27 and of the p27T197A mutant lacking the SGK1 phosphorylation site, but not of the phosphomimetic p27T197D mutant. In sgk1+/+ mice, TAC increased significantly SGK1 and Acta1 mRNA levels and decreased the nuclear to cytoplasmic protein ratio of p27 in cardiac tissue, effects blunted in the sgk1-/- mice. Conclusion: SGK1-induced hypertrophy of cardiomyocytes involves p27 phosphorylation at T197, which fosters cytoplasmic p27 localization.

  6. AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets

    OpenAIRE

    Neveu, Gregory; Ziv-Av, Amotz; Barouch-Bentov, Rina; Berkerman, Elena; Mulholland, Jon; Einav, Shirit

    2015-01-01

    Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the μ subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved ant...

  7. Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, plays an important role in cell-to-cell communication during colitis.

    Directory of Open Access Journals (Sweden)

    Saravanan Ayyadurai

    Full Text Available PepT1 is a member of the proton-oligopeptide cotransporter family SLC15, which mediates the transport of di/tripeptides from intestinal lumen into epithelial cells. MicroRNAs (miRNAs, a small noncoding RNAs (21-23 nucleotides, post-transcriptionally regulate gene expression by binding to the 3'-untranslated regions (UTRs of their target mRNAs. Although the role of most miRNAs remains elusive, they have been implicated in vital cellular functions such as intestinal epithelial cells differentiation, proliferation, and apoptosis. In the present study, we investigated the effect of intestinal epithelial PepT1 expression on microRNA (miRNA expression/secretion in the colons of control mice and in mice with experimentally induced colonic inflammation (colitis. The colonic miRNA expression was deregulated in both colitis and control mice but the deregulation of miRNA expression/secretion was specific to colonic tissue and did not affect other tissues such as spleen and liver. Intestinal epithelial PepT1-dependent deregulation of colonic miRNA expression not only affects epithelial cells but also other cell types, such as intestinal macrophages. Importantly, we found the miRNA 23b which was known to be involved in inflammatory bowel disease was secreted and transported between cells to impose a gene-silencing effect on recipient intestinal macrophages. Based on our data, we may conclude that the expression of a specific protein, PepT1, in the intestine affects local miRNA expression/secretion in the colon on a tissue specific manner and may play an important role during the induction and progression of colitis. Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, could play a crucial role in cell-to-cell communication during colitis.

  8. AML1/RUNX1 Phosphorylation by Cyclin-Dependent Kinases Regulates the Degradation of AML1/RUNX1 by the Anaphase-Promoting Complex‡

    Science.gov (United States)

    Biggs, Joseph R.; Peterson, Luke F.; Zhang, Youhong; Kraft, Andrew S.; Zhang, Dong-Er

    2006-01-01

    AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Here, we report that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein which cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree. PMID:17015473

  9. Endothelin—1 promoted proliferation of vascular smooth muscle cell through pathway of extracellular signal—regulated kinase and cyclin D1

    Institute of Scientific and Technical Information of China (English)

    ZHANGYing-Min; WANGKe-Qiang; ZHOUGuo-Min; ZHOJi; GEJun-Bo

    2003-01-01

    AIM:To investigate whether endothelin-1(ET-1) can promote human umbilical artery smooth muscle artery smooth muscle cell (HUASMC) proliferation through pathway of extracellular signal-regulated kinase (ERK) and cyclin D1.METHODS: The effects of ET-1 and PD98059 on HUASMC were evaluated by MTT assay. The content of DNA was defined by [3H]TdR assay and cell cycle was analyzed by flow cytomerty. Western blot analysis was employed to detect the active phosphorylated state of ERK and the expression of cylin D1.RESULTS:Firstly, ET-1(100nmol/L) stimulated HUASMC proliferation compared with the group withou ET-1(P<0.05) and PD98059 group (P<0.05). PD98059 inhibited the HUASMC proliferation stimulated by ET-1(P<0.05). Secondly, ET-1 stimulated DNA synthesis of HUASMC compared with the group without ET-1(P<0.05). Thirdly, ET-1 promoted the cell cycle transition from G0/G1 phase to S phase. G0/G1 phase cell percentage was obviously decreased compared with the group without ET-1(P<0.05). S phase cell percentage was increased compared with the group without ET-1(P<0.05). Fourthly, ET-1 increased the phosphorylated level of ERK and the expression of cylin D1, an inhibitor of ERK blocked phosphorylated level of ERK and cyclin D1 expression. ERK phosphorylated level of ET-1 group was evidently increased compared with PD98059 group (P<0.05), Cyclin D1 protein expression also was increased compared with PD98059 group (P<0.05). While nonphosphorylated ERK expression remained unchanged. CONCLUSION:Endothelin-1 promoted vascular smooth muscle cell proliferation through pathway of ERK and cyclin D1.

  10. Matrine promotes G0/G1 arrest and down-regulates cyclin D1 expression in human rhabdomyosarcoma cells.

    Science.gov (United States)

    Guo, L; Xue, T Y; Xu, W; Gao, J Z

    2013-09-01

    Matrine has a broad-spectrum of anti-cancer effects and is efficient in the inhibition of proliferation of hepatoma cells, leukemia cells and neuroblastoma cell. However, its efficacy and tentative mechanisms in rhabdomyosarcoma have not been addressed before. This study aimed to investigate the effects of Matrine on cell cycle and expression of cyclin D1 in human rhabdomyosarcoma cells (RD cell line). RD cell line was treated with different concentrations (0, 0.5, 1.0, and 1.5 mg/mL) of Matrine, and cell proliferation and cell cycle were evaluated using, respectively, MTT assay and flow cytometry. The effect of Matrine on cyclin D1 mRNA levels was measured by RT-PCR. There was a dose-dependent inhibition of proliferation in the matrine-treated group (inhibition of proliferation rate in control cells 12.70 ± 0.35%; Matrine-treated cells [0.5, 1.0, and 1.5 mg/mL]: 31.16 ± 0.11%, 42.96 ± 0.9%, and 57.26 ± 0.8%). The G0 / G1 ratio in study groups were, respectively, 58.44 ± 3.57%, 64.79 ± 2.03%, 69.97 ± 2.89% and 75.03 ± 1.23%.Cyclin D1 mRNA levels progressively diminished (control group ratio of cyclin D1 / β-actin: 0.59 ± 0.06; Matrine: 0.35 ± 0.05, 0.27 ± 0.02 and 0.04 ± 0.03). All aforementioned changes were significant (PMatrine markedly suppresses cell proliferation in RD cells by decreasing expression of cyclin D1 mRNA and blocking the cell cycle at the G0 / G1 stage.

  11. Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells.

    Science.gov (United States)

    Zhang, Xi; Liu, Long; Chen, Chao; Chi, Ya-Li; Yang, Xiang-Qun; Xu, Yan; Li, Xiao-Tong; Guo, Shi-Lei; Xiong, Shao-Hu; Shen, Man-Ru; Sun, Yu; Zhang, Chuan-Sen; Hu, Kai-Meng

    2013-10-14

    The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined. The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H₂O₂, high glucose/U0126 or normal glucose/H₂O₂/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions. We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H₂O₂ stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor

  12. P120ctn overexpression enhances β-catenin-E-cadherin binding and down regulates expression of survivin and cyclin D1 in BEL-7404 hepatoma cells

    Institute of Scientific and Technical Information of China (English)

    Chao-Zan Nong; Li-Li Pan; Wei-Sheng He; Xi-Liang Zha; Hai-Hong Ye; Hua-Yi Huang

    2006-01-01

    AIM: To understand the role of P120ctn in E-cadherin-mediated cell-cell adhesion and signaling as well as in hepatoma cell biological function.METHODS: We stably overexpressed p120ctn isoform 3A in BEL-7404 human hepatoma cells and studied the effect of p120ctn on β-catenin and E-cadherin binding as well as p120ctn and β-catenin subcellular localization using immunoprecipitation, Western blotting and confocalmicroscopy. We also investigated the inhibitory effect of p120ctn transfection on the expression of apoptotic protein survivin survivin and cell cycle regulator cyclin D1in the cells.RERULTS: Western blotting indicated that p120ctn expression increased after cells were transfected with p120ctn isoform 3A. The protein was located mainly at membrane under immunofluorescent microscope.β-catenin nuclear expression was reduced after overexpression of p120ctn isoform 3A. The p120ctn-E-cadherin binding increased after transfection of p120ctn isoform 3A. Furthermore, overexpression of p120ctn down regulated the expression of apoptotic protein survivin and cell cycle regulator cyclin D1. These effects led to reduction of cell proliferation.CONCLUSION: Our results indicate that p120ctn plays an important role in regulating the formation of E-cadherin and -catenin complex, cell apoptosis, cell cycle and cancer cell biological function.

  13. Post-transcriptional regulation of cyclins D1, D3 and G1 and proliferation of human cancer cells depend on IMP-3 nuclear localization.

    Science.gov (United States)

    Rivera Vargas, T; Boudoukha, S; Simon, A; Souidi, M; Cuvellier, S; Pinna, G; Polesskaya, A

    2014-05-29

    RNA-binding proteins of the IMP family (insulin-like growth factor 2 (IGF2) mRNA-binding proteins 1-3) are important post-transcriptional regulators of gene expression. Multiple studies have linked high expression of IMP proteins, and especially of IMP-3, to an unfavorable prognosis in numerous types of cancer. The specific importance of IMP-3 for cancer transformation remains poorly understood. We here show that all three IMPs can directly bind the mRNAs of cyclins D1, D3 and G1 (CCND1, D3 and G1) in vivo and in vitro, and yet only IMP-3 regulates the expression of these cyclins in a significant manner in six human cancer cell lines of different origins. In the absence of IMP-3, the levels of CCND1, D3 and G1 proteins fall dramatically, and the cells accumulate in the G1 phase of the cell cycle, leading to almost complete proliferation arrest. Our results show that, compared with IMP-1 and IMP-2, IMP-3 is enriched in the nucleus, where it binds the transcripts of CCND1, D3 and G1. The nuclear localization of IMP-3 depends on its protein partner HNRNPM and is indispensable for the post-transcriptional regulation of expression of the cyclins. Cytoplasmic retention of IMP-3 and HNRNPM in human cancer cells leads to significant drop in proliferation. In conclusion, a nuclear IMP-3-HNRNPM complex is important for the efficient synthesis of CCND1, D3 and G1 and for the proliferation of human cancer cells.

  14. Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription

    Science.gov (United States)

    Murray, Thomas V.A.; Smyrnias, Ioannis; Schnelle, Moritz; Mistry, Rajesh K.; Zhang, Min; Beretta, Matteo; Martin, Daniel; Anilkumar, Narayana; de Silva, Shana M.; Shah, Ajay M.; Brewer, Alison C.

    2015-01-01

    Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1–3 week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2′-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell

  15. CARMA3 is overexpressed in colon cancer and regulates NF-{kappa}B activity and cyclin D1 expression

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Zhifeng; Zhao, Tingting; Wang, Zhenning; Xu, Yingying; Song, Yongxi; Wu, Jianhua [Department of General Surgery, First Affiliated Hospital of China Medical University, Shenyang (China); Xu, Huimian, E-mail: xuhuimianpaper@yahoo.com.cn [Department of General Surgery, First Affiliated Hospital of China Medical University, Shenyang (China)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer CARMA3 expression is elevated in colon cancers. Black-Right-Pointing-Pointer CARMA3 promotes proliferation and cell cycle progression in colon cancer cells. Black-Right-Pointing-Pointer CARMA3 upregulates cyclinD1 through NF-{kappa}B activation. -- Abstract: CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression and TNM stage (p = 0.0383), lymph node metastasis (p = 0.0091) and Ki67 proliferation index (p = 0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-I{kappa}B levels and NF-{kappa}B activity and its overexpression increased p-I{kappa}B expression and NF-{kappa}B activity. NF-{kappa}B inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-{kappa}B mediated upregulation of cyclin D1.

  16. Mechanisms of MEOX1 and MEOX2 regulation of the cyclin dependent kinase inhibitors p21 and p16 in vascular endothelial cells.

    Science.gov (United States)

    Douville, Josette M; Cheung, David Y C; Herbert, Krista L; Moffatt, Teri; Wigle, Jeffrey T

    2011-01-01

    Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21(CIP1/WAF1) and p16(INK4a) govern the G(1)/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21(CIP1/WAF1) and p16(INK4a). MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21(CIP1/WAF1) and p16(INK4a) expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21(CIP1/WAF1) and p16(INK4a). In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16(INK4a) in a DNA binding dependent manner, whereas they induce p21(CIP1/WAF1) in a DNA binding independent manner.

  17. Mechanisms of MEOX1 and MEOX2 regulation of the cyclin dependent kinase inhibitors p21 and p16 in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Josette M Douville

    Full Text Available Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21(CIP1/WAF1 and p16(INK4a govern the G(1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21(CIP1/WAF1 and p16(INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21(CIP1/WAF1 and p16(INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21(CIP1/WAF1 and p16(INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16(INK4a in a DNA binding dependent manner, whereas they induce p21(CIP1/WAF1 in a DNA binding independent manner.

  18. The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

    Directory of Open Access Journals (Sweden)

    Nicole Dölker

    2014-10-01

    Full Text Available Regulation of the c-Abl (ABL1 tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL. Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2 domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

  19. The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

    Science.gov (United States)

    Dölker, Nicole; Górna, Maria W; Sutto, Ludovico; Torralba, Antonio S; Superti-Furga, Giulio; Gervasio, Francesco L

    2014-10-01

    Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

  20. Human Papillomavirus Type 16 Mutant E7 Protein Induces Oncogenic Transformation via Up-regulation of Cyclin A and cdc25A

    Institute of Scientific and Technical Information of China (English)

    Jin-hua LIU; Yu-liang ZHANG; Li-qin ZHU; Yin-yu XU; Min ZHAO; Xin-xing WU

    2008-01-01

    A new mutant human papiUomavirus type 16 E7 gene, termed HPV16 HBE7, was isolated from cervical carcinoma biopsy samples from patients in an area with high incidence of cervical cancer (Hubei province, China). A previous study showed that the HPVI6 HBE7 protein was primarily cytoplasmic while wild-type HPV16 E7 protein, termed HPV16 WET, was concentrated in the nucleus. With the aim of studying the biological functions of HPV16 HBE7, the transforming potential of HPV16 HBE7 in NIH/3T3 cells was detected through observation of cell morphology, cell proliferation assay and anchorage-independent growth assay. The effect of HPVI6 HBE7 on cell cycle was examined by flow cytometry. Dual-luciferase reporter assay and RT-PCR were used to investigate the influence of HPVI6 HBE7 protein on the expression of regulation factors associated with GI/S checkpoint. The results showed that HPV16 HBE7 protein, as well as HPV16 WE7 protein, held transformation activity. NIH/3T3 cells expressing HPV16 HBE7 could easily transition from G1 phase into S phase and expressed high level of cyclin A and cdc25A. These results indicated HPV16 mutant E7 protein, located in the cytoplasm, induces oncogenic transformation of NIH/3T3 cells via up-regulation of cyclin A and cdc25A.

  1. The periodic down regulation of Cyclin E gene expression from exit of mitosis to end of G(1) is controlled by a deacetylase- and E2F-associated bipartite repressor element.

    Science.gov (United States)

    Polanowska, J; Fabbrizio, E; Le Cam, L; Trouche, D; Emiliani, S; Herrera, R; Sardet, C

    2001-07-12

    The expression of cyclin E and that of a few other bona fide cell cycle regulatory genes periodically oscillates every cycle in proliferating cells. Although numerous experiments have documented the role of E2F sites and E2F activities in the control of these genes as cells exit from G(0) to move through the initial G(1)/S phase transition, almost nothing is known on the role of E2Fs during the subsequent cell cycles. Here we show that a variant E2F-site that is part of the Cyclin E Repressor Module (CERM) (Le Cam et al., 1999b) accounts for the periodic down regulation of the cyclin E promoter observed between the exit from mitosis until the mid/late G(1) phase in exponentially cycling cells. This cell cycle-dependent repression correlates with the periodic binding of an atypical G(1)-specific high molecular weight p107-E2F complex (Cyclin E Repressor Complex: CERC2) that differs in both size and DNA binding behaviors from known p107-E2F complexes. Notably, affinity purified CERC2 displays a TSA-sensitive histone deacetylase activity and, consistent with this, derepression of the cyclin E promoter by trichostatin A depends on the CERM element. Altogether, this shows that the cell cycle-dependent control of cyclin E promoter in cycling cells is embroiled in acetylation pathways via the CERM-like E2F element.

  2. T1R3 homomeric sweet taste receptor regulates adipogenesis through Gαs-mediated microtubules disassembly and Rho activation in 3T3-L1 cells.

    Science.gov (United States)

    Masubuchi, Yosuke; Nakagawa, Yuko; Medina, Johan; Nagasawa, Masahiro; Kojima, Itaru; Rasenick, Mark M; Inagaki, Takeshi; Shibata, Hiroshi

    2017-01-01

    We previously reported that 3T3-L1 cells express a functional sweet taste receptor possibly as a T1R3 homomer that is coupled to Gs and negatively regulates adipogenesis by a Gαs-mediated but cAMP-independent mechanism. Here, we show that stimulation of this receptor with sucralose or saccharin induced disassembly of the microtubules in 3T3-L1 preadipocytes, which was attenuated by overexpression of the dominant-negative mutant of Gαs (Gαs-G226A). In contrast, overexpression of the constitutively active mutant of Gαs (Gαs-Q227L) as well as treatment with cholera toxin or isoproterenol but not with forskolin caused disassembly of the microtubules. Sweetener-induced microtubule disassembly was accompanied by activation of RhoA and Rho-associated kinase (ROCK). This was attenuated with by knockdown of GEF-H1, a microtubule-localized guanine nucleotide exchange factor for Rho GTPase. Furthermore, overexpression of the dominant-negative mutant of RhoA (RhoA-T19N) blocked sweetener-induced dephosphorylation of Akt and repression of PPARγ and C/EBPα in the early phase of adipogenic differentiation. These results suggest that the T1R3 homomeric sweet taste receptor negatively regulates adipogenesis through Gαs-mediated microtubule disassembly and consequent activation of the Rho/ROCK pathway.

  3. Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

    Directory of Open Access Journals (Sweden)

    Schwarzenbach Heidi

    2010-06-01

    Full Text Available Abstract Background The aim of the current study was to analyze the involvement of methyl-CpG binding proteins (MBDs and histone modifications on the regulation of CD44, Cyclin D2, GLIPR1 and PTEN in different cellular contexts such as the prostate cancer cells DU145 and LNCaP, and the breast cancer cells MCF-7. Since global chromatin changes have been shown to occur in tumours and regions of tumour-associated genes are affected by epigenetic modifications, these may constitute important regulatory mechanisms for the pathogenesis of malignant transformation. Methods In DU145, LNCaP and MCF-7 cells mRNA expression levels of CD44, Cyclin D2, GLIPR1 and PTEN were determined by quantitative RT-PCR at the basal status as well as after treatment with demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor Trichostatin A. Furthermore, genomic DNA was bisulfite-converted and sequenced. Chromatin immunoprecipitation was performed with the stimulated and unstimulated cells using antibodies for MBD1, MBD2 and MeCP2 as well as 17 different histone antibodies. Results Comparison of the different promoters showed that MeCP2 and MBD2a repressed promoter-specifically Cyclin D2 in all cell lines, whereas in MCF-7 cells MeCP2 repressed cell-specifically all methylated promoters. Chromatin immunoprecipitation showed that all methylated promoters associated with at least one MBD. Treatment of the cells by the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR caused dissociation of the MBDs from the promoters. Only MBD1v1 bound and repressed methylation-independently all promoters. Real-time amplification of DNA immunoprecipitated by 17 different antibodies showed a preferential enrichment for methylated lysine of histone H3 (H3K4me1, H3K4me2 and H3K4me3 at the particular promoters. Notably, the silent promoters were associated with unmodified histones which were acetylated following treatment by 5-aza-CdR. Conclusions This study is one

  4. Regulation of the Candida albicans Hypha-Inducing Transcription Factor Ume6 by the CDK1 Cyclins Cln3 and Hgc1

    Science.gov (United States)

    Mendelsohn, Sigal; Pinsky, Mariel; Weissman, Ziva

    2017-01-01

    ABSTRACT The ability to switch between proliferation as yeast cells and development into hyphae is a hallmark of Candida albicans. The switch to hyphal morphogenesis depends on external inducing conditions, but its efficiency is augmented in stationary-phase cells. Ume6, a transcription factor that is itself transcriptionally induced under hypha-promoting conditions, is both necessary and sufficient for hyphal morphogenesis. We found that Ume6 is regulated posttranslationally by the cell cycle kinase Cdc28/Cdk1, which reduces Ume6 activity via different mechanisms using different cyclins. Together with the cyclin Hgc1, Cdk1 promotes degradation of Ume6 via the SCFCDC4 ubiquitin ligase. Since HGC1 is a key transcriptional target of Ume6, this results in a negative-feedback loop between Hgc1 and Ume6. In addition, we found that Cln3, a G1 cyclin that is essential for cell cycle progression and yeast proliferation, suppresses hyphal morphogenesis and that Cln3 suppresses Ume6 activity both in the heterologous Saccharomyces cerevisiae system and in C. albicans itself. This activity of Cln3 may provide the basis for the antagonistic relationship between yeast proliferation and hyphal development in C. albicans. IMPORTANCE The yeast to hypha (mold) morphogenetic switch of Candida albicans plays a role in its virulence and constitutes a diagnostic trait for this organism, the most prevalent systemic fungal pathogen in industrialized countries. It has long been known that hyphae are most efficiently induced from stationary cultures. Here, a molecular basis for this observation is provided. The G1 cyclin Cln3, an essential promoter of yeast proliferation, was found to suppress hyphal induction. Suppression of hyphal induction is achieved by inhibition of the activity of the central activator of hyphal morphogenesis, the transcription factor Ume6. Thus, levels of Cln3 control the switch between proliferation of C. albicans as individual yeast cells and development into

  5. Regulation of proliferation in developing human tooth germs by MSX muscle segment homeodomain proteins and cyclin-dependent kinase inhibitor p19(INK4d).

    Science.gov (United States)

    Kero, Darko; Vukojevic, Katarina; Stazic, Petra; Sundov, Danijela; Brakus, Snjezana Mardesic; Saraga-Babic, Mirna

    2017-09-21

    Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19(INK4d). p19(INK4d) induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19(INK4d) by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19(INK4d) in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19(INK4d) throughout the investigated period indicates that p19(INK4d) plays active role during human tooth development. Furthermore, comparison of expression domains of p19(INK4d) with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.

  6. A Cyclin Dependent Kinase Regulatory Subunit (CKS) Gene of Pigeonpea Imparts Abiotic Stress Tolerance and Regulates Plant Growth and Development in Arabidopsis.

    Science.gov (United States)

    Tamirisa, Srinath; Vudem, Dashavantha R; Khareedu, Venkateswara R

    2017-01-01

    Frequent climatic changes in conjunction with other extreme environmental factors are known to affect growth, development and productivity of diverse crop plants. Pigeonpea, a major grain legume of the semiarid tropics, endowed with an excellent deep-root system, is known as one of the important drought tolerant crop plants. Cyclin dependent kinases (CDKs) are core cell cycle regulators and play important role in different aspects of plant growth and development. The cyclin-dependent kinase regulatory subunit gene (CKS) was isolated from the cDNA library of pigeonpea plants subjected to drought stress. Pigeonpea CKS (CcCKS) gene expression was detected in both the root and leaf tissues of pigeonpea and was upregulated by polyethylene glycol (PEG), mannitol, NaCl and abscisic acid (ABA) treatments. The overexpression of CcCKS gene in Arabidopsis significantly enhanced tolerance of transgenics to drought and salt stresses as evidenced by different physiological parameters. Under stress conditions, transgenics showed higher biomass, decreased rate of water loss, decreased MDA levels, higher free proline contents, and glutathione levels. Moreover, under stress conditions transgenics exhibited lower stomatal conductance, lower transpiration, and higher photosynthetic rates. However, under normal conditions, CcCKS-transgenics displayed decreased plant growth rate, increased cell size and decreased stomatal number compared to those of wild-type plants. Real-time polymerase chain reaction revealed that CcCKS could regulate the expression of both ABA-dependent and ABA-independent genes associated with abiotic stress tolerance as well as plant growth and development. As such, the CcCKS seems promising and might serve as a potential candidate gene for enhancing the abiotic stress tolerance of crop plants.

  7. Rescue of cyclin D1 deficiency by knockin cyclin E

    NARCIS (Netherlands)

    Geng, Y.; Whoriskey, W.; Park, M.Y.; Bronson, R.T.; Medema, R.H.; Li, T.; Weinberg, R.A.; Sicinski, P.

    1999-01-01

    D-type cyclins and cyclin E represent two very distinct classes of mammalian G1 cyclins. We have generated a mouse strain in which the coding sequences of the cyclin D1 gene (Ccnd1) have been deleted and replaced by those of human cyclin E (CCNE). In the tissues and cells of these mice, the expressi

  8. Timing of cyclin E gene expression depends on the regulated association of a bipartite repressor element with a novel E2F complex.

    OpenAIRE

    Le Cam, L; Polanowska, J.; Fabbrizio, E; Olivier, M.; Philips, A.; Ng Eaton, E; Classon, M; Geng, Y; Sardet, C.

    1999-01-01

    Transient induction of the cyclin E gene in late G1 gates progression into S. We show that this event is controlled via a cyclin E repressor module (CERM), a novel bipartite repressor element located near the cyclin E transcription start site. CERM consists of a variant E2F-binding site and a contiguous upstream AT-rich sequence which cooperate during G0/G1 to delay cyclin E expression until late G1. CERM binds the protein complex CERC, which disappears upon progression through G0-G1 and reap...

  9. Hepatitis C virus non-structural 5B protein interacts with cyclin A2 and regulates viral propagation

    DEFF Research Database (Denmark)

    Pham, Long; Ngo, HT; Lim, YS

    2012-01-01

    Background & Aims Hepatitis C virus (HCV) requires host cellular proteins for its own propagation. To identify the cellular factors necessary for HCV propagation, we have recently screened the small interfering RNA (siRNA) library targeting cell cycle genes using cell culture grown HCV (HCVcc......, in vitro and in vivo protein binding assays, luciferase reporter gene assay, and immunoblot assay. Results We showed that siRNA-mediated depletion of CycA2 significantly inhibited HCV replication in both HCV subgenomic replicon cells and HCVcc-infected cells. Furthermore, HCV non-structural 5B (NS5B......) specifically interacted with CycA2 in vitro and in vivo. Protein interaction was mediated through the cyclin box of CycA2 and the palm domain of NS5B. We further showed that R/HxL motif in the palm domain of HCV NS5B mediated protein interaction with CycA2 and this interaction was necessary for HCV replication...

  10. Cyclin-Dependent Kinase 5 Regulates Dendritic Spine Formation and Maintenance of Cortical Neuron in the Mouse Brain.

    Science.gov (United States)

    Mita, Naoki; He, Xiaojuan; Sasamoto, Kodai; Mishiba, Tomohide; Ohshima, Toshio

    2016-03-01

    Cyclin-dependent kinase 5 (Cdk5) activity is dependent on its association with 1 of 2 neuron-specific activators, p35 or p39. Cdk5 and its activators play an important role in brain development as well as higher functions like synaptic plasticity, learning, and memory. Reduction in p35 was reported in postmortem schizophrenia brain, in which reduced dendritic spine density was observed. Previous in vitro experiments have shown that Cdk5 is involved in dendritic spine formation, although in vivo evidence is limited. We examined dendritic spine formation in inducible-p35 conditional knockout (p35 cKO); p39 KO mice. When we deleted the p35 gene either during early postnatal days or at adult stage, we observed reduced spine densities of layer V neurons in the cerebral cortex and CA1 pyramidal neurons in the hippocampus. We further generated CA1-specific p35 conditional knockout (CA1-p35 cKO) mice and also CA1-p35 cKO; p39 KO mice in which have specific deletion of p35 in the CA1 region of hippocampus. We found a greater reduction in spine densities in CA1 pyramidal neurons in CA1-p35 cKO; p39 KO mice than in CA1-p35 cKO mice. These results indicate that dendritic spine formation and neuronal maintenance are dependent on Cdk5 activity.

  11. Cellular inhibitor of apoptosis protein-1 (cIAP1) can regulate E2F1 transcription factor-mediated control of cyclin transcription.

    Science.gov (United States)

    Cartier, Jessy; Berthelet, Jean; Marivin, Arthur; Gemble, Simon; Edmond, Valérie; Plenchette, Stéphanie; Lagrange, Brice; Hammann, Arlette; Dupoux, Alban; Delva, Laurent; Eymin, Béatrice; Solary, Eric; Dubrez, Laurence

    2011-07-29

    The inhibitor of apoptosis protein cIAP1 (cellular inhibitor of apoptosis protein-1) is a potent regulator of the tumor necrosis factor (TNF) receptor family and NF-κB signaling pathways in the cytoplasm. However, in some primary cells and tumor cell lines, cIAP1 is expressed in the nucleus, and its nuclear function remains poorly understood. Here, we show that the N-terminal part of cIAP1 directly interacts with the DNA binding domain of the E2F1 transcription factor. cIAP1 dramatically increases the transcriptional activity of E2F1 on synthetic and CCNE promoters. This function is not conserved for cIAP2 and XIAP, which are cytoplasmic proteins. Chromatin immunoprecipitation experiments demonstrate that cIAP1 is recruited on E2F binding sites of the CCNE and CCNA promoters in a cell cycle- and differentiation-dependent manner. cIAP1 silencing inhibits E2F1 DNA binding and E2F1-mediated transcriptional activation of the CCNE gene. In cells that express a nuclear cIAP1 such as HeLa, THP1 cells and primary human mammary epithelial cells, down-regulation of cIAP1 inhibits cyclin E and A expression and cell proliferation. We conclude that one of the functions of cIAP1 when localized in the nucleus is to regulate E2F1 transcriptional activity.

  12. The plasticizer BBP selectively inhibits epigenetic regulator sirtuin during differentiation of C3H10T1/2 stem cell line.

    Science.gov (United States)

    Zhang, Jian; Choudhury, Mahua

    2017-03-01

    Exposure to environmental chemicals can perturb an individual's metabolic set point, especially during critical periods of development, and as a result increase his or her propensity towards obesity that is manifested later in life and possibly in successive generations. We hypothesized that benzyl butyl phthalate (BBP), a widespread endocrine disruptor, may impair one important epigenetic regulator, sirtuin, in mesenchymal stem cells and induce adipogenesis. Our results showed that gene expression of two well-known adipogenic markers, aP2 and PPARγ, were significantly increased from day 2 to day 8 under 50μM BBP exposure when compared to control in C3H10T1/2 stem cells (p<0.05) and induced adipogenesis. Sirt1 gene expression was also significantly decreased at day 2, 4, 6, and 8 (p<0.05). However, Sirt7 gene expression was decreased only at day 2 and 8 (p<0.05) while other sirtuin transcriptional levels remained unaltered throughout. Furthermore, Sirt1 and Sirt3 protein expression was decreased (p<0.05) and overall protein hyperacetylation was observed at day 8. Furthermore, FOXO1 and β-catenin, Sirt1 targets and adipogenesis regulators, were hyperacetylated at day 8. PGC1α, NRF1, NRF2, and Tfam, were also significantly decreased (p<0.05). In conclusion, our study suggests for the first time that BBP, a potential epigenetic disruptor, can lead to increased adipogenesis and metabolic dysregulation by impairing vital epigenetic regulators. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma

    Directory of Open Access Journals (Sweden)

    Eberhart Charles G

    2010-11-01

    Full Text Available Abstract Background The Sonic hedgehog (Shh signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors. Methods We silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples. Results Silencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63% and primary astrocytoma tumor samples (32%, but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas. Conclusions Our results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.

  14. Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.

    Directory of Open Access Journals (Sweden)

    Carla Manuela Abreu

    2013-04-01

    Full Text Available The mediators of the DNA damage response (DDR are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR-specific protein kinases.

  15. Lack of sik1 in mouse embryonic stem cells impairs cardiomyogenesis by down-regulating the cyclin-dependent kinase inhibitor p57kip2.

    Directory of Open Access Journals (Sweden)

    Antonio Romito

    Full Text Available Sik1 (salt inducible kinase 1 is a serine/threonine kinase that belongs to the stress- and energy-sensing AMP-activated protein kinase family. During murine embryogenesis, sik1 marks the monolayer of future myocardial cells that will populate first the primitive ventricle, and later the primitive atrium suggesting its involvement in cardiac cell differentiation and/or heart development. Despite that observation, the involvement of sik1 in cardiac differentiation is still unknown. We examined the sik1 function during cardiomyocyte differentiation using the ES-derived embryoid bodies. We produced a null embryonic stem cell using a gene-trap cell line carrying an insertion in the sik1 locus. In absence of the sik1 protein, the temporal appearance of cardiomyocytes is delayed. Expression profile analysis revealed sik1 as part of a genetic network that controls the cell cycle, where the cyclin-dependent kinase inhibitor p57(Kip2 is directly involved. Collectively, we provided evidence that sik1-mediated effects are specific for cardiomyogenesis regulating cardiomyoblast cell cycle exit toward terminal differentiation.

  16. Cyclin-dependent kinase 5 modulates the transcriptional activity of the mineralocorticoid receptor and regulates expression of brain-derived neurotrophic factor.

    Science.gov (United States)

    Kino, Tomoshige; Jaffe, Howard; Amin, Niranjana D; Chakrabarti, Mayukh; Zheng, Ya-Li; Chrousos, George P; Pant, Harish C

    2010-05-01

    Glucocorticoids, major end effectors of the stress response, play an essential role in the homeostasis of the central nervous system (CNS) and contribute to memory consolidation and emotional control through their intracellular receptors, the glucocorticoid and mineralocorticoid receptors. Cyclin-dependent kinase 5 (CDK5), on the other hand, plays important roles in the morphogenesis and functions of the central nervous system, and its aberrant activation has been associated with development of neurodegenerative disorders. We previously reported that CDK5 phosphorylated the glucocorticoid receptor and modulated its transcriptional activity. Here we found that CDK5 also regulated mineralocorticoid receptor-induced transcriptional activity by phosphorylating multiple serine and threonine residues located in its N-terminal domain through physical interaction. Aldosterone and dexamethasone, respectively, increased and suppressed mRNA/protein expression of brain-derived neurotrophic factor (BDNF) in rat cortical neuronal cells, whereas the endogenous glucocorticoid corticosterone showed a biphasic effect. CDK5 enhanced the effect of aldosterone and dexamethasone on BDNF expression. Because this neurotrophic factor plays critical roles in neuronal viability, synaptic plasticity, consolidation of memory, and emotional changes, we suggest that aberrant activation of CDK5 might influence these functions through corticosteroid receptors/BDNF.

  17. The upstream open reading frame of cyclin-dependent kinase inhibitor 1A mRNA negatively regulates translation of the downstream main open reading frame

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyoung Mi; Cho, Hana [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Yoon Ki, E-mail: yk-kim@korea.ac.kr [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer CDKN1A mRNA is a bona fide NMD substrate. Black-Right-Pointing-Pointer The uORF of CDKN1A mRNA is efficiently translated. Black-Right-Pointing-Pointer Translation of downstream main ORF is negatively regulated by translation of uORF in CDKN1A mRNA. -- Abstract: The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein 80 and 20 (CBP80/20). During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here, our microarray analysis reveals that the level of cyclin-dependent kinase inhibitor 1A (CDKN1A; also known as Waf1/p21) mRNAs increases in cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is a NMD-inducing feature. Using chimeric reporter constructs, we find that the uORF of CDKN1A mRNA negatively modulates translation of the main downstream ORF. These findings provide biological insights into the possible role of NMD in diverse biological pathways mediated by CDKN1A.

  18. Timing of cyclin E gene expression depends on the regulated association of a bipartite repressor element with a novel E2F complex.

    Science.gov (United States)

    Le Cam, L; Polanowska, J; Fabbrizio, E; Olivier, M; Philips, A; Ng Eaton, E; Classon, M; Geng, Y; Sardet, C

    1999-04-01

    Transient induction of the cyclin E gene in late G1 gates progression into S. We show that this event is controlled via a cyclin E repressor module (CERM), a novel bipartite repressor element located near the cyclin E transcription start site. CERM consists of a variant E2F-binding site and a contiguous upstream AT-rich sequence which cooperate during G0/G1 to delay cyclin E expression until late G1. CERM binds the protein complex CERC, which disappears upon progression through G0-G1 and reappears upon entry into the following G1. CERC disappearance correlates kinetically with the liberation of the CERM module in vivo and cyclin E transcriptional induction. CERC contains E2F4/DP1 and a pocket protein, and sediments faster than classical E2F complexes in a glycerol gradient, suggesting the presence of additional components in a novel high molecular weight complex. Affinity purified CERC binds to CERM but not to canonical E2F sites, thus displaying behavior different from known E2F complexes. In cells nullizygous for members of the Rb family, CERC is still detectable and CERM-dependent repression is functional. Thus p130, p107 and pRb function interchangeably in CERC. Notably, the CERC-CERM complex dissociates prematurely in pRb-/- cells in correspondence with the premature expression of cyclin E. Thus, we identify a new regulatory module that controls repression of G1-specific genes in G0/G1.

  19. The effect of fasting and refeeding on mRNA expression of PepT1 and gastrointestinal hormones regulating digestion and food intake in zebrafish (Danio rerio).

    Science.gov (United States)

    Koven, William; Schulte, Patricia

    2012-12-01

    In vertebrates, a significant part of ingested protein is absorbed as di- and tripeptides through a brush border membrane proton/oligopeptide transporter protein called PepT1. The aim of the present study was to determine the effect of short-term food deprivation and refeeding in adult zebrafish (Danio rerio) on gastrointestinal mRNA expression of PepT1 as well as on the satiety hormones cholecystokinin (CCK), gastrin-releasing peptide (GRP) and ghrelin (GHR) in order to elucidate a potential mechanism driving compensatory growth. Sixty adult zebrafish were stocked in a 40-L aquarium and fed daily a commercial flake diet to satiation for 10 days where the digestive tracts (DT) of sampled fish (n = 5) were dissected out. Samplings were repeated following 1, 2 and 5 days of food deprivation and after 1, 2 and 5 days of refeeding. The RNA was extracted from all sampled DTs and analyzed by quantitative real-time PCR for the mRNA expression of PepT1, rRNA 18S, CCK, GRP and GHR. PepT1 mRNA expression increased with successive refeedings reaching a level approximately 8 times higher than pre-fast levels. CCK, GRP and GHR mRNA levels also decreased during fasting, but increased only to pre-fasting levels with refeeding. Overall, the results suggest that PepT1 may be a contributing mechanism to compensatory growth that could influence CCK secretion and GRP and GHR activity.

  20. Aspirin Breaks the Crosstalk between 3T3-L1 Adipocytes and 4T1 Breast Cancer Cells by Regulating Cytokine Production.

    Directory of Open Access Journals (Sweden)

    Chia-Chien Hsieh

    Full Text Available Breast cancer is one of the most common cancers in women worldwide. The obesity process is normally accompanied by chronic, low-grade inflammation. Infiltration by inflammatory cytokines and immune cells provides a favorable microenvironment for tumor growth, migration, and metastasis. Epidemiological evidence has shown that aspirin is an effective agent against several types of cancer. The aim of this study is to investigate the anti-inflammatory and anti-cancer effects of aspirin on 3T3-L1 adipocytes, 4T1 murine breast cancer cells, and their crosstalk. The results showed that aspirin treatment inhibited differentiation and lipid accumulation by 3T3-L1 preadipocytes, and decreased the secretion of the inflammatory adipokine MCP-1 after stimulation with tumor necrosis factor (TNF-α or conditioned medium from RAW264.7 cells. In 4T1 cells, treatment with aspirin decreased cell viability and migration, possibly by suppressing MCP-1 and VEGF secretion. Subsequently, culture of 4T1 cells in 3T3-L1 adipocyte-conditioned medium (Ad-CM and co-culture of 3T3-L1 and 4T1 cells using a transwell plate were performed to clarify the relationship between these two cell lines. Aspirin exerted its inhibitory effects in the transwell co-culture system, as well as the conditioned-medium model. Aspirin treatment significantly inhibited the proliferation of 4T1 cells, and decreased the production of MCP-1 and PAI-1 in both the Ad-CM model and co-culture system. Aspirin inhibited inflammatory MCP-1 adipokine production by 3T3-L1 adipocytes and the cell growth and migration of 4T1 cells. It also broke the crosstalk between these two cell lines, possibly contributing to its chemopreventive properties in breast cancer. This is the first report that aspirin's chemopreventive activity supports the potential application in auxiliary therapy against obesity-related breast cancer development.

  1. Aspirin Breaks the Crosstalk between 3T3-L1 Adipocytes and 4T1 Breast Cancer Cells by Regulating Cytokine Production.

    Science.gov (United States)

    Hsieh, Chia-Chien; Huang, Yu-Shan

    2016-01-01

    Breast cancer is one of the most common cancers in women worldwide. The obesity process is normally accompanied by chronic, low-grade inflammation. Infiltration by inflammatory cytokines and immune cells provides a favorable microenvironment for tumor growth, migration, and metastasis. Epidemiological evidence has shown that aspirin is an effective agent against several types of cancer. The aim of this study is to investigate the anti-inflammatory and anti-cancer effects of aspirin on 3T3-L1 adipocytes, 4T1 murine breast cancer cells, and their crosstalk. The results showed that aspirin treatment inhibited differentiation and lipid accumulation by 3T3-L1 preadipocytes, and decreased the secretion of the inflammatory adipokine MCP-1 after stimulation with tumor necrosis factor (TNF)-α or conditioned medium from RAW264.7 cells. In 4T1 cells, treatment with aspirin decreased cell viability and migration, possibly by suppressing MCP-1 and VEGF secretion. Subsequently, culture of 4T1 cells in 3T3-L1 adipocyte-conditioned medium (Ad-CM) and co-culture of 3T3-L1 and 4T1 cells using a transwell plate were performed to clarify the relationship between these two cell lines. Aspirin exerted its inhibitory effects in the transwell co-culture system, as well as the conditioned-medium model. Aspirin treatment significantly inhibited the proliferation of 4T1 cells, and decreased the production of MCP-1 and PAI-1 in both the Ad-CM model and co-culture system. Aspirin inhibited inflammatory MCP-1 adipokine production by 3T3-L1 adipocytes and the cell growth and migration of 4T1 cells. It also broke the crosstalk between these two cell lines, possibly contributing to its chemopreventive properties in breast cancer. This is the first report that aspirin's chemopreventive activity supports the potential application in auxiliary therapy against obesity-related breast cancer development.

  2. Cinnamon and its Components Suppress Vascular Smooth Muscle Cell Proliferation by Up-Regulating Cyclin-Dependent Kinase Inhibitors.

    Science.gov (United States)

    Kwon, Hyeeun; Lee, Jung-Jin; Lee, Ji-Hye; Cho, Won-Kyung; Gu, Min Jung; Lee, Kwang Jin; Ma, Jin Yeul

    2015-01-01

    Cinnamomum cassia bark has been used in traditional herbal medicine to treat a variety of cardiovascular diseases. However, the antiproliferative effect of cinnamon extract on vascular smooth muscle cells (VSMCs) and the corresponding restenosis has not been explored. Hence, after examining the effect of cinnamon extract on VSMC proliferation, we investigated the possible involvement of signal transduction pathways associated with early signal and cell cycle analysis, including regulatory proteins. Besides, to identify the active components, we investigated the components of cinnamon extract on VSMC proliferation. Cinnamon extract inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and suppressed the PDGF-stimulated early signal transduction. In addition, cinnamon extract arrested the cell cycle and inhibited positive regulatory proteins. Correspondingly, the protein levels of p21 and p27 not only were increased in the presence of cinnamon extract, also the expression of proliferating cell nuclear antigen (PCNA) was inhibited by cinnamon extract. Besides, among the components of cinnamon extract, cinnamic acid (CA), eugenol (EG) and cinnamyl alcohol significantly inhibited the VSMC proliferation. Overall, the present study demonstrates that cinnamon extract inhibited the PDGF-BB-induced proliferation of VSMCs through a G0/G1 arrest, which down-regulated the expression of cell cycle positive regulatory proteins by up-regulating p21 and p27 expression.

  3. Regulation of GATA-binding protein 2 levels via ubiquitin-dependent degradation by Fbw7: involvement of cyclin B-cyclin-dependent kinase 1-mediated phosphorylation of THR176 in GATA-binding protein 2.

    Science.gov (United States)

    Nakajima, Tomomi; Kitagawa, Kyoko; Ohhata, Tatsuya; Sakai, Satoshi; Uchida, Chiharu; Shibata, Kiyoshi; Minegishi, Naoko; Yumimoto, Kanae; Nakayama, Keiichi I; Masumoto, Kazuma; Katou, Fuminori; Niida, Hiroyuki; Kitagawa, Masatoshi

    2015-04-17

    A GATA family transcription factor, GATA-binding protein 2 (GATA2), participates in cell growth and differentiation of various cells, such as hematopoietic stem cells. Although its expression level is controlled by transcriptional induction and proteolytic degradation, the responsible E3 ligase has not been identified. Here, we demonstrate that F-box/WD repeat-containing protein 7 (Fbw7/Fbxw7), a component of Skp1, Cullin 1, F-box-containing complex (SCF)-type E3 ligase, is an E3 ligase for GATA2. GATA2 contains a cell division control protein 4 (Cdc4) phosphodegron (CPD), a consensus motif for ubiquitylation by Fbw7, which includes Thr(176). Ectopic expression of Fbw7 destabilized GATA2 and promoted its proteasomal degradation. Substitution of threonine 176 to alanine in GATA2 inhibited binding with Fbw7, and the ubiquitylation and degradation of GATA2 by Fbw7 was suppressed. The CPD kinase, which mediates the phosphorylation of Thr(176), was cyclin B-cyclin-dependent kinase 1 (CDK1). Moreover, depletion of endogenous Fbw7 stabilized endogenous GATA2 in K562 cells. Conditional Fbw7 depletion in mice increased GATA2 levels in hematopoietic stem cells and myeloid progenitors at the early stage. Increased GATA2 levels in Fbw7-conditional knock-out mice were correlated with a decrease in a c-Kit high expressing population of myeloid progenitor cells. Our results suggest that Fbw7 is a bona fide E3 ubiquitin ligase for GATA2 in vivo.

  4. p14ARF post-transcriptional regulation of nuclear cyclin D1 in MCF-7 breast cancer cells: discrimination between a good and bad prognosis?

    Directory of Open Access Journals (Sweden)

    Eileen M McGowan

    Full Text Available As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation, and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late

  5. miR-338-3p Is Down-Regulated by Hepatitis B Virus X and Inhibits Cell Proliferation by Targeting the 3′-UTR Region of CyclinD1

    Directory of Open Access Journals (Sweden)

    Xiaoyu Fu

    2012-07-01

    Full Text Available Hepatitis B virus X protein (HBx is recognized as an oncogene in hepatocellular carcinoma (HCC. HBx regulates microRNA expression, including down-regulating miR-338-3p in LO2 cells. Here, we investigated miR-338-3p function in HBx-mediated hepatocarcinogenesis. In 23 HBV-infected HCC clinical patient tumor and adjacent non-tumor control tissues, 17 and 19 tumors expressed HBx mRNA and protein, respectively. When considered as a group, HBV-infected HCC tumors had lower miR-338-3p expression than controls; however, miR-338-3p was only significantly down-regulated in HBx-positive tumors, indicating that HBx inversely correlated with miR-338-3p. Functional characterization of miR-338-3p indicated that miR-338-3p mimics inhibited cell proliferation by inducing cell cycle arrest at the G1/S phase as assessed by EdU and cell cycle assays in HBx-expressing LO2 cells. CyclinD1, containing two putative miR-338-3p targets, was confirmed as a direct target using 3′-UTR luciferase reporter assays from cells transfected with mutated binding sites. Mutating the 2397–2403 nt binding site conferred the greatest resistance to miR-338-3p suppression of CyclinD1, indicating that miR-338-3p suppresses CyclinD1 at this site. Overall, this study demonstrates that miR-338-3p inhibits proliferation by regulating CyclinD1, and HBx down-regulates miR-338-3p in HCC. This newly identified miR-338-3p/CyclinD1 interaction provides novel insights into HBx-mediated hepatocarcinogenesis and may facilitate therapeutic development against HCC.

  6. AKAP95 promotes cell cycle progression via interactions with cyclin E and low molecular weight cyclin E

    Science.gov (United States)

    Kong, Xiang-Yu; Zhang, Deng-Cheng; Zhuang, Wen-Xin; Hua, Su-Hang; Dai, Yue; Yuan, Yang-Yang; Feng, Li-Li; Huang, Qian; Teng, Bo-Gang; Yu, Xiu-Yi; Liu, Wen-Zhi; Zhang, Yong-Xing

    2016-01-01

    AKAP95 in lung cancer tissues showed higher expression than in paracancerous tissues. AKAP95 can bind with cyclin D and cyclin E during G1/S cell cycle transition, but its molecular mechanisms remain unclear. To identify the mechanism of AKAP95 in cell cycle progression, we performed AKAP95 transfection and silencing in A549 cells, examined AKAP95, cyclin E1 and cyclin E2 expression, and the interactions of AKAP95 with cyclins E1 and E2. Results showed that over-expression of AKAP95 promoted cell growth and AKAP95 bound cyclin E1 and E2, low molecular weight cyclin E1 (LWM-E1) and LWM-E2. Additionally AKAP95 bound cyclin E1 and LMW-E2 in the nucleus during G1/S transition, bound LMW-E1 during G1, S and G2/M, and bound cyclin E2 mainly on the nuclear membrane during interphase. Cyclin E2 and LMW-E2 were also detected. AKAP95 over-expression increased cyclin E1 and LMW-E2 expression but decreased cyclin E2 levels. Unlike cyclin E1 and LMW-E2 that were nuclear located during the G1, S and G1/S phases, cyclin E2 and LMW-E1 were expressed in all cell cycle phases, with cyclin E2 present in the cytoplasm and nuclear membrane, with traces in the nucleus. LMW-E1 was present in both the cytoplasm and nucleus. The 20 kDa form of LMW-E1 showed only cytoplasmic expression, while the 40 kDa form was nuclear expressed. The expression of AKAP95, cyclin E1, LMW-E1 and -E2, might be regulated by cAMP. We conclude that AKAP95 might promote cell cycle progression by interacting with cyclin E1 and LMW-E2. LMW-E2, but not cyclin E2, might be involved in G1/S transition. The binding of AKAP95 and LMW-E1 was found throughout cell cycle. PMID:27158371

  7. Reduced hepatic tumor incidence in cyclin G1-deficient mice

    DEFF Research Database (Denmark)

    Jensen, Michael Rugaard; Factor, Valentina M; Fantozzi, Anna

    2003-01-01

    Cyclin G1 is a transcriptional target of the tumor suppressor p53, and its expression is increased after DNA damage. Recent data show that cyclin G1 can regulate the levels of p53 by a mechanism that involves dephosphorylation of Mdm2 by protein phosphatase 2A. To understand the biologic role of ...

  8. Self-Renewal and High Proliferative Colony Forming Capacity of Late-Outgrowth Endothelial Progenitors Is Regulated by Cyclin-Dependent Kinase Inhibitors Driven by Notch Signaling.

    Science.gov (United States)

    Patel, Jatin; Wong, Ho Yi; Wang, Weili; Alexis, Josue; Shafiee, Abbas; Stevenson, Alexander J; Gabrielli, Brian; Fisk, Nicholas M; Khosrotehrani, Kiarash

    2016-04-01

    Since the discovery of endothelial colony forming cells (ECFC), there has been significant interest in their therapeutic potential to treat vascular injuries. ECFC cultures display significant heterogeneity and a hierarchy among cells able to give rise to high proliferative versus low proliferative colonies. Here we aimed to define molecularly this in vitro hierarchy. Based on flow cytometry, CD34 expression levels distinguished two populations. Only CD34 + ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD34- ECFCs formed only small clusters. CD34 + ECFCs were the only ones to self-renew in stringent single-cell cultures and gave rise to both CD34 + and CD34- cells. Upon replating, CD34 + ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 + ECFC were superior at restoring perfusion and better engrafted when injected into ischemic hind limbs. Transcriptomic analysis identified cyclin-dependent kinase (CDK) cell cycle inhibiting genes (p16, p21, and p57), the Notch signaling pathway (dll1, dll4, hes1, and hey1), and the endothelial cytokine il33 as highly expressed in CD34 + ECFC. Blocking the Notch pathway using a γ-secretase inhibitor (DAPT) led to reduced expression of cell cycle inhibitors, increased cell proliferation followed by a loss of self-renewal, and HPP colony formation capacity reflecting progenitor exhaustion. Similarly shRNA knockdown of p57 strongly affected self-renewal of ECFC colonies. ECFC hierarchy is defined by Notch signalling driving cell cycle regulators, progenitor quiescence and self-renewal potential.

  9. Expression and signification of cell cycle regulation protein Cyclin D1-CDK4-p21 in scar cancer%细胞周期调控系统相关因子 Cyclin D1-CDK4-p21在瘢痕癌中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    林宇静; 郭瑞珍; 王海青

    2014-01-01

    Objective Dysfunction of cell cycle regulation is one of the key factors for cellular carcinogenesis .This paper aimed to study the expression and significance of cell cycle regulation protein Cyclin D 1-CDK4-p21 in scar cancer . Methods The expressions of Cyclin D1, CDK4 and p21 protains were detected in scar cancer group , pathological scar group and normal skin group respectively by using immunohistochemical staining (SP).The mRNA expression levels of Cyclin D1, CDK4 and p21 were detected by the use of nucleic acid-mediated in-situ hybridization .Correlation analysis was made on the indexes , and the average optical density and positive area were analyzed using image analysis . Results The expressions of Cyclin D1, CDK4 and p21 protains and the mRNA ex-pression levels of cyclin D1, CDK4 and p21 were high in scar cancer group, low in pathological scar group , and negative in normal skin group.The mean optical density and positive area in scar cancer group were significantly different from pathological scar group and normal skin group (P0.05).In terms of correlation analysis , the expressions of Cyclin D 1 and CDK4 as well as p21 and CDK4 in scar cancer tissue were both in posi-tive correlations. Conclusion The occurrence of scar cancer is related to the abnormal expression of Cyclin D 1 and CDK4.The complex formed by Cyclin D1 and CDK4 may promote the G1/S transition, proliferation and tumorigenesis of scar cancer .In scar canc-er, the inhibition of Cyclin D1-CDK4 complex might be caused by other members of CKI family or even inbibitors of other families apart from CDK family.%目的:细胞周期调控机制失调是细胞增生肿瘤发生的重要因素。文中探讨细胞周期调控系统相关因子Cyclin D1-CDK4-p21在瘢痕癌中的表达及意义。方法选取遵义医学院病理教研室和中山大学附属第五医院病理科2005-2011年石蜡包埋标本,分为瘢痕癌组、病理性瘢痕组和正常皮肤组。应

  10. Bile acid-regulated peroxisome proliferator-activated receptor-α (PPARα) activity underlies circadian expression of intestinal peptide absorption transporter PepT1/Slc15a1.

    Science.gov (United States)

    Okamura, Ayako; Koyanagi, Satoru; Dilxiat, Adila; Kusunose, Naoki; Chen, Jia Jun; Matsunaga, Naoya; Shibata, Shigenobu; Ohdo, Shigehiro

    2014-09-05

    Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor α (PPARα). Nocturnally active mice mainly consumed their food during the dark phase. PPARα activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPARα-mediated transactivation of Slc15a1. The time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal peptide transporter.

  11. Transgenic cyclin E triggers dysplasia and multiple pulmonary adenocarcinomas.

    Science.gov (United States)

    Ma, Yan; Fiering, Steven; Black, Candice; Liu, Xi; Yuan, Ziqiang; Memoli, Vincent A; Robbins, David J; Bentley, Heather A; Tsongalis, Gregory J; Demidenko, Eugene; Freemantle, Sarah J; Dmitrovsky, Ethan

    2007-03-06

    Cyclin E is a critical G(1)-S cell cycle regulator aberrantly expressed in bronchial premalignancy and lung cancer. Cyclin E expression negatively affects lung cancer prognosis. Its role in lung carcinogenesis was explored. Retroviral cyclin E transduction promoted pulmonary epithelial cell growth, and small interfering RNA targeting of cyclin E repressed this growth. Murine transgenic lines were engineered to mimic aberrant cyclin E expression in the lung. Wild-type and proteasome degradation-resistant human cyclin E transgenic lines were independently driven by the human surfactant C (SP-C) promoter. Chromosome instability (CIN), pulmonary dysplasia, sonic hedgehog (Shh) pathway activation, adenocarcinomas, and metastases occurred. Notably, high expression of degradation-resistant cyclin E frequently caused dysplasia and multiple lung adenocarcinomas. Thus, recapitulation of aberrant cyclin E expression as seen in human premalignant and malignant lung lesions reproduces in the mouse frequent features of lung carcinogenesis, including CIN, Shh pathway activation, dysplasia, single or multiple lung cancers, or presence of metastases. This article reports unique mouse lung cancer models that replicate many carcinogenic changes found in patients. These models provide insights into the carcinogenesis process and implicate cyclin E as a therapeutic target in the lung.

  12. Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

    Science.gov (United States)

    Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

    2014-01-01

    The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.

  13. Regulation of APCCdh1 E3 ligase activity by the Fbw7/cyclin E signaling axis contributes to the tumor suppressor function of Fbw7

    Institute of Scientific and Technical Information of China (English)

    Alan W Lau; Hiroyuki Inuzuka; Hidefumi Fukushima; Lixin Wan; Pengda Liu; Daming Gao; Yi Sun

    2013-01-01

    Fbw7 and Cdh1 are substrate-recognition subunits of the SCF-and APC-type E3 ubiquitin ligases,respectively.There is emerging evidence suggesting that both Fbw7 and Cdh1 function as tumor suppressors by targeting oncoproteins for destruction.Loss of Fbw7,but not Cdh1,is frequently observed in various human tumors.However,it remains largely unknown how Fbw7 mechanistically functions as a tumor suppressor and whether there is a signaling crosstalk between Fbw7 and Cdh1.Here,we report that Fbw7-deficient cells not only display elevated expression levels of SCFFbw7 substrates,including cyclin E,but also have increased expression of various APCdh1 substrates.We further defined cyclin E as the critical signaling link by which Fbw7 governs APCCdh1 activity,as depletion of cyclin E in Fbw7-deficient cells results in decreased expression of APCdh1 substrates to levels comparable to those in wildtype (WT) cells.Conversely,ectopic expression of cyclin E recapitulates the aberrant APCdh1 substrate expression observed in Fbw7-deficient cells.More importantly,4A-Cdh1 that is resistant to Cdk2/cyclin E-mediated phosphorylation,but not WT-Cdh1,reversed the elevated expression of various APCCdh1 substrates in Fbw7-deficient cells.Overexpression of 4A-Cdh1 also resulted in retarded cell growth and decreased anchorage-independent colony formation.Altogether,we have identified a novel regulatory mechanism by which Fbw7 governs Cdh1 activity in a cyclin E-dependent manner.As a result,loss of Fbw7 can lead to aberrant increase in the expression of both SCFFbw7and APCCdh1 substrates.Our study provides a better understanding of the tumor suppressor function of Fbw7,and suggests that Cdk2/cyclin E inhibitors could serve as effective therapeutic agents for treating Fbw7-deficient tumors.

  14. miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2.

    Science.gov (United States)

    Yadav, Sanjay; Pandey, Ankita; Shukla, Aruna; Talwelkar, Sarang S; Kumar, Ashutosh; Pant, Aditya B; Parmar, Devendra

    2011-10-28

    In chronic alcoholism, brain shrinkage and cognitive defects because of neuronal death are well established, although the sequence of molecular events has not been fully explored yet. We explored the role of microRNAs (miRNAs) in ethanol-induced apoptosis of neuronal cells. Ethanol-sensitive miRNAs in SH-SY5Y, a human neuroblastoma cell line, were identified using real-time PCR-based TaqMan low-density arrays. Long-term exposure to ethanol (0.5% v/v for 72 h) produced a maximum increase in expression of miR-497 (474-fold) and miR-302b (322-fold). Similar to SH-SY5Y, long-term exposure to ethanol induced miR-497 and miR-302b in IMR-32, another human neuroblastoma cell line. Using in silico approaches, BCL2 and cyclin D2 (CCND2) were identified as probable target genes of these miRNAs. Cotransfection studies with 3'-UTR of these genes and miRNA mimics have demonstrated that BCL2 is a direct target of miR-497 and that CCND2 is regulated negatively by either miR-302b or miR-497. Overexpression of either miR-497 or miR-302b reduced expression of their identified target genes and increased caspase 3-mediated apoptosis of SH-SY5Y cells. However, overexpression of only miR-497 increased reactive oxygen species formation, disrupted mitochondrial membrane potential, and induced cytochrome c release (mitochondria-related events of apoptosis). Moreover, ethanol induced changes in miRNAs, and their target genes were substantially prevented by pre-exposure to GSK-3B inhibitors. In conclusion, our studies have shown that ethanol-induced neuronal apoptosis follows both the mitochondria-mediated (miR-497- and BCL2-mediated) and non-mitochondria-mediated (miR-302b- and CCND2-mediated) pathway.

  15. Down-Regulation of mir-424 Contributes to the Abnormal Angiogenesis via MEK1 and Cyclin E1 in Senile Hemangioma: Its Implications to Therapy

    Science.gov (United States)

    Nakashima, Taiji; Jinnin, Masatoshi; Etoh, Tomomi; Fukushima, Satoshi; Masuguchi, Shinichi; Maruo, Keishi; Inoue, Yuji; Ishihara, Tsuyoshi; Ihn, Hironobu

    2010-01-01

    Background Senile hemangioma, so-called cherry angioma, is known as the most common vascular anomalies specifically seen in the aged skin. The pathogenesis of its abnormal angiogenesis is still unclear. Methodology/Principal Findings In this study, we found that senile hemangioma consisted of clusters of proliferated small vascular channels in upper dermis, indicating that this tumor is categorized as a vascular tumor. We then investigated the mechanism of endothelial proliferation in senile hemangioma, focusing on microRNA (miRNA). miRNA PCR array analysis revealed the mir-424 level in senile hemangioma was lower than in other vascular anomalies. Protein expression of MEK1 and cyclin E1, the predicted target genes of mir-424, was increased in senile hemangioma compared to normal skin or other anomalies, but their mRNA levels were not. The inhibition of mir-424 in normal human dermal microvascular ECs (HDMECs) using specific inhibitor in vitro resulted in the increase of protein expression of MEK1 or cyclin E1, while mRNA levels were not affected by the inhibitor. Specific inhibitor of mir-424 also induced the cell proliferation of HDMECs significantly, while the cell number was decreased by the transfection of siRNA for MEK1 or cyclin E1. Conclusions/Significance Taken together, decreased mir-424 expression and increased levels of MEK1 or cyclin E1 in senile hemangioma may cause abnormal cell proliferation in the tumor. Senile hemangioma may be the good model for cutaneous angiogenesis. Investigation of senile hemangioma and the regulatory mechanisms of angiogenesis by miRNA in the aged skin may lead to new treatments using miRNA by the transfection into senile hemangioma. PMID:21179471

  16. Transforming growth factor-β1 induces cell cycle arrest by activating atypical cyclin-dependent kinase 5 through up-regulation of Smad3-dependent p35 expression in human MCF10A mammary epithelial cells.

    Science.gov (United States)

    Park, Seong Ji; Yang, Sun Woo; Kim, Byung-Chul

    2016-04-01

    Cyclin-dependent kinases (Cdks) play important roles in control of cell division. Cdk5 is an atypical member of Cdk family with non-cyclin-like regulatory subunit, p35, but its role in cell cycle progression is still unclear. In the present study, we investigated the role of Cdk5/p35 on transforming growth factor-β1 (TGF-β1)-induced cell cycle arrest. In human MCF10A mammary epithelial cells, TGF-β1 induced cell cycle arrest at G1 phase and increased p27KIP1 expression. Interestingly, pretreatment with roscovitine, an inhibitor of Cdk5, or transfection with small interfering (si) RNAs specific to Cdk5 and p35 significantly attenuated the TGF-β1-induced p27KIP1 expression and cell cycle arrest. TGF-β1 increased Cdk5 activity via up-regulation of p35 gene at transcriptional level, and these effects were abolished by transfection with Smad3 siRNA or infection of adenovirus carrying Smad3 mutant at the C-tail (3SA). Chromatin immunoprecipitation assay further revealed that wild type Smad3, but not mutant Smad3 (3SA), binds to the region of the p35 promoter region (-1000--755) in a TGF-β1-dependent manner. These results for the first time demonstrate a role of Cdk5/p35 in the regulation of cell cycle progression modulated by TGF-β1.

  17. Rising cyclin-CDK levels order cell cycle events.

    Directory of Open Access Journals (Sweden)

    Catherine Oikonomou

    Full Text Available BACKGROUND: Diverse mitotic events can be triggered in the correct order and time by a single cyclin-CDK. A single regulator could confer order and timing on multiple events if later events require higher cyclin-CDK than earlier events, so that gradually rising cyclin-CDK levels can sequentially trigger responsive events: the "quantitative model" of ordering. METHODOLOGY/PRINCIPAL FINDINGS: This 'quantitative model' makes predictions for the effect of locking cyclin at fixed levels for a protracted period: at low cyclin levels, early events should occur rapidly, while late events should be slow, defective, or highly variable (depending on threshold mechanism. We titrated the budding yeast mitotic cyclin Clb2 within its endogenous expression range to a stable, fixed level and measured time to occurrence of three mitotic events: growth depolarization, spindle formation, and spindle elongation, as a function of fixed Clb2 level. These events require increasingly more Clb2 according to their normal order of occurrence. Events occur efficiently and with low variability at fixed Clb2 levels similar to those observed when the events normally occur. A second prediction of the model is that increasing the rate of cyclin accumulation should globally advance timing of all events. Moderate (<2-fold overexpression of Clb2 accelerates all events of mitosis, resulting in consistently rapid sequential cell cycles. However, this moderate overexpression also causes a significant frequency of premature mitoses leading to inviability, suggesting that Clb2 expression level is optimized to balance the fitness costs of variability and catastrophe. CONCLUSIONS/SIGNIFICANCE: We conclude that mitotic events are regulated by discrete cyclin-CDK thresholds. These thresholds are sequentially triggered as cyclin increases, yielding reliable order and timing. In many biological processes a graded input must be translated into discrete outputs. In such systems, expression of

  18. Apoptosis of Glioblastoma U251 Cells Induced by Carmustine Combined All-trans Retinoic Acid via Regulating Cyclin E and p27kip 1

    Institute of Scientific and Technical Information of China (English)

    QI Bin; WEIJun; HU Guo-zhang; YANG Hong-fa; BI Chun-hua; SUN Zhi-gang; TIAN Yu

    2011-01-01

    The effect and mechanism of carmustine(BCNU) combined with all-trans retinoic acid(ATRA) on the apoptosis of human glioblastoma U251 cells were investigated by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay,flow cytometry,reverse transcription-polymerase chain reaction(RT-PCR) and Western blot analysis.The results show that BCNU or ATRA shows time- and dose-dependent inhibition effects on human glioblastoma U251 cells and the combination of BCNU with ATRA shows an synergistic inhibition effect on human glioblastoma U251 cells,and the combined BCNU and ATRA can significantly inhibit the proliferation of human glioblastoma U251 cells,and induce the apoptosis of them,making the cells arrest in the stage of G1 phase,the stage of S and G2 phases decline,the rate of the apoptosis of human glioblastoma U251 cells increase,the corresponding mRNA expression of cyclin E and cyclin-dependent kinase 2(CDK2) downregulated and the corresponding mRNA expression of p27kip 1 unregulated.In addition,the combined BCNU and ATRA reduced the protein expression of nuclear factor kappa B(NF-kB).Taken together,these results suggest that the treatment of human glioblastoma U251 cells with a combination application of ATRA and BCNU can exert synergistic effect,the course of this kind of combination chemotherapy may likely be associated with multiple molecular mechanisms for apoptosis,furthermore,the cyclin E and p27kip 1 should be considered as novel targets for controlling the growth of glioblastoma cells.

  19. TReP-132 Controls Cell Proliferation by Regulating the Expression of the Cyclin-Dependent Kinase Inhibitors p21WAF1/Cip1 and p27Kip1

    Science.gov (United States)

    Gizard, Florence; Robillard, Romain; Barbier, Olivier; Quatannens, Brigitte; Faucompré, Anne; Révillion, Françoise; Peyrat, Jean-Philippe; Staels, Bart; Hum, Dean W.

    2005-01-01

    The transcriptional regulating protein of 132 kDa (TReP-132) has been identified in steroidogenic tissues, where it acts as a coactivator of steroidogenic factor 1 (SF-1). We show here that TReP-132 plays a role in the control of cell proliferation. In human HeLa cells, TReP-132 knockdown by using small interfering RNA resulted in increased G1→S cell cycle progression. The growth-inhibitory effects of TReP-132 was further shown to be mediated by induction of G1 cyclin-dependent kinase inhibitors p21WAF1 (p21) and p27KIP1 (p27) expression levels. As a consequence, G1 cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G1 phase. The stimulatory effect of TReP-132 on p21 and p27 gene transcription involved interaction of TReP-132 with the transcription factor Sp1 at proximal Sp1-binding sites in their promoters. Moreover, in different breast tumor cell lines, endogenous TReP-132 expression was positively related with a lower proliferation rate. In addition, TReP-132 knockdown resulted in enhanced cell proliferation and lowered p21 and p27 mRNA levels in the steroid-responsive and nonresponsive T-47D and MDA-MB-231 cell lines, respectively. Finally, a statistic profiling of human breast tumor samples highlighted that expression of TReP-132 is correlated with p21 and p27 levels and is associated with lower tumor incidence and aggressiveness. Together, these results identify TReP-132 as a basal cell cycle regulatory protein acting, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters. PMID:15899840

  20. Lin-28 homologue A (LIN28A) promotes cell cycle progression via regulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 (CCND1), and cell division cycle 25 homolog A (CDC25A) expression in cancer.

    Science.gov (United States)

    Li, Ning; Zhong, Xiaomin; Lin, Xiaojuan; Guo, Jinyi; Zou, Lian; Tanyi, Janos L; Shao, Zhongjun; Liang, Shun; Wang, Li-Ping; Hwang, Wei-Ting; Katsaros, Dionyssios; Montone, Kathleen; Zhao, Xia; Zhang, Lin

    2012-05-18

    The RNA-binding protein LIN28A regulates the translation and stability of a large number of mRNAs as well as the biogenesis of certain miRNAs in embryonic stem cells and developing tissues. Increasing evidence indicates that LIN28A functions as an oncogene promoting cancer cell growth. However, little is known about its molecular mechanism of cell cycle regulation in cancer. Using tissue microarrays, we found that strong LIN28A expression was reactivated in about 10% (7.1-17.1%) of epithelial tumors (six tumor types, n = 369). Both in vitro and in vivo experiments demonstrate that LIN28A promotes cell cycle progression in cancer cells. Genome-wide RNA-IP-chip experiments indicate that LIN28A binds to thousands of mRNAs, including a large group of cell cycle regulatory mRNAs in cancer and embryonic stem cells. Furthermore, the ability of LIN28A to stimulate translation of LIN28A-binding mRNAs, such as CDK2, was validated in vitro and in vivo. Finally, using a combined gene expression microarray and bioinformatics approach, we found that LIN28A also regulates CCND1 and CDC25A expression and that this is mediated by inhibiting the biogenesis of let-7 miRNA. Taken together, these results demonstrate that LIN28A is reactivated in about 10% of epithelial tumors and promotes cell cycle progression by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent).

  1. Lin-28 Homologue A (LIN28A) Promotes Cell Cycle Progression via Regulation of Cyclin-dependent Kinase 2 (CDK2), Cyclin D1 (CCND1), and Cell Division Cycle 25 Homolog A (CDC25A) Expression in Cancer*

    Science.gov (United States)

    Li, Ning; Zhong, Xiaomin; Lin, Xiaojuan; Guo, Jinyi; Zou, Lian; Tanyi, Janos L.; Shao, Zhongjun; Liang, Shun; Wang, Li-Ping; Hwang, Wei-Ting; Katsaros, Dionyssios; Montone, Kathleen; Zhao, Xia; Zhang, Lin

    2012-01-01

    The RNA-binding protein LIN28A regulates the translation and stability of a large number of mRNAs as well as the biogenesis of certain miRNAs in embryonic stem cells and developing tissues. Increasing evidence indicates that LIN28A functions as an oncogene promoting cancer cell growth. However, little is known about its molecular mechanism of cell cycle regulation in cancer. Using tissue microarrays, we found that strong LIN28A expression was reactivated in about 10% (7.1–17.1%) of epithelial tumors (six tumor types, n = 369). Both in vitro and in vivo experiments demonstrate that LIN28A promotes cell cycle progression in cancer cells. Genome-wide RNA-IP-chip experiments indicate that LIN28A binds to thousands of mRNAs, including a large group of cell cycle regulatory mRNAs in cancer and embryonic stem cells. Furthermore, the ability of LIN28A to stimulate translation of LIN28A-binding mRNAs, such as CDK2, was validated in vitro and in vivo. Finally, using a combined gene expression microarray and bioinformatics approach, we found that LIN28A also regulates CCND1 and CDC25A expression and that this is mediated by inhibiting the biogenesis of let-7 miRNA. Taken together, these results demonstrate that LIN28A is reactivated in about 10% of epithelial tumors and promotes cell cycle progression by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent). PMID:22467868

  2. Phosphate-Activated Cyclin-Dependent Kinase Stabilizes G1 Cyclin To Trigger Cell Cycle Entry

    Science.gov (United States)

    Menoyo, S.; Ricco, N.; Bru, S.; Hernández-Ortega, S.; Escoté, X.; Aldea, M.

    2013-01-01

    G1 cyclins, in association with a cyclin-dependent kinase (CDK), are universal activators of the transcriptional G1-S machinery during entry into the cell cycle. Regulation of cyclin degradation is crucial for coordinating progression through the cell cycle, but the mechanisms that modulate cyclin stability to control cell cycle entry are still unknown. Here, we show that a lack of phosphate downregulates Cln3 cyclin and leads to G1 arrest in Saccharomyces cerevisiae. The stability of Cln3 protein is diminished in strains with low activity of Pho85, a phosphate-sensing CDK. Cln3 is an in vitro substrate of Pho85, and both proteins interact in vivo. More interestingly, cells that carry a CLN3 allele encoding aspartic acid substitutions at the sites of Pho85 phosphorylation maintain high levels of Cln3 independently of Pho85 activity. Moreover, these cells do not properly arrest in G1 in the absence of phosphate and they die prematurely. Finally, the activity of Pho85 is essential for accumulating Cln3 and for reentering the cell cycle after phosphate refeeding. Taken together, our data indicate that Cln3 is a molecular target of the Pho85 kinase that is required to modulate cell cycle entry in response to environmental changes in nutrient availability. PMID:23339867

  3. Immune regulation in T1D and T2D: prospective role of Foxp3+ Treg cells in disease pathogenesis and treatment

    Directory of Open Access Journals (Sweden)

    Mara eKornete

    2013-06-01

    Full Text Available There is increasing evidence that dysregulated immune responses play key roles in the pathogenesis and complications of type 1 but also type 2 diabetes. Indeed, chronic inflammation and autoimmunity, which are salient features of type 1 diabetes, are now believed to actively contribute to the pathogenesis of type 2 diabetes. The accumulation of activated innate and adaptive immune cells in various metabolic tissues results in the release of inflammatory mediators, which promote insulin resistance and β-cell damage. Moreover, these dysregulated immune responses can also mutually influence the prevalence of both type 1 and 2 diabetes. In this review article, we discuss the central role of immune responses in the patho-physiology and complications of type 1 and 2 diabetes, and provide evidence that regulation of these responses, particularly through the action of regulatory T cells, may be a possible therapeutic avenue for the treatment of these disease and their respective complications.

  4. A roller coaster ride with the mitotic cyclins.

    Science.gov (United States)

    Fung, Tsz Kan; Poon, Randy Y C

    2005-06-01

    Cyclins are discovered as proteins that accumulate progressively through interphase and disappear abruptly at mitosis during each cell cycle. In mammalian cells, cyclin A accumulates from late G1 phase and is destroyed before metaphase, and cyclin B is destroyed slightly later at anaphase. The abundance of the mitotic cyclins is mainly regulated at the levels of transcription and proteolysis. Transcription is stimulated and repressed by several transcription factors, including B-MYB, E2F, FOXM1, and NF-Y. Elements in the promoter, including CCRE/CDE and CHR, are in part responsible for the cell cycle oscillation of transcription. Destruction of the mitotic cyclins is carried out by the ubiquitin ligases APC/C(CDC20) and APC/C(CDH1). Central to our knowledge is the understanding of how APC/C is turned on from anaphase to early G1 phase, and turned off from late G1 till the spindle-assembly checkpoint is deactivated in metaphase. Reciprocal actions of cyclin-dependent kinases (CDKs) on APC/C, as well as on the SCF complexes ensure that the mitotic cyclins are destroyed only at the proper time.

  5. Prognostic Importance of Cell Cycle Regulators Cyclin D1 (CCND1) and Cyclin-Dependent Kinase Inhibitor 1B (CDKN1B/p27) in Sporadic Gastric Cancers

    Science.gov (United States)

    Minarikova, Petra; Halkova, Tereza; Belsanova, Barbora; Tuckova, Inna; Belina, Frantisek; Dusek, Ladislav; Zavoral, Miroslav

    2016-01-01

    Background. Gastric cancer is known for a notable variety in the course of the disease. Clinical factors, such as tumor stage, grade, and localization, are key in patient survival. It is expected that molecular factors such as somatic mutations and gene amplifications are also underlying tumor biological behavior and may serve as factors for prognosis estimation. Aim. The purpose of this study was to examine gene amplifications from a panel of genes to uncover potential prognostic marker candidates. Methods. A panel of gene amplifications including 71 genes was tested by multiplex ligation-dependent probe amplification (MLPA) technique in 76 gastric cancer samples from a Caucasian population. The correlation of gene amplification status with patient survival was determined by the Kaplan-Meier method. Results. The amplification of two cell cycle regulators, CCND1 and CDKN1B, was identified to have a negative prognostic role. The medial survival of patients with gastric cancer displaying amplification compared to patients without amplification was 192 versus 725 days for CCND1 (P = 0.0012) and 165 versus 611 days for CDKN1B (P = 0.0098). Conclusion. Gene amplifications of CCND1 and CDKN1B are potential candidates to serve as prognostic markers for the stratification of patients based on the estimate of survival in the management of gastric cancer patients.

  6. Making a Short Story Long: Regulation of P-TEFb and HIV-1 Transcriptional Elongation in CD4+ T Lymphocytes and Macrophages

    Directory of Open Access Journals (Sweden)

    Andrew P. Rice

    2012-06-01

    Full Text Available Productive transcription of the integrated HIV-1 provirus is restricted by cellular factors that inhibit RNA polymerase II elongation. The viral Tat protein overcomes this by recruiting a general elongation factor, P-TEFb, to the TAR RNA element that forms at the 5’ end of nascent viral transcripts. P-TEFb exists in multiple complexes in cells, and its core consists of a kinase, Cdk9, and a regulatory subunit, either Cyclin T1 or Cyclin T2. Tat binds directly to Cyclin T1 and thereby targets the Cyclin T1/P-TEFb complex that phosphorylates the CTD of RNA polymerase II and the negative factors that inhibit elongation, resulting in efficient transcriptional elongation. P-TEFb is tightly regulated in cells infected by HIV-1—CD4+ T lymphocytes and monocytes/macrophages. A number of mechanisms have been identified that inhibit P-TEFb in resting CD4+ T lymphocytes and monocytes, including miRNAs that repress Cyclin T1 protein expression and dephosphorylation of residue Thr186 in the Cdk9 T-loop. These repressive mechanisms are overcome upon T cell activation and macrophage differentiation when the permissivity for HIV-1 replication is greatly increased. This review will summarize what is currently known about mechanisms that regulate P-TEFb and how this regulation impacts HIV-1 replication and latency.

  7. A distinct expression pattern of cyclin K in mammalian testes suggests a functional role in spermatogenesis.

    Directory of Open Access Journals (Sweden)

    Xiaocong Xiang

    Full Text Available Germ cell and embryonic stem cells are inextricably linked in many aspects. Remarkably both can generate all somatic cell types in organisms. Yet the molecular regulation accounting for these similarities is not fully understood. Cyclin K was previously thought to associate with CDK9 to regulate gene expression. However, we and others have recently shown that its cognate interacting partners are CDK12 and CDK13 in mammalian cells. We further demonstrated that cyclin K is essential for embryonic stem cell maintenance. In this study, we examined the expression of cyclin K in various murine and human tissues. We found that cyclin K is highly expressed in mammalian testes in a developmentally regulated manner. During neonatal spermatogenesis, cyclin K is highly expressed in gonocytes and spermatogonial stem cells. In adult testes, cyclin K can be detected in spermatogonial stem cells but is absent in differentiating spermatogonia, spermatids and spermatozoa. Interestingly, the strongest expression of cyclin K is detected in primary spermatocytes. In addition, we found that cyclin K is highly expressed in human testicular cancers. Knockdown of cyclin K in a testicular cancer cell line markedly reduces cell proliferation. Collectively, we suggest that cyclin K may be a novel molecular link between germ cell development, cancer development and embryonic stem cell maintenance.

  8. Functional Variants at the 11q13 Risk Locus for Breast Cancer Regulate Cyclin D1 Expression through Long-Range Enhancers

    Science.gov (United States)

    French, Juliet D.; Ghoussaini, Maya; Edwards, Stacey L.; Meyer, Kerstin B.; Michailidou, Kyriaki; Ahmed, Shahana; Khan, Sofia; Maranian, Mel J.; O’Reilly, Martin; Hillman, Kristine M.; Betts, Joshua A.; Carroll, Thomas; Bailey, Peter J.; Dicks, Ed; Beesley, Jonathan; Tyrer, Jonathan; Maia, Ana-Teresa; Beck, Andrew; Knoblauch, Nicholas W.; Chen, Constance; Kraft, Peter; Barnes, Daniel; González-Neira, Anna; Alonso, M. Rosario; Herrero, Daniel; Tessier, Daniel C.; Vincent, Daniel; Bacot, Francois; Luccarini, Craig; Baynes, Caroline; Conroy, Don; Dennis, Joe; Bolla, Manjeet K.; Wang, Qin; Hopper, John L.; Southey, Melissa C.; Schmidt, Marjanka K.; Broeks, Annegien; Verhoef, Senno; Cornelissen, Sten; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A.; Loehberg, Christian R.; Ekici, Arif B.; Beckmann, Matthias W.; Peto, Julian; dos Santos Silva, Isabel; Johnson, Nichola; Aitken, Zoe; Sawyer, Elinor J.; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Marme, Frederik; Schneeweiss, Andreas; Sohn, Christof; Burwinkel, Barbara; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Menegaux, Florence; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Milne, Roger L.; Zamora, M. Pilar; Arias Perez, Jose Ignacio; Benitez, Javier; Anton-Culver, Hoda; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Lichtner, Peter; Schmutzler, Rita K.; Engel, Christoph; Brauch, Hiltrud; Hamann, Ute; Justenhoven, Christina; Aaltonen, Kirsimari; Heikkilä, Päivi; Aittomäki, Kristiina; Blomqvist, Carl; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Sueta, Aiko; Bogdanova, Natalia V.; Antonenkova, Natalia N.; Dörk, Thilo; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Wu, Anna H.; Tseng, Chiu-chen; Van Den Berg, David; Stram, Daniel O.; Lambrechts, Diether; Peeters, Stephanie; Smeets, Ann; Floris, Giuseppe; Chang-Claude, Jenny; Rudolph, Anja; Nickels, Stefan; Flesch-Janys, Dieter; Radice, Paolo; Peterlongo, Paolo; Bonanni, Bernardo; Sardella, Domenico; Couch, Fergus J.; Wang, Xianshu; Pankratz, Vernon S.; Lee, Adam; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Teo, Soo Hwang; Yip, Cheng Har; Ng, Char-Hong; Vithana, Eranga Nishanthie; Kristensen, Vessela; Zheng, Wei; Deming-Halverson, Sandra; Shrubsole, Martha; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Grip, Mervi; Andrulis, Irene L.; Knight, Julia A.; Glendon, Gord; Mulligan, Anna Marie; Devilee, Peter; Seynaeve, Caroline; García-Closas, Montserrat; Figueroa, Jonine; Chanock, Stephen J.; Lissowska, Jolanta; Czene, Kamila; Klevebring, Daniel; Schoof, Nils; Hooning, Maartje J.; Martens, John W.M.; Collée, J. Margriet; Tilanus-Linthorst, Madeleine; Hall, Per; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cox, Angela; Balasubramanian, Sabapathy P.; Blot, William; Signorello, Lisa B.; Cai, Qiuyin; Pharoah, Paul D.P.; Healey, Catherine S.; Shah, Mitul; Pooley, Karen A.; Kang, Daehee; Yoo, Keun-Young; Noh, Dong-Young; Hartman, Mikael; Miao, Hui; Sng, Jen-Hwei; Sim, Xueling; Jakubowska, Anna; Lubinski, Jan; Jaworska-Bieniek, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; McKay, James; Toland, Amanda E.; Ambrosone, Christine B.; Yannoukakos, Drakoulis; Godwin, Andrew K.; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Chen, Shou-Tung; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Schoemaker, Minouk J.; Ponder, Bruce A.J.; Nevanlinna, Heli; Brown, Melissa A.; Chenevix-Trench, Georgia; Easton, Douglas F.; Dunning, Alison M.

    2013-01-01

    Analysis of 4,405 variants in 89,050 European subjects from 41 case-control studies identified three independent association signals for estrogen-receptor-positive tumors at 11q13. The strongest signal maps to a transcriptional enhancer element in which the G allele of the best candidate causative variant rs554219 increases risk of breast cancer, reduces both binding of ELK4 transcription factor and luciferase activity in reporter assays, and may be associated with low cyclin D1 protein levels in tumors. Another candidate variant, rs78540526, lies in the same enhancer element. Risk association signal 2, rs75915166, creates a GATA3 binding site within a silencer element. Chromatin conformation studies demonstrate that these enhancer and silencer elements interact with each other and with their likely target gene, CCND1. PMID:23540573

  9. Cyclin D2 is sufficient to drive beta cell self-renewal and regeneration.

    Science.gov (United States)

    Tschen, Shuen-Ing; Zeng, Chun; Field, Loren; Dhawan, Sangeeta; Bhushan, Anil; Georgia, Senta

    2017-08-01

    Diabetes results from an inadequate mass of functional beta cells, due to either beta cell loss caused by autoimmune destruction (Type I diabetes) or beta cell failure in response to insulin resistance (Type II diabetes). Elucidating the mechanisms that regulate beta cell mass may be key to developing new techniques that foster beta cell regeneration as a cellular therapy to treat diabetes. While previous studies concluded that cyclin D2 is required for postnatal beta cell self-renewal in mice, it is not clear if cyclin D2 is sufficient to drive beta cell self-renewal. Using transgenic mice that overexpress cyclin D2 specifically in beta cells, we show that cyclin D2 overexpression increases beta cell self-renewal post-weaning and results in increased beta cell mass. Beta cells that overexpress cyclin D2 are responsive to glucose stimulation, suggesting they are functionally mature. Beta cells that overexpress cyclin D2 demonstrate an enhanced regenerative capacity after injury induced by streptozotocin toxicity. To understand if cyclin D2 overexpression is sufficient to drive beta cell self-renewal, we generated a novel mouse model where cyclin D2 is only expressed in beta cells of cyclin D2(-/-) mice. Transgenic overexpression of cyclin D2 in cyclin D2(-)(/)(-) beta cells was sufficient to restore beta cell mass, maintain normoglycaemia, and improve regenerative capacity when compared to cyclin D2(-/-) littermates. Taken together, our results indicate that cyclin D2 is sufficient to regulate beta cell self-renewal and that manipulation of its expression could be used to enhance beta cell regeneration.

  10. 长牡蛎细胞周期调控关键基因cyclin B3 的克隆及其在性腺发育中的作用%Molecular cloning and characterization of the key regulator of cell cycle cyclin B3 in Pacific Oyster (Crassostrea gigas), and its role in gonad development

    Institute of Scientific and Technical Information of China (English)

    王桐; 李莉; 阙华勇; 张国范

    2011-01-01

    首次在长牡蛎(Crassostrea gigas)中克隆得到细胞周期蛋白B3(cyclin B3)的cDNA 全长序列和基因组结构序列。cyclin B3 基因cDNA 全长2383 bp, 其中编码区长度为1293 bp, 编码一条含430 个氨基酸的多肽链, 氨基酸序列比对和结构域分析均表明其为其他物种cyclin B3 的同源蛋白, 预测的蛋白大小为47.8 kD。cyclin B3 基因含有10 个外显子和9 个内含子, 外显子和内含子的数目和大小与其物种的进化地位相符。Real Time PCR 分析表明, 该基因表达有较强的组织特异性, cyclin B3 mRNA 在性腺中的含量最高, 而在外套膜中的含量最少, 前者是后者的92 倍左右; 对处于不同发育阶段性腺中cyclin B3 基因表达分析结果显示, 性腺中cyclin B3 mRNA 含量随性腺发育程度的提高而提高, 这与其在细胞分裂和减数分裂中的作用相符, 同时也有助于满足早期胚胎发育时期旺盛的细胞分裂对cyclin B3 蛋白需求。%The full length cDNA sequence of cyclin B3 was cloned from Pacific oyster (Crassostrea gigas) for the first time. Cyclin B3 cDNA was 2383 bp in length, containing a 1293bp CDS that encoded a peptide of 430 amino acids with a calculated molecular weight of 47.8 kD. Multiple alignment and converted domain analysis showed that this peptide was a homolog of cyclin B3. The gDNA sequence of cyclin B3 contained 10 exons and 9 introns, which was consistent with the position in evolution of C. gigas. Expression of cyclin B3 gene was found in all tissues, but also showed a very strong tissue-specific feature; the amount of cyclin B3 in gonad was the highest among tissues, 100 times of mantle, the lowest expression tissue. The mRNA amount of cyclin B3 in gonad increased with the maturation of gonad. The expression implied the important role of cyclin B3 in mitosis, meiosis and early development of embryo.

  11. JunB breakdown in mid-/late G2 is required for down-regulation of cyclin A2 levels and proper mitosis.

    Science.gov (United States)

    Farràs, Rosa; Baldin, Véronique; Gallach, Sandra; Acquaviva, Claire; Bossis, Guillaume; Jariel-Encontre, Isabelle; Piechaczyk, Marc

    2008-06-01

    JunB, a member of the AP-1 family of dimeric transcription factors, is best known as a cell proliferation inhibitor, a senescence inducer, and a tumor suppressor, although it also has been attributed a cell division-promoting activity. Its effects on the cell cycle have been studied mostly in G1 and S phases, whereas its role in G2 and M phases still is elusive. Using cell synchronization experiments, we show that JunB levels, which are high in S phase, drop during mid- to late G2 phase due to accelerated phosphorylation-dependent degradation by the proteasome. The forced expression of an ectopic JunB protein in late G2 phase indicates that JunB decay is necessary for the subsequent reduction of cyclin A2 levels in prometaphase, the latter event being essential for proper mitosis. Consistently, abnormal JunB expression in late G2 phase entails a variety of mitotic defects. As these aberrations may cause genetic instability, our findings contrast with the acknowledged tumor suppressor activity of JunB and reveal a mechanism by which the deregulation of JunB might contribute to tumorigenesis.

  12. Cyclin-dependent kinases 7 and 9 specifically regulate neutrophil transcription and their inhibition drives apoptosis to promote resolution of inflammation

    Science.gov (United States)

    Leitch, A E; Lucas, C D; Marwick, J A; Duffin, R; Haslett, C; Rossi, A G

    2012-01-01

    Terminally differentiated neutrophils are short-lived but the key effector cells of the innate immune response, and have a prominent role in the pathogenesis and propagation of many inflammatory diseases. Delayed apoptosis, which is responsible for their extended longevity, is critically dependent on a balance of intracellular survival versus pro-apoptotic proteins. Here, we elucidate the mechanism by which the cyclin-dependent kinase (CDK) inhibitor drugs such as R-roscovitine and DRB (5,6-dichloro-1-beta-𝒟-ribofuranosylbenzimidazole) mediate neutrophil apoptosis. We demonstrate (by a combination of microarray, confocal microscopy, apoptosis assays and western blotting) that the phosphorylation of RNA polymerase II by CDKs 7 and 9 is inhibited by R-roscovitine and that specific effects on neutrophil transcriptional capacity are responsible for neutrophil apoptosis. Finally, we show that specific CDK7 and 9 inhibition with DRB drives resolution of neutrophil-dominant inflammation. Thus, we highlight a novel mechanism that controls both primary human neutrophil transcription and apoptosis that could be targeted by selective CDK inhibitor drugs to resolve established inflammation. PMID:22743999

  13. Some cyclin-dependent kinase inhibitors-related genes are regulated by vitamin C in a model of diet-induced obesity.

    Science.gov (United States)

    Boqué, Noemí; Campión, Javier; Milagro, Fermín Ignacio; Moreno-Aliaga, Maria-Jesús; Martinez, José Alfredo

    2009-08-01

    The aim of this research was to investigate differential gene expression of cyclin-dependent kinase inhibitors (CKIs) in white adipose tissue (WAT) and liver from high-fat fed male Wistar rats with or without vitamin C (VC) supplementation (750 mg/kg of body weight). After 56 d of experimentation, animals fed on a cafeteria diet increased significantly body weights and total body fat. Reverse transcription-polymerase chain reaction (RT-PCR) studies showed that cafeteria diet decreased p21 and p57 mRNA expression in subcutaneous WAT and increased p21 mRNA in liver. Overall, these data provide new information about the role of high fat intake on mRNA levels of several CKIs with implications in adipogenesis, cell metabolism and weight homeostasis. Interestingly, VC supplementation partially prevented diet-induced adiposity and increased p27 mRNA in liver without any changes in the other tissues and genes analyzed. Thus, hepatic mRNA changes induced by ascorbic acid indicate a possible role of these genes in diet-induced oxidative stress processes.

  14. Cyclin F/FBXO1 interacts with HIV-1 Vif and restricts progeny virion infectivity by ubiquitination and proteasomal degradation of Vif through SCF (Cyclin F) E3 ligase machinery.

    Science.gov (United States)

    Augustine, Tracy; Chaudhary, Priyanka; Gupta, Kailash; Islam, Sehbanul; Ghosh, Payel; Santra, Manas Kumar; Mitra, Debashis

    2017-02-09

    Cyclin F, also known as FBXO1, is the largest among all cyclins which oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, Cyclin F functions as the substrate binding subunit of SCFCyclin F E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4+ T cells, we observed down-regulation of Cyclin F (CCNF) gene. Later, using gene over expression and knockdown studies, we identified that Cyclin F negatively influences HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that Cyclin F negatively regulates the expression of viral protein, Vif (Viral infectivity factor), at the protein level. We also identified a novel host-pathogen interaction between Cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a Cyclin F-specific amino acid motif in the C-terminal region of Vif shows rescue of the protein from Cyclin F-mediated down-regulation. Subsequently, we have shown that Vif is a novel substrate of the SCFCyclin F E3 ligase, where Cyclin F mediates ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we have shown that Cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate Cyclin F as a novel F-box protein which functions as an intrinsic cellular regulator of HIV-1 Vif and imparts a negative regulatory effect on maintenance of viral infectivity by restoring APOBEC3G expression.

  15. T(1,1,t+1,t,1)和T(1,1,t+2,t,1)的色唯一性%Chromatic Uniqueness of T(1,1,t+1,t,1)and T(1,1,t+2,t,1)

    Institute of Scientific and Technical Information of China (English)

    毛建树; 刘儒英

    2009-01-01

    In this paper,let h(G,x)denote adjoining polynomial of graph G,and β(G)denote the minimum negative real roots of h(G,,x),we prove that T(1,1,t+1,t,1)andT(1,1,t+2,t,1)with t≥3 are chromatically unique.%h(G,x)表示图G的伴随多项式,β(G)表示h(G,x)的最小负实根.证明了当t≥3时,T(1,1,t+1,t,1)和T(1,1,t+2,t,1)的补图的色唯一性.

  16. Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control

    DEFF Research Database (Denmark)

    Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K

    2015-01-01

    Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays...... an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme...... that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein...

  17. A novel function for Cyclin A2: Control of cell invasion via RhoA signaling

    Science.gov (United States)

    Arsic, Nikola; Bendris, Nawal; Peter, Marion; Begon-Pescia, Christina; Rebouissou, Cosette; Gadéa, Gilles; Bouquier, Nathalie; Bibeau, Frédéric

    2012-01-01

    Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2–depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration. PMID:22232705

  18. Potential gene regulatory role for cyclin D3 in muscle cells

    Indian Academy of Sciences (India)

    Fathima Athar; Veena K Parnaik

    2015-09-01

    Cyclin D3 is important for muscle development and regeneration, and is involved in post-mitotic arrest of muscle cells. Cyclin D3 also has cell-cycle-independent functions such as regulation of specific genes in other tissues. Ectopic expression of cyclin D3 in myoblasts, where it is normally undetectable, promotes muscle gene expression and faster differentiation kinetics upon serum depletion. In the present study, we investigated the mechanistic role of cyclin D3 in muscle gene regulation. We initially showed by mutational analysis that a stable and functional cyclin D3 was required for promoting muscle differentiation. Using chromatin immunoprecipitation assays, we demonstrated that expression of cyclin D3 in undifferentiated myoblasts altered histone epigenetic marks at promoters of muscle-specific genes like MyoD, Pax7, myogenin and muscle creatine kinase but not non-muscle genes. Cyclin D3 expression also reduced the mRNA levels of certain epigenetic modifier genes. Our data suggest that epigenetic modulation of muscle-specific genes in cyclin-D3-expressing myoblasts may be responsible for faster differentiation kinetics upon serum depletion. Our results have implications for a regulatory role for cyclin D3 in muscle-specific gene activation.

  19. Promoter de-methylation of cyclin D2 by sulforaphane in prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Hsu Anna

    2011-10-01

    Full Text Available Abstract Sulforaphane (SFN, an isothiocyanate derived from cruciferous vegetables, induces potent anti-proliferative effects in prostate cancer cells. One mechanism that may contribute to the anti-proliferative effects of SFN is the modulation of epigenetic marks, such as inhibition of histone deacetylase (HDAC enzymes. However, the effects of SFN on other common epigenetic marks such as DNA methylation are understudied. Promoter hyper-methylation of cyclin D2, a major regulator of cell cycle, is correlated with prostate cancer progression, and restoration of cyclin D2 expression exerts anti-proliferative effects on LnCap prostate cancer cells. Our study aimed to investigate the effects of SFN on DNA methylation status of cyclin D2 promoter, and how alteration in promoter methylation impacts cyclin D2 gene expression in LnCap cells. We found that SFN significantly decreased the expression of DNA methyltransferases (DNMTs, especially DNMT1 and DNMT3b. Furthermore, SFN significantly decreased methylation in cyclin D2 promoter regions containing c-Myc and multiple Sp1 binding sites. Reduced methlyation of cyclin D2 promoter corresponded to an increase in cyclin D2 transcript levels, suggesting that SFN may de-repress methylation-silenced cyclin D2 by impacting epigenetic pathways. Our results demonstrated the ability of SFN to epigenetically modulate cyclin D2 expression, and provide novel insights into the mechanisms by which SFN may regulate gene expression as a prostate cancer chemopreventive agent.

  20. Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis

    Institute of Scientific and Technical Information of China (English)

    Qiang Chen; Xiaoyan Zhang; Qing Jiang; Paul R Clarke; Chuanmao Zhang

    2008-01-01

    Cyclin Bl is a key regulatory protein controlling cell cycle progression in vertebrates. Cyclin Bl binds CDK1, a cyclin-dependent kinase catalytic subunit, forming a complex that orchestrates mitosis through phosphorylation of key proteins. Cyclin Bl regulates both the activation of CDK1 and its subcellular localization, which may be critical for substrate selection. Here, we demonstrate that cyclin Bl is concentrated on the outer plate of the kinetochore during prometaphase. This localization requires the cyclin box region of the protein. Cyclin Bl is displaced from individual kinetochores to the spindle poles by microtubule attachment to the kinetochores, and this displacement is dependent on the dynein/dynactin complex. Depletion of cyclin Bl by vector-based siRNA causes inefficient attachment between kinetochores and microtubules, and chromosome alignment defects, and delays the onset of anaphase. We conclude that cyclin Bl accumulates at kinetochores during prometaphase, where it contributes to the correct attachment of microtubules to kinetochores and efficient alignment of the chromosomes, most likely through localized phosphorylation of specific substrates by cyclin B1-CDK1. Cyclin Bl is then transported from each kinetochore as microtubule attachment is completed, and this relocalization may redirect the activity of cyclin B1-CDK1 and contribute to inactivation of the spindle assembly checkpoint.

  1. Maternal gestational betaine supplementation-mediated suppression of hepatic cyclin D2 and presenilin1 gene in newborn piglets is associated with epigenetic regulation of the STAT3-dependent pathway.

    Science.gov (United States)

    Cai, Demin; Yuan, Mengjie; Jia, Yimin; Liu, Haoyu; Hu, Yun; Zhao, Ruqian

    2015-12-01

    Betaine, which donates methyl groups through methionine metabolism for DNA and protein methylation, is critical for epigenetic gene regulation, especially during fetal development. Here we fed gestational sows with control or betaine supplemented diets (3 g/kg) throughout the pregnancy to explore the effects of maternal betaine on hepatic cell proliferation in neonatal piglets. Neonatal piglets born to betaine-supplemented sows demonstrated a reduction of cell number and DNA content in the liver, which was associated with significantly down-regulated hepatic expression of cell cycle regulatory genes, cyclin D2 (CCND2) and presenilin1 (PSEN1). Moreover, STAT3 binding to the promoter of CCND2 and PSEN1 was also lower in betaine-exposed piglets, accompanied by strong reduction of STAT3 mRNA and protein expression, along with its phosphorylation at Tyr705 and Ser727 residues. Also, prenatal betaine exposure significantly attenuated upstream kinases of STAT3 signaling pathway (phospho-ERK1/2, phospho-SRC and phospho-JAK2) in the livers of neonates. Furthermore, the repressed STAT3 expression in the liver of betaine-exposed piglets was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3 on its promoter, together with significantly up-regulated expression of H3K27me3 and enhancer of zeste homolog 2 (EZH2) proteins, as well as miR-124a, which targets STAT3. Taken together, our results suggest that maternal dietary betaine supplementation during gestation inhibits hepatic cell proliferation in neonatal piglets, at least partly, through epigenetic regulation of hepatic CCND2 and PSEN1 genes via a STAT3-dependent pathway. These neonatal changes in cell cycle and proliferation regulation may lead to lower liver weight and hepatic DNA content at weaning.

  2. Cyclin A2 Is Required for Sister Chromatid Segregation, But Not Separase Control, in Mouse Oocyte Meiosis

    Directory of Open Access Journals (Sweden)

    Sandra A. Touati

    2012-11-01

    Full Text Available In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis.

  3. Cyclin A2 is required for sister chromatid segregation, but not separase control, in mouse oocyte meiosis.

    Science.gov (United States)

    Touati, Sandra A; Cladière, Damien; Lister, Lisa M; Leontiou, Ioanna; Chambon, Jean-Philippe; Rattani, Ahmed; Böttger, Franziska; Stemmann, Olaf; Nasmyth, Kim; Herbert, Mary; Wassmann, Katja

    2012-11-29

    In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis.

  4. miR-340 inhibits glioblastoma cell proliferation by suppressing CDK6, cyclin-D1 and cyclin-D2

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xuesong; Gong, Xuhai [Department of Neurology, Daqing Oilfield General Hospital, Daqing, Heilongjiang 163001 (China); Chen, Jing [Department of Neurology, Daqing Longnan Hospital, Daqing, Heilongjiang, 163001 China (China); Zhang, Jinghui [Department of Cardiology, The Fourth Hospital of Harbin City, Harbin, Heilongjiang 150026 (China); Sun, Jiahang [Department of Neurosurgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086 (China); Guo, Mian, E-mail: guomian_hyd@163.com [Department of Neurosurgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086 (China)

    2015-05-08

    Glioblastoma development is often associated with alteration in the activity and expression of cell cycle regulators, such as cyclin-dependent kinases (CKDs) and cyclins, resulting in aberrant cell proliferation. Recent studies have highlighted the pivotal roles of miRNAs in controlling the development and growth of glioblastoma. Here, we provide evidence for a function of miR-340 in the inhibition of glioblastoma cell proliferation. We found that miR-340 is downregulated in human glioblastoma tissue samples and several established glioblastoma cell lines. Proliferation and neurosphere formation assays revealed that miR-340 plays an oncosuppressive role in glioblastoma, and that its ectopic expression causes significant defect in glioblastoma cell growth. Further, using bioinformatics, luciferase assay and western blot, we found that miR-340 specifically targets the 3′UTRs of CDK6, cyclin-D1 and cyclin-D2, leading to the arrest of glioblastoma cells in the G0/G1 cell cycle phase. Confirming these results, we found that re-introducing CDK6, cyclin-D1 or cyclin-D2 expression partially, but significantly, rescues cells from the suppression of cell proliferation and cell cycle arrest mediated by miR-340. Collectively, our results demonstrate that miR-340 plays a tumor-suppressive role in glioblastoma and may be useful as a diagnostic biomarker and/or a therapeutic avenue for glioblastoma. - Highlights: • miR-340 is downregulated in glioblastoma samples and cell lines. • miR-340 inhibits glioblastoma cell proliferation. • miR-340 directly targets CDK6, cyclin-D1, and cyclin-D2. • miR-340 regulates glioblastoma cell proliferation via CDK6, cyclin-D1 and cyclin-D2.

  5. Chromosomal and Extrachromosomal Instability of the cyclin D2 Gene is Induced by Myc Overexpression

    Directory of Open Access Journals (Sweden)

    Sabine Mai

    1999-08-01

    Full Text Available We examined the expression of cyclins D1, D2, D3, and E in mouse B-lymphocytic tumors. Cyclin D2 mRNA was consistently elevated in plasmacytomas, which characteristically contain Myc-activating chromosome translocations and constitutive c-Myc mRNA and protein expression. We examined the nature of cyclin D2 overexpression in plasmacytomas and other tumors. Human and mouse tumor cell lines that exhibited c-Myc dysregulation displayed instability of the cyclin D2 gene, detected by Southern blot, fluorescent in situ hybridization (FISH, and in extrachromosomal preparations (Hirt extracts. Cyclin D2 instability was not seen in cells with low levels of c-Myc protein. To unequivocally demonstrate a role of c-Myc in the instability of the cyclin D2 gene, a Myc-estrogen receptor chimera was activated in two mouse cell lines. After 3 to 4 days of Myc-ERTm activation, instability at the cyclin D2 locus was seen in the form of extrachromosomal elements, determined by FISH of metaphase and interphase nuclei and of purified extrachromosomal elements. At the same time points, Northern and Western blot analyses detected increased cyclin D2 mRNA and protein levels. These data suggest that Myc-induced genomic instability may contribute to neoplasia by increasing the levels of a cell cycle—regulating protein, cyclin D2, via intrachromosomal amplification of its gene or generation of extrachromosomal copies.

  6. Translokin (Cep57) interacts with cyclin D1 and prevents its nuclear accumulation in quiescent fibroblasts.

    Science.gov (United States)

    Ruiz-Miró, Maria; Colomina, Neus; Fernández, Rita M H; Garí, Eloi; Gallego, Carme; Aldea, Martí

    2011-05-01

    Nuclear accumulation of cyclin D1 because of altered trafficking or degradation is thought to contribute directly to neoplastic transformation and growth. Mechanisms of cyclin D1 localization in S phase have been studied in detail, but its control during exit from the cell cycle and quiescence is poorly understood. Here we report that translokin (Tlk), a microtubule-associated protein also termed Cep57, interacts with cyclin D1 and controls its nucleocytoplasmic distribution in quiescent cells. Tlk binds to regions of cyclin D1 also involved in binding to cyclin-dependent kinase 4 (Cdk4), and a fraction of cyclin D1 associates to the juxtanuclear Tlk network in the cell. Downregulation of Tlk levels results in undue nuclear accumulation of cyclin D1 and increased Cdk4-dependent phosphorylation of pRB under quiescence conditions. In turn, overexpression of Tlk prevents proper cyclin D1 accumulation in the nucleus of proliferating cells in an interaction-dependent manner, inhibits Cdk4-dependent phosphorylation of pRB and hinders cell cycle progression to S phase. We propose that the Tlk acts as a key negative regulator in the pathway that drives nuclear import of cyclin D1, thus contributing to prevent pRB inactivation and to maintain cellular quiescence.

  7. A critical role for FBXW8 and MAPK in cyclin D1 degradation and cancer cell proliferation.

    Directory of Open Access Journals (Sweden)

    Hiroshi Okabe

    Full Text Available Cyclin D1 regulates G1 progression. Its transcriptional regulation is well understood. However, the mechanism underlying cyclin D1 ubiquitination and its subsequent degradation is not yet clear. We report that cyclin D1 undergoes increased degradation in the cytoplasm during S phase in a variety of cancer cells. This is mediated by phosphorylation at Thr286 through the activity of the Ras/Raf/MEK/ERK cascade and the F-box protein FBXW8, which is an E3 ligase. The majority of FBXW8 is expressed in the cytoplasm during G1 and S phase. In contrast, cyclin D1 accumulates in the nucleus during G1 phase and exits into the cytoplasm in S phase. Increased cyclin D1 degradation is linked to association with FBXW8 in the cytoplasm, and enhanced phosphorylation of cyclin D1 through sustained ERK1/2 signaling. Depletion of FBXW8 caused a significant accumulation of cyclin D1, as well as sequestration of CDK1 in the cytoplasm. This resulted in a severe reduction of cell proliferation. These effects could be rescued by constitutive nuclear expression of cyclin D1-T286A. Thus, FBXW8 plays an essential role in cancer cell proliferation through proteolysis of cyclin D1. It may present new opportunities to develop therapies targeting destruction of cyclin D1 or its regulator E3 ligase selectively.

  8. Aortic endothelial cells regulate proliferation of human monocytes in vitro via a mechanism synergistic with macrophage colony-stimulating factor. Convergence at the cyclin E/p27(Kip1) regulatory checkpoint.

    Science.gov (United States)

    Antonov, A S; Munn, D H; Kolodgie, F D; Virmani, R; Gerrity, R G

    1997-06-15

    Monocyte-derived macrophages (Mphis) are pivotal participants in the pathogenesis of atherosclerosis. Evidence from both animal and human plaques indicates that local proliferation may contribute to accumulation of lesion Mphis, and the major Mphi growth factor, macrophage colony stimulating factor (MCSF), is present in atherosclerotic plaques. However, most in vitro studies have failed to demonstrate that human monocytes/Mphis possess significant proliferative capacity. We now report that, although human monocytes cultured in isolation showed only limited MCSF-induced proliferation, monocytes cocultured with aortic endothelial cells at identical MCSF concentrations underwent enhanced (up to 40-fold) and prolonged (21 d) proliferation. In contrast with monocytes in isolation, this was optimal at low seeding densities, required endothelial cell contact, and could not be reproduced by coculture with smooth muscle cells. Intimal Mphi isolated from human aortas likewise showed endothelial cell contact-dependent, MCSF-induced proliferation. Consistent with a two-signal mechanism governing Mphi proliferation, the cell cycle regulatory protein, cyclin E, was rapidly upregulated by endothelial cell contact in an MCSFindependent fashion, but MCSF was required for successful downregulation of the cell cycle inhibitory protein p27(Kip1) before cell cycling. Thus endothelial cells and MCSF differentially and synergistically regulate two Mphi genes critical for progression through the cell cycle.

  9. Cyclin D1 inhibits whereas c-Myc enhances the cytotoxicity of cisplatin in mouse pancreatic cancer cells via regulation of several members of the NF-κB and Bcl-2 families

    Directory of Open Access Journals (Sweden)

    Ayman El-Kady

    2011-01-01

    Full Text Available Background: Cisplatin (CDDP is a drug used for treatment of many types of malignancy but pancreatic cancer is relatively resistant to it. This study aims to determine whether and how cyclin D1 (D1 and c-Myc influence the response of pancreatic cancer cells to CDDP. Materials and Methods: Ela-mycPT mouse pancreatic cancer cells were transfected with D1 or c-myc cDNA and treated with CDDP alone or together with NPCD, an inhibitor of cyclin dependent ckinase (CDK 4 and 6. Reverse transcription followed by polymerase chain reaction (RT-PCR and western blot assays were used to determine the mRNA and protein levels of interested genes. Cell viability was determined using 3-(4, 5-Dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT assay. Results: Treatment of Ela-mycPT1 cells with CDDP caused an increase in c-myc expression but a slightly latent decrease in D1 expression, whereas D1 and c-Myc proteins repressed each other. D1 or c-Myc rendered Ela-mycPT1 cells resistant or sensitive, respectively, to CDDP. D1 induced the expression of several members of the NF-κB family, including RelA, RelB, Nfκb1 and Nfκb2. D1 also induced BIRC5 and several pro-survival members of the Bcl-2 gene family, including Bcl-2, Mcl-1 and Bad while it decreased the level of the pro-apoptotic Noxa. Inhibition of CDK4 or CDK6 kinase activity by NPCD did not affect these effects of D1. In contrast, c-Myc in Ela-mycPT1 and Ela-mycPT4 cells has the opposite effects to D1 on the expression of most of these apoptosis regulating genes. Conclusion: Our results suggest that induction of c-Myc and inhibition of D1 may be mechanisms for CDDP to elicit cytotoxicity. On the other hand, D1 induces whereas c-Myc represses the expression of key NF-κB family members to induce and repress, respectively, the expression of BIRC5 and several Bcl-2 family members, in turn inhibiting or enhancing the response to CDDP.

  10. Cyclin E-p27 opposition and regulation of the G1 phase of the cell cycle in the murine neocortical PVE: a quantitative analysis of mRNA in situ hybridization

    Science.gov (United States)

    Delalle, I.; Takahashi, T.; Nowakowski, R. S.; Tsai, L. H.; Caviness, V. S. Jr

    1999-01-01

    We have analyzed the expression patterns of mRNAs of five cell cycle related proteins in the ventricular zone of the neocortical cerebral wall over the course of the neuronogenetic interval in the mouse. One set of mRNAs (cyclin E and p21) are initially expressed at high levels but expression then falls to a low asymptote. A second set (p27, cyclin B and cdk2) are initially expressed at low levels but ascend to peak levels only to decline again. These patterns divide the overall neuronogenetic interval into three phases. In phase 1 cyclin E and p21 levels of mRNA expression are high, while those of mRNAs of p27, cdk2 and cyclin B are low. In this phase the fraction of cells leaving the cycle after each mitosis, Q, is low and the duration of the G1 phase, TG1, is short. In phase 2 levels of expression of cyclin E and p21 fall to asymptote while levels of expression of mRNA of the other three proteins reach their peaks. Q increases to approach 0.5 and TG1 increases even more rapidly to approach its maximum length. In phase 3 levels of expression of cyclin E and p21 mRNAs remain low and those of the mRNAs of the other three proteins fall. TG1 becomes maximum and Q rapidly increases to 1.0. The character of these phases can be understood in part as consequences of the reciprocal regulatory influence of p27 and cyclin E and of the rate limiting functions of p27 at the restriction point and of cyclin E at the G1 to S transition.

  11. Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Sang Hyeok; Seo, Sung-Keum [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); An, Sungkwan; Choe, Tae-Boo [Department of Microbiological Engineering, Kon-Kuk University, Gwangjin-gu, Seoul (Korea, Republic of); Hong, Seok-Il [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Lee, Yun-Han, E-mail: yhlee87@yuhs.ac [Department of Radiation Oncology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Park, In-Chul, E-mail: parkic@kcch.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of)

    2014-10-24

    Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

  12. E-type cyclins modulate telomere integrity in mammalian male meiosis.

    Science.gov (United States)

    Manterola, Marcia; Sicinski, Piotr; Wolgemuth, Debra J

    2016-06-01

    We have shown that E-type cyclins are key regulators of mammalian male meiosis. Depletion of cyclin E2 reduced fertility in male mice due to meiotic defects, involving abnormal pairing and synapsis, unrepaired DNA, and loss of telomere structure. These defects were exacerbated by additional loss of cyclin E1, and complete absence of both E-type cyclins produces a meiotic catastrophe. Here, we investigated the involvement of E-type cyclins in maintaining telomere integrity in male meiosis. Spermatocytes lacking cyclin E2 and one E1 allele (E1+/-E2-/-) displayed a high rate of telomere abnormalities but can progress to pachytene and diplotene stages. We show that their telomeres exhibited an aberrant DNA damage repair response during pachynema and that the shelterin complex proteins TRF2 and RAP2 were significantly decreased in the proximal telomeres. Moreover, the insufficient level of these proteins correlated with an increase of γ-H2AX foci in the affected telomeres and resulted in telomere associations involving TRF1 and telomere detachment in later prophase-I stages. These results suggest that E-type cyclins are key modulators of telomere integrity during meiosis by, at least in part, maintaining the balance of shelterin complex proteins, and uncover a novel role of E-type cyclins in regulating chromosome structure during male meiosis.

  13. Regulation of human endometrial stromal proliferation and differentiation by C/EBPβ involves cyclin E-cdk2 and STAT3.

    Science.gov (United States)

    Wang, Wei; Taylor, Robert N; Bagchi, Indrani C; Bagchi, Milan K

    2012-12-01

    During each menstrual cycle, the human uterus undergoes a unique transformation, known as decidualization, which involves endometrial stromal proliferation and differentiation. During this process, the stromal cells are transformed into decidual cells, which produce factors that prepare the uterus for potential embryo implantation. We previously identified the transcription factor CCAAT/enhancer-binding protein (C/EBP)β as a regulator of endometrial stromal proliferation and differentiation in mice. In this study, we addressed the role of C/EBPβ in human endometrial decidualization. Using small interfering RNA targeted to C/EBPβ mRNA, we demonstrated that C/EBPβ controls the proliferation of primary human endometrial stromal cells (HESCs) by regulating the expression of several key cell cycle-regulatory factors during the G(1)-S phase transition. Additionally, loss of C/EBPβ expression blocked the differentiation of HESCs in response to estrogen, progesterone, and cyclic AMP. Gene expression profiling of normal and C/EBPβ-deficient HESCs revealed that the receptor for the cytokine IL-11 and its downstream signal transducer signal transducer and activator of transcription 3 (STAT3) are targets of regulation by C/EBPβ. Chromatin immunoprecipitation analysis indicated that C/EBPβ controls the expression of STAT3 gene by directly interacting with a distinct regulatory sequence in its 5'-flanking region. Attenuation of STAT3 mRNA expression in HESCs resulted in markedly reduced differentiation of these cells, indicating an important role for STAT3 in decidualization. Gene expression profiling, using STAT3-deficient HESCs, showed an extensive overlap of pathways downstream of STAT3 and C/EBPβ during stromal cell differentiation. Collectively, these findings revealed a novel functional link between C/EBPβ and STAT3 that is a critical regulator of endometrial differentiation in women.

  14. Regulation of the G1/S Transition in Hepatocytes: Involvement of the Cyclin-Dependent Kinase Cdk1 in the DNA Replication

    Directory of Open Access Journals (Sweden)

    Anne Corlu

    2012-01-01

    Full Text Available A singular feature of adult differentiated hepatocytes is their capacity to proliferate allowing liver regeneration. This review emphasizes the literature published over the last 20 years that established the most important pathways regulating the hepatocyte cell cycle. Our article also aimed at illustrating that many discoveries in this field benefited from the combined use of in vivo models of liver regeneration and in vitro models of primary cultures of human and rodent hepatocytes. Using these models, our laboratory has contributed to decipher the different steps of the progression into the G1 phase and the commitment to S phase of proliferating hepatocytes. We identified the mitogen dependent restriction point located at the two-thirds of the G1 phase and the concomitant expression and activation of both Cdk1 and Cdk2 at the G1/S transition. Furthermore, we demonstrated that these two Cdks contribute to the DNA replication. Finally, we provided strong evidences that Cdk1 expression and activation is correlated to extracellular matrix degradation upon stimulation by the pro-inflammatory cytokine TNFα leading to the identification of a new signaling pathway regulating Cdk1 expression at the G1/S transition. It also further confirms the well-orchestrated regulation of liver regeneration via multiple extracellular signals and pathways.

  15. Concurrent Deletion of Cyclin E1 and Cyclin-Dependent Kinase 2 in Hepatocytes Inhibits DNA Replication and Liver Regeneration in Mice

    OpenAIRE

    Hu W; Nevzorova YA; Haas U; Moro N.; Sicinski P; Geng Y; Barbacid M; Trautwein C; Liedtke C.

    2014-01-01

    The liver has a strong regenerative capacity. After injury, quiescent hepatocytes can reenter the mitotic cell cycle to restore tissue homeostasis. This G0/G1-S cell-cycle transition of primed hepatocytes is regulated by complexes of cyclin-dependent kinase 2 (Cdk2) with E-type cyclins (CcnE1 or CcnE2). However, single genetic ablation of either E-cyclin or Cdk2 does not affect overall liver regeneration. Here, we systematically investigated the contribution of CcnE1, CcnE2, and Cdk2 for live...

  16. Cyclin D1 represses gluconeogenesis via inhibition of the transcriptional coactivator PGC1α.

    Science.gov (United States)

    Bhalla, Kavita; Liu, Wan-Ju; Thompson, Keyata; Anders, Lars; Devarakonda, Srikripa; Dewi, Ruby; Buckley, Stephanie; Hwang, Bor-Jang; Polster, Brian; Dorsey, Susan G; Sun, Yezhou; Sicinski, Piotr; Girnun, Geoffrey D

    2014-10-01

    Hepatic gluconeogenesis is crucial to maintain normal blood glucose during periods of nutrient deprivation. Gluconeogenesis is controlled at multiple levels by a variety of signal transduction and transcriptional pathways. However, dysregulation of these pathways leads to hyperglycemia and type 2 diabetes. While the effects of various signaling pathways on gluconeogenesis are well established, the downstream signaling events repressing gluconeogenic gene expression are not as well understood. The cell-cycle regulator cyclin D1 is expressed in the liver, despite the liver being a quiescent tissue. The most well-studied function of cyclin D1 is activation of cyclin-dependent kinase 4 (CDK4), promoting progression of the cell cycle. We show here a novel role for cyclin D1 as a regulator of gluconeogenic and oxidative phosphorylation (OxPhos) gene expression. In mice, fasting decreases liver cyclin D1 expression, while refeeding induces cyclin D1 expression. Inhibition of CDK4 enhances the gluconeogenic gene expression, whereas cyclin D1-mediated activation of CDK4 represses the gluconeogenic gene-expression program in vitro and in vivo. Importantly, we show that cyclin D1 represses gluconeogenesis and OxPhos in part via inhibition of peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) activity in a CDK4-dependent manner. Indeed, we demonstrate that PGC1α is novel cyclin D1/CDK4 substrate. These studies reveal a novel role for cyclin D1 on metabolism via PGC1α and reveal a potential link between cell-cycle regulation and metabolic control of glucose homeostasis.

  17. Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells

    Directory of Open Access Journals (Sweden)

    Choi Yoon

    2009-05-01

    Full Text Available Abstract Background Maintaining the correct balance of proliferation versus differentiation in retinal progenitor cells (RPCs is essential for proper development of the retina. The cell cycle regulator cyclin D1 is expressed in RPCs, and mice with a targeted null allele at the cyclin D1 locus (Ccnd1-/- have microphthalmia and hypocellular retinas, the latter phenotype attributed to reduced RPC proliferation and increased photoreceptor cell death during the postnatal period. How cyclin D1 influences RPC behavior, especially during the embryonic period, is unclear. Results In this study, we show that embryonic RPCs lacking cyclin D1 progress through the cell cycle at a slower rate and exit the cell cycle at a faster rate. Consistent with enhanced cell cycle exit, the relative proportions of cell types born in the embryonic period, such as retinal ganglion cells and photoreceptor cells, are increased. Unexpectedly, cyclin D1 deficiency decreases the proportions of other early born retinal neurons, namely horizontal cells and specific amacrine cell types. We also found that the laminar positioning of horizontal cells and other cell types is altered in the absence of cyclin D1. Genetically replacing cyclin D1 with cyclin D2 is not efficient at correcting the phenotypes due to the cyclin D1 deficiency, which suggests the D-cyclins are not fully redundant. Replacement with cyclin E or inactivation of cyclin-dependent kinase inhibitor p27Kip1 restores the balance of RPCs and retinal cell types to more normal distributions, which suggests that regulation of the retinoblastoma pathway is an important function for cyclin D1 during embryonic retinal development. Conclusion Our findings show that cyclin D1 has important roles in RPC cell cycle regulation and retinal histogenesis. The reduction in the RPC population due to a longer cell cycle time and to an enhanced rate of cell cycle exit are likely to be the primary factors driving retinal hypocellularity

  18. Histone deacetylase inhibitor, Trichostatin A induces ubiquitin-dependent cyclin D1 degradation in MCF-7 breast cancer cells

    Directory of Open Access Journals (Sweden)

    Charles Coombes R

    2006-02-01

    Full Text Available Abstract Background Cyclin D1 is an important regulator of G1-S phase cell cycle transition and has been shown to be important for breast cancer development. GSK3β phosphorylates cyclin D1 on Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of the cyclin in the cytoplasm. Recent findings suggest that the development of small-molecule cyclin D1 ablative agents is of clinical relevance. We have previously shown that the histone deacetylase inhibitor trichostatin A (TSA induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells prior to repression of cyclin D1 gene (CCND1 transcription. TSA treatment also resulted in accumulation of polyubiquitylated GFP-cyclin D1 species and reduced levels of the recombinant protein within the nucleus. Results Here we provide further evidence for TSA-induced ubiquitin-dependent degradation of cyclin D1 and demonstrate that GSK3β-mediated nuclear export facilitates this activity. Our observations suggest that TSA treatment results in enhanced cyclin D1 degradation via the GSK3β/CRM1-dependent nuclear export/26S proteasomal degradation pathway in MCF-7 cells. Conclusion We have demonstrated that rapid TSA-induced cyclin D1 degradation in MCF-7 cells requires GSK3β-mediated Thr-286 phosphorylation and the ubiquitin-dependent 26S proteasome pathway. Drug induced cyclin D1 repression contributes to the inhibition of breast cancer cell proliferation and can sensitize cells to CDK and Akt inhibitors. In addition, anti-cyclin D1 therapy may be highly specific for treating human breast cancer. The development of potent and effective cyclin D1 ablative agents is therefore of clinical relevance. Our findings suggest that HDAC inhibitors may have therapeutic potential as small-molecule cyclin D1 ablative agents.

  19. D-type cyclins in adult human testis and testicular cancer

    DEFF Research Database (Denmark)

    Bartkova, J; Rajpert-de Meyts, E; Skakkebaek, N E

    1999-01-01

    on immunohistochemical and immunochemical analysis of human adult testis and 32 testicular tumours to examine the differential expression and abundance of cyclins D1, D2, and D3 in relation to cell type, proliferation, differentiation, and malignancy. In normal testis, the cell type-restricted expression patterns were...... D2 but not D1 or D3, while the invasive testicular tumours showed variable positivity for cyclins D2 and D3, but rarely D1. An unexpected correlation with differentiation rather than proliferation was found particularly for cyclin D3 in teratomas, a conceptually significant observation confirmed...... by massive up-regulation of cyclin D3 in the human teratocarcinoma cell line NTera2/D1 induced to differentiate along the neuronal lineage. These results suggest a possible involvement of cyclin D2 in the early stages of testicular oncogenesis and the striking examples of proliferation-independent expression...

  20. Control of cyclin C levels during development of Dictyostelium.

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    David M Greene

    Full Text Available BACKGROUND: Cdk8 and its partner cyclin C form part of the mediator complex which links the basal transcription machinery to regulatory proteins. The pair are required for correct regulation of a subset of genes and have been implicated in control of development in a number of organisms including the social amoeba Dictyostelium discoideum. When feeding, Dictyostelium amoebae are unicellular but upon starvation they aggregate to form a multicellular structure which develops into a fruiting body containing spores. Cells in which the gene encoding Cdk8 has been deleted fail to enter aggregates due to a failure of early gene expression. PRINCIPAL FINDINGS: We have monitored the expression levels of cyclin C protein during development and find levels decrease after the multicellular mound is formed. This decrease is triggered by extracellular cAMP that, in turn, is working in part through an increase in intracellular cAMP. The loss of cyclin C is coincident with a reduction in the association of Cdk8 with a high molecular weight complex in the nucleus. Overexpression of cyclin C and Cdk8 lead to an increased rate of early development, consistent with the levels being rate limiting. CONCLUSIONS: Overall these results show that both cyclin C and Cdk8 are regulated during development in response to extracellular signals and the levels of these proteins are important in controlling the timing of developmental processes. These findings have important implications for the role of these proteins in controlling development, suggesting that they are targets for developmental signals to regulate gene expression.

  1. The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast.

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    Richard C Silva

    Full Text Available The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle.

  2. Active Component of Danshen (Salvia miltiorrhiza Bunge, Tanshinone I, Attenuates Lung Tumorigenesis via Inhibitions of VEGF, Cyclin A, and Cyclin B Expressions

    Directory of Open Access Journals (Sweden)

    Yu-Tang Tung

    2013-01-01

    Full Text Available Tanshinone I (T1 and tanshinone II (T2 are the major diterpenes isolated from Danshen (Salvia miltiorrhiza Bunge. Three human lung adenocarcinoma cell lines, A549, CL1-0, and CL1-5, were treated with T1 and T2 for the in vitro antitumor test. Results showed that T1 was more effective than T2 in inhibiting the growth of lung cancer cells via suppressing the expression of VEGF, Cyclin A, and Cyclin B proteins in a dose-dependent manner. Moreover, a transgenic mice model of the human vascular endothelial growth factor-A165 (hVEGF-A165 gene-induced pulmonary tumor was further treated with T1 for the in vivo lung cancer therapy test. T1 significantly attenuated hVEGF-A165 overexpression to normal levels of the transgenic mice (Tg that were pretreated with human monocytic leukemia THP-1 cell-derived conditioned medium (CM. It also suppressed the formation of lung adenocarcinoma tumors (16.7% compared with two placebo groups (50% for Tg/Placebo and 83.3% for Tg/CM/Placebo; P<0.01. This antitumor effect is likely to slow the progression of cells through the S and G2/M phases of the cell cycle. Blocking of the tumor-activated cell cycle pathway may be a critical mechanism for the observed antitumorigenic effects of T1 treatment on vasculogenesis and angiogenesis.

  3. Cyc17, a meiosis-specific cyclin, is essential for anaphase initiation and chromosome segregation in Tetrahymena thermophila.

    Science.gov (United States)

    Yan, Guan-Xiong; Dang, Huai; Tian, Miao; Zhang, Jing; Shodhan, Anura; Ning, Ying-Zhi; Xiong, Jie; Miao, Wei

    2016-07-17

    Although the role of cyclins in controlling nuclear division is well established, their function in ciliate meiosis remains unknown. In ciliates, the cyclin family has undergone massive expansion which suggests that diverse cell cycle systems exist, and this warrants further investigation. A screen for cyclins in the model ciliate Tetrahymena thermophila showed that there are 34 cyclins in this organism. Only 1 cyclin, Cyc17, contains the complete cyclin core and is specifically expressed during meiosis. Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings. Further investigation suggested that Cyc17 is involved in regulating spindle pole attachment, and is thus essential for chromosome segregation at meiosis. These findings suggest a simple model in which chromosome segregation is influenced by Cyc17.

  4. Molecular cloning and functional characterization of cyclin E and CDK2 from Penaeus monodon.

    Science.gov (United States)

    Zhao, C; Fu, M J; Qiu, L H

    2016-09-16

    Reduced reproductive performance of the black tiger shrimp (Penaeus monodon) has caused economic losses and hampered the fishing industry. Detailed investigation of the molecular mechanism by which the cell cycle is regulated in this organism is needed to understand the development and maturation of ovaries and oocytes, with a view to improving reproductive capacity. Cell cycle progression is mainly determined by cyclin-dependent kinase (CDK) and cyclin complexes, the cyclin E/CDK2 complex playing a key role in G1/S transition. However, knowledge of the interplay between cyclin E and CDK2 in invertebrates remains limited. In this study, full-length P. monodon cyclin E (Pmcyclin E) and CDK2 (PmCDK2) sequences were cloned. The open reading frame of Pmcyclin E was 1263 bp in length and encoded a 47.9-kDa protein, while that of PmCDK2 was 921 bp, encoding a protein of 34.9 kDa. Recombinant cyclin E and CDK2 proteins were expressed in Escherichia coli and purified by Ni-chelating affinity chromatography. In addition, a pull-down assay was performed to identify any interaction between Pmcyclin E and PmCDK2. This research provides a basis for the study of the functional mechanisms of the cyclin E/CDK2 complex in shrimp, further enriching our knowledge of invertebrate cell cycle regulation.

  5. Altered expression of cyclin A 1 in muscle of patients with facioscapulohumeral muscle dystrophy (FSHD-1.

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    Anna Pakula

    Full Text Available OBJECTIVES: Cyclin A1 regulates cell cycle activity and proliferation in somatic and germ-line cells. Its expression increases in G1/S phase and reaches a maximum in G2 and M phases. Altered cyclin A1 expression might contribute to clinical symptoms in facioscapulohumeral muscular dystrophy (FSHD. METHODS: Muscle biopsies were taken from the Vastus lateralis muscle for cDNA microarray, RT-PCR, immunohistochemistry and Western blot analyses to assess RNA and protein expression of cyclin A1 in human muscle cell lines and muscle tissue. Muscle fibers diameter was calculated on cryosections to test for hypertrophy. RESULTS: cDNA microarray data showed specifically elevated cyclin A1 levels in FSHD vs. other muscular disorders such as caveolinopathy, dysferlinopathy, four and a half LIM domains protein 1 deficiency and healthy controls. Data could be confirmed with RT-PCR and Western blot analysis showing up-regulated cyclin A1 levels also at protein level. We found also clear signs of hypertrophy within the Vastus lateralis muscle in FSHD-1 patients. CONCLUSIONS: In most somatic human cell lines, cyclin A1 levels are low. Overexpression of cyclin A1 in FSHD indicates cell cycle dysregulation in FSHD and might contribute to clinical symptoms of this disease.

  6. A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression

    DEFF Research Database (Denmark)

    Lukas, C; Kramer, E R; Peters, J M;

    2001-01-01

    Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits...... the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism...... of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified...

  7. Cyclin B1 Vaccine Delays Spontaneous Tumors

    Science.gov (United States)

    Laura A, Vella; Min, Yu; Amy, Phillips; Olivera J, Finn

    2012-01-01

    We previously identified cyclin B1-specific T cells and antibodies in cancer patients with cyclin B1+ tumors and also in some healthy individuals. We also demonstrated that these responses may be important in cancer immunosurveillance by showing that vaccination against cyclin B1 prevents growth of transplantable cyclin B1+ tumors in mice. Constitutive overexpression of cyclin B1 was determined to correlate with the lack of p53 function. This allowed us to use p53−/− mice as a model that better approximates human disease. p53−/− mice spontaneously develop cyclin B1+ tumors. At 5–6 weeks of age, when the mice were still healthy with no evidence of tumor, they received the cyclin B1 vaccine and were then observed for tumor growth. We demonstrate that cyclin B1 vaccination can delay spontaneous cyclin B1+ tumor growth and increases median survival of tumor bearing p53−/− mice. PMID:19769738

  8. Cyclin A2:At the crossroads of cell cycle and cell invasion

    Institute of Scientific and Technical Information of China (English)

    Abdelhalim; Loukil; Caroline; T; Cheung; Nawal; Bendris; Bénédicte; Lemmers; Marion; Peter; Jean; Marie; Blanchard

    2015-01-01

    Cyclin A2 is an essential regulator of the cell division cycle through the activation of kinases that participate to the regulation of S phase as well as the mitotic entry. However,whereas its degradation by the proteasome in mid mitosis was thought to be essential for mitosis to proceed,recent observations show that a small fraction of cyclin A2 persists beyond metaphase and is degraded by autophagy. Its implication in the control of cytoskeletal dynamics and cell movement has unveiled its role in the modulation of Rho A activity. Since this GTPase is involved in both cell rounding early in mitosis and later,in the formation of the cleavage furrow,this suggests that cyclin A2 is a novel actor in cytokinesis. Taken together,these data point to this cyclin as a potential mediator of cell-niche interactions whose dysregulation could be taken as a hallmark of metastasis.

  9. Cdc2/cyclin B1 对中心体蛋白Nlp的调控在细胞生长中的作用%The Effect of Cdc2/cyclinB1 on Regulating Centrosome Protein Nlp in Cell Growth

    Institute of Scientific and Technical Information of China (English)

    赵雪莲; 宋咏梅; 金顺钱; 詹启敏

    2010-01-01

    目的 研究中心体蛋白Nlp 受Cdc2/cyclinB1磷酸化调控对细胞生长的影响.方法 Northern blot检测不同组织中Nlp的表达;EGFP-Nlp转染HeLa细胞,采用Double-Thymidine方法细胞同步化后释放,细胞免疫荧光方法观察Nlp在整个细胞周期中的亚细胞定位;构建Nlp磷酸化位点突变细胞系,采用人工计数和MTT法检测磷酸化位点突变对细胞生长的影响.结果 Nlp在不同组织中存在表达差异,Nlp在不同细胞周期呈现不同的亚细胞定位,磷酸化位点Ser185和Ser589的突变促进细胞的体外生长.结论 Cdc2/cyclin B1磷酸化位点突变后,Nlp获得了更强的促进细胞生长的能力.

  10. Cyclin-CDK-CKI and UPP Participate in the Regulation of Reproduction and the Progression of Gonad Development in Crustacean%Cyclin-CDK-CKI及UPP参与生殖调控及在甲壳动物性腺发育中的研究进展

    Institute of Scientific and Technical Information of China (English)

    韩坤煌; 张子平; 王艺磊; 邹志华

    2010-01-01

    Cyclin-CDK-CKI是参与真核细胞周期调控的3个重要的调节因子.泛素蛋白酶体途径(ubiquitin proteasome pathway,UPP)是真核细胞内非溶酶体途径的蛋白质选择性降解的重要途径.细胞周期中许多时相特异性周期蛋白通过泛素化被周期性的降解对于细胞分裂的调控进程具有重要的作用.主要综述了Cyclin-CDK-CKI和UPP的组成、生理生化特性及其参与生殖调控的作用机制,同时阐述了这两大系统之间的联系及其在甲壳动物性腺发育中的研究进展.

  11. Cyclin D1 expression in prostate carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, R.A.; Ravinal, R.C.; Costa, R.S.; Lima, M.S. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Patologia, Ribeirão Preto, SP, Brasil, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Tucci, S. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Cirurgia e Anatomia, Divisão de Urologia, Ribeirão Preto, SP, Brasil, Divisão de Urologia, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Muglia, V.F. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Medicina Interna (Centro de Ciência da Imagem), Ribeirão Preto, SP, Brasil, Departamento de Medicina Interna (Centro de Ciência da Imagem), Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Reis, R.B. Dos [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Cirurgia e Anatomia, Divisão de Urologia, Ribeirão Preto, SP, Brasil, Divisão de Urologia, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, G.E.B. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Patologia, Ribeirão Preto, SP, Brasil, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2014-05-09

    The purpose of this study was to investigate the relationship between cyclin D1 expression and clinicopathological parameters in patients with prostate carcinoma. We assessed cyclin D1 expression by conventional immunohistochemistry in 85 patients who underwent radical prostatectomy for prostate carcinoma and 10 normal prostate tissue samples retrieved from autopsies. We measured nuclear immunostaining in the entire tumor area and based the results on the percentage of positive tumor cells. The preoperative prostate-specific antigen (PSA) level was 8.68±5.16 ng/mL (mean±SD). Cyclin D1 staining was positive (cyclin D1 expression in >5% of tumor cells) in 64 cases (75.4%) and negative (cyclin D1 expression in ≤5% of tumor cells) in 21 cases (including 15 cases with no immunostaining). Normal prostate tissues were negative for cyclin D1. Among patients with a high-grade Gleason score (≥7), 86% of patients demonstrated cyclin D1 immunostaining of >5% (P<0.05). In the crude analysis of cyclin D1 expression, the high-grade Gleason score group showed a mean expression of 39.6%, compared to 26.9% in the low-grade Gleason score group (P<0.05). Perineural invasion tended to be associated with cyclin D1 expression (P=0.07), whereas cyclin D1 expression was not associated with PSA levels or other parameters. Our results suggest that high cyclin D1 expression could be a potential marker for tumor aggressiveness.

  12. Correlation between Cyclin Dependent Kinases and Artemisinin-Induced Dormancy in Plasmodium falciparum In Vitro.

    Directory of Open Access Journals (Sweden)

    Karen-Ann Gray

    Full Text Available Artemisinin-induced dormancy provides a plausible explanation for recrudescence following artemisinin monotherapy. This phenomenon shares similarities with cell cycle arrest where cyclin dependent kinases (CDKs and cyclins play an important role.Transcription profiles of Plasmodium falciparum CDKs and cyclins before and after dihydroartemisinin (DHA treatment in three parasite lines, and the effect of CDK inhibitors on parasite recovery from DHA-induced dormancy were investigated.After DHA treatment, parasites enter a dormancy phase followed by a recovery phase. During the dormancy phase parasites up-regulate pfcrk1, pfcrk4, pfcyc2 and pfcyc4, and down-regulate pfmrk, pfpk5, pfpk6, pfcrk3, pfcyc1 and pfcyc3. When entering the recovery phase parasites immediately up-regulate all CDK and cyclin genes. Three CDK inhibitors, olomoucine, WR636638 and roscovitine, produced distinct effects on different phases of DHA-induced dormancy, blocking parasites recovery.The up-regulation of PfCRK1 and PfCRK4, and down regulation of other CDKs and cyclins correlate with parasite survival in the dormant state. Changes in CDK expression are likely to negatively regulate parasite progression from G1 to S phase. These findings provide new insights into the mechanism of artemisinin-induced dormancy and cell cycle regulation of P. falciparum, opening new opportunities for preventing recrudescence following artemisinin treatment.

  13. Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

    Science.gov (United States)

    Roques, Magali; Wall, Richard J; Douglass, Alexander P; Ramaprasad, Abhinay; Ferguson, David J P; Kaindama, Mbinda L; Brusini, Lorenzo; Joshi, Nimitray; Rchiad, Zineb; Brady, Declan; Guttery, David S; Wheatley, Sally P; Yamano, Hiroyuki; Holder, Anthony A; Pain, Arnab; Wickstead, Bill; Tewari, Rita

    2015-11-01

    Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

  14. Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes

    KAUST Repository

    Roques, Magali

    2015-11-13

    Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

  15. Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2.

    Science.gov (United States)

    Makarević, Jasmina; Rutz, Jochen; Juengel, Eva; Kaulfuss, Silke; Reiter, Michael; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A

    2014-01-01

    Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25-10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug.

  16. Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2.

    Directory of Open Access Journals (Sweden)

    Jasmina Makarević

    Full Text Available Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25-10 mg/ml on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP. Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug.

  17. A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins.

    Science.gov (United States)

    Fisher, D L; Nurse, P

    1996-02-15

    Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin-dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis.

  18. Cyclin F: A component of an E3 ubiquitin ligase complex with roles in neurodegeneration and cancer.

    Science.gov (United States)

    Galper, Jasmin; Rayner, Stephanie L; Hogan, Alison L; Fifita, Jennifer A; Lee, Albert; Chung, Roger S; Blair, Ian P; Yang, Shu

    2017-08-01

    Cyclin F, encoded by CCNF, is the substrate recognition component of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCF(cyclin F). E3 ubiquitin ligases play a key role in ubiquitin-proteasome mediated protein degradation, an essential component of protein homeostatic mechanisms within the cell. By recognising and regulating the availability of several protein substrates, SCF(cyclin F) plays a role in regulating various cellular processes including replication and repair of DNA and cell cycle checkpoint control. Cyclin F dysfunction has been implicated in various forms of cancer and CCNF mutations were recently linked to familial and sporadic amyotrophic lateral sclerosis and frontotemporal dementia, offering a new lead to understanding the pathogenic mechanisms underlying neurodegeneration. In this review, we evaluate the current literature on the function of cyclin F with an emphasis on its roles in cancer and neurodegeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. T1 difficulty modulates the attentional blink only when T1 is unmasked

    DEFF Research Database (Denmark)

    Nielsen, Simon; Andersen, Tobias S

    processing resources being occupied by T1 when T2 is presented. If so, it is expected that varying T1 difficulty should modulate the AB magnitude. Previous findings however are inconsistent: Christmann & Leuthold (2004) manipulated T1 difficulty by contrast and found that an easy T1 (high contrast) decreased...... with contrast and exposure. We suggested that the use of pattern masks might have compromised ours, and similar studies. In a new set of experiments we test this hypothesis and vary T1 difficulty with contrast, only this time we omit T1's mask. We find significant AB interference from manipulating T1....... We hypothesize that the rapid onset of T1 induces an attentional capture effect, which increases with contrast. This challenges the use of contrast to manipulate T1 in studies examining how an easy T1 affects the AB – any positive effects may be compromised by the increased capture effect....

  20. Trichosanthin inhibiting PC3 proil ferationv ai down-regulation the expression of p-ERK and Cyc lin D1%天花粉蛋白通过下调p-ERK及Cyclin D1表达抑制PC3细胞增殖

    Institute of Scientific and Technical Information of China (English)

    黄益玲; 胡火军; 尤程程; 黄迎娣; 黄利鸣

    2015-01-01

    目的:探讨天花粉蛋白(TCS)体外对前列腺癌细胞(PC3)生长的抑制作用及可能机制。方法采用噻唑蓝(MTT)检测TCS对PC3细胞的抑制作用。流式细胞术( FCM)检测TCS对PC3细胞周期的影响,Western印迹检测TCS对ERK、p-ERK及细胞周期调节蛋白Cyclin D1表达的影响。结果 MTT 结果显示 TCS 能有效抑制 PC3细胞的生长,具有时间及剂量依赖性。流式细胞检测发现 TCS能够将 PC3细胞阻滞于 G1期, Western 印迹检测TCS能抑制ERK磷酸化及细胞周期调节蛋白Cyclin D1的表达。结论 TCS对PC3细胞的增殖具有明显的抑制作用,其作用机制可能与抑制丝裂原活化蛋白激酶( MAPK)细胞增殖信号通路及降低Cyclin D1表达,从而诱导细胞发生G1期阻滞有关。%[Abstrca t] Obj ective To explore the inhibition effect of Trichosanthin (TCS) on prostate cancer PC3 cells and investigate the pos-sible mechanism .Methods MTT assay was used to detect the proliferation inhibition effect of TCS on PC 3 cells,cell cycle was analyzed by flow cytometry.Expressions of p-ERK and Cyclin D1 protein were detected by Western blot .Results TCS could inhibit the growth of PC 3 cell in a time and dose dependent manner .FCM detection showed that TCS could arrest PC 3 cells in G1 Phase.Western blot found that the protein expressions of p-ERK and Cyclin D1 were decreased after treated with TCS .Conclusions TCS could inhibit the proliferation of PC 3 cells by down-regulating the mitogen activated protein kinase pathway and inducing the G 1 phase arrest.

  1. Cyclin-dependent kinases in C. elegans

    Directory of Open Access Journals (Sweden)

    Boxem Mike

    2006-05-01

    Full Text Available Abstract Cell division is an inherent part of organismal development, and defects in this process can lead to developmental abnormalities as well as cancerous growth. In past decades, much of the basic cell-cycle machinery has been identified, and a major challenge in coming years will be to understand the complex interplay between cell division and multicellular development. Inevitably, this requires the use of more complex multicellular model systems. The small nematode Caenorhabditis elegans is an excellent model system to study the regulation of cell division in a multicellular organism, and is poised to make important contributions to this field. The past decade has already seen a surge in cell-cycle research in C. elegans, yielding information on the function of many basic cell-cycle regulators, and making inroads into the developmental control of cell division. This review focuses on the in vivo roles of cyclin-dependent kinases in C. elegans, and highlights novel findings implicating CDKs in coupling development to cell-cycle progression.

  2. Stage-specific requirement for cyclin D1 in glial progenitor cells of the cerebral cortex.

    Science.gov (United States)

    Nobs, Lionel; Baranek, Constanze; Nestel, Sigrun; Kulik, Akos; Kapfhammer, Josef; Nitsch, Cordula; Atanasoski, Suzana

    2014-05-01

    Despite the vast abundance of glial progenitor cells in the mouse brain parenchyma, little is known about the molecular mechanisms driving their proliferation in the adult. Here we unravel a critical role of the G1 cell cycle regulator cyclin D1 in controlling cell division of glial cells in the cortical grey matter. We detect cyclin D1 expression in Olig2-immunopositive (Olig2+) oligodendrocyte progenitor cells, as well as in Iba1+ microglia and S100β+ astrocytes in cortices of 3-month-old mice. Analysis of cyclin D1-deficient mice reveals a cell and stage-specific molecular control of cell cycle progression in the various glial lineages. While proliferation of fast dividing Olig2+ cells at early postnatal stages becomes gradually dependent on cyclin D1, this particular G1 regulator is strictly required for the slow divisions of Olig2+/NG2+ oligodendrocyte progenitors in the adult cerebral cortex. Further, we find that the population of mature oligodendrocytes is markedly reduced in the absence of cyclin D1, leading to a significant decrease in the number of myelinated axons in both the prefrontal cortex and the corpus callosum of 8-month-old mutant mice. In contrast, the pool of Iba1+ cells is diminished already at postnatal day 3 in the absence of cyclin D1, while the number of S100β+ astrocytes remains unchanged in the mutant.

  3. Cyclin G2 suppresses estrogen-mediated osteogenesis through inhibition of Wnt/β-catenin signaling.

    Directory of Open Access Journals (Sweden)

    Jinlan Gao

    Full Text Available Estrogen plays an important role in the maintenance of bone formation, and deficiency in the production of estrogen is directly linked to postmenopausal osteoporosis. To date, the underlying mechanisms of estrogen-mediated osteogenic differentiation are not well understood. In this study, a pluripotent mesenchymal precursor cell line C2C12 was used to induce osteogenic differentiation and subjected to detection of gene expressions or to manipulation of cyclin G2 expressions. C57BL/6 mice were used to generate bilateral ovariectomized and sham-operated mice for analysis of bone mineral density and protein expression. We identified cyclin G2, an unconventional member of cyclin, is involved in osteoblast differentiation regulated by estrogen in vivo and in vitro. In addition, the data showed that ectopic expression of cyclin G2 suppressed expression of osteoblast transcription factor Runx2 and osteogenic differentiation marker genes, as well as ALP activity and in vitro extracellular matrix mineralization. Mechanistically, Wnt/β-catenin signaling pathway is essential for cyclin G2 to inhibit osteogenic differentiation. To the best of our knowledge, the current study presents the first evidence that cyclin G2 serves as a negative regulator of both osteogenesis and Wnt/β-catenin signaling. Most importantly, the basal and 17β-estradiol-induced osteogenic differentiation was restored by overexpression of cyclin G2. These results taken together suggest that cyclin G2 may function as an endogenous suppressor of estrogen-induced osteogenic differentiation through inhibition of Wnt/β-catenin signaling.

  4. The Role of the Low Molecular Weight (LMW) Isoforms of Cyclin E in Breast Cancer Tumorigenesis

    Science.gov (United States)

    2007-06-01

    clones include: 1) dicentric chromosomes , chromosomes which contain two copies of the same centromere, 2) ring chromosomes , which results from 2...cells also had more metaphases with chromosome fragments and dicentric chromosomes than the metaphases of EL or empty vector overexpressing cells...fragments or dicentric chromosomes . Overall our results show that the full-length cyclin E does generate genomic instability in 76NE6 cells, however, T1

  5. Phosphorylation of Rad9 at serine 328 by cyclin A-Cdk2 triggers apoptosis via interfering Bcl-xL.

    Directory of Open Access Journals (Sweden)

    Zhuo Zhan

    Full Text Available Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.

  6. Cyclin A1 shows age-related expression in benign tonsils, HPV16-dependent overexpression in HNSCC and predicts lower recurrence rate in HNSCC independently of HPV16

    Directory of Open Access Journals (Sweden)

    Weiss Daniel

    2012-06-01

    Full Text Available Abstract Background Promoter methylation of the tumor suppressor gene Cyclin A1 could be associated with Human Papillomavirus 16 (HPV16 induced Head and Neck Squamous Cell Carcinoma (HNSCC and Cervical Carcinoma. There is disagreement about the impact of this epigenetic event on protein expression of Cyclin A1 in malignant and non-malignant tissue and there hardly exists any information about possible relationships between Cyclin A1 expression and clinicopathological characteristics in HNSCC. Methods We analyzed protein expression of Cyclin A1 in 81 HNSCC and 74 benign tonsils by immunohistochemistry and correlated it to Cyclin A1 methylation status, presence of HPV16 infection and other clinicopathological characteristics. Results Overexpression of Cyclin A1 was more present in HNSCC than in tonsils (p Cyclin A1 significantly correlated with the expression of Cyclin-dependent kinase-inhibitor p16 (p = 0.000672 and 0.00495. In tonsils, expression of Cyclin A1 was inversely proportional to age (p = 0.00000396, and further correlated with expression of tumor suppressor gene p53 (p = 0.000228. In HNSCC Cyclin A1 expression was associated with the presence of HPV16 DNA (p = 0.0014 and a lower recurrence rate in univariate and multivariate analysis (p = 0.002 and 0.013. Neither in HNSCC nor in tonsils Cyclin A1 expression correlated with promoter methylation. Conclusions Cyclin A1 is an important cell cycle regulator with age-related increased expression in tonsils of children. HPV16 induces overexpression of Cyclin A1 in HNSCC despite promoter methylation. Overexpression of Cyclin A1 predicts a lower recurrence rate in HNSCC independently of HPV16.

  7. DNA methyltrans-ferase 3b regulating the Cyclin D1 gene expression by microRNA-145 in the human hepatocellular carcinoma cell line%肝癌细胞DNA甲基转移酶3b通过微小RNA-145调节细胞周期素D1基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    王佳辰; 司亚卿; 秦贯军

    2014-01-01

    Objective To discuss the mechanism of the DNA methyltrans-ferase 3b (DNMT3b) regulating the Cyclin D1 gene expression in SMMC7721 cell line.Methods DNMT3b small interfering RNA (siRNA) was transfected into SMMC-7721 cells.Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of the microRNA (miR)-145 and Cyclin D1 mRNA.MSP was used to detect whether the promoter of miR-145 was methylated.Results DNMT3b has been successfully suppressed in the culture cells transfected by miR-145 with a transfection efficiency over 90% ; The methylation status of miR-145 gene showed no significant differences between two groups (P >0.05) ; The expression of miR-145 was significantly higher than in control group (P < 0.01) and the change between two groups had no difference (P > 0.05).Conclusion DNMT3b can regulate the expression of Cyclin D1 by miR-145,affect the cell cycle and cause a large number of apoptosis of SMMC-7721 cells.%目的 探讨DNA甲基转移酶3b(DNMT3b)在人肝癌细胞株(SMMC7721)中调节细胞周期素(Cyclin) D1基因表达的机制.方法 用DNMT3b的小干扰RNA(siRNA)表达载体转染SMMC7721细胞抑制DNMT3b的表达;采用逆转录-聚合酶链反应(RT-PCR)检测微小RNA(miR)-145、Cyclin D1 mRNA表达的变化;用甲基化特异性PCR (MSP)技术检测miR-145基因启动子区甲基化状态的变化.结果 siRNA转染肝癌细胞的效率可达90%以上,DNMT3b成功被抑制;两组中miR-145基因启动子区甲基化水平的差异无统计学意义(P>0.05);实验组miR-145表达水平明显高于对照组(P<0.01),但Cylin D1 mRNA表达水平的差异无统计学意义(P>0.05).结论 DNMT3b可以通过miR-145调节Cyclin D1的表达,从而影响细胞周期,进而引起肝癌细胞的大量凋亡.

  8. Cyclin B1和survivin在非小细胞肺癌中的表达和意义%The expression and significance of cyclin B1 and survivin in human non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    Guosheng Li; Xuhan Liu; Daorong Zhang; Dong Liu; Zhiyong Li

    2011-01-01

    Objective: We studied the expression of cyclin B1 and survivin in human non-small cell lung cancer (NSCLC), and the relationship between such expression and clinicopathological features of NSCLC. Methods: One hundred cases of tissue specimen including NSCLC, neighboring noncancerous tissue and normal lung tissue were collected at random. These specimens were detected by immunohistochemical methods. Results: The expression of cyclin B1 and survivin showed significant difference (P 0.05) in NSCLC. Statistical significance was marked between different clinical stages of NSCLC and the expression of cyclin B1 and survivin (P < 0.05). Conclusion: The overexpression of cyclin B1 and survivin was found in NSCLC. The expression of cyclin B1 and survivin might be up-regulated during an early step of tumorigenesis and during the development of NSCLC. The progression of cell cycle could be efficiently connected with the control of apoptosis by the interrelations between the overexpression of cyclin B1 and that of survivin in NSCLC during the G2/M phase. The overexpression of cyclin B1 and survivin might be used as marker in showing the dividing and proliferating ability, and the inhibiting apoptosis ability (lengthening cell lifespan) of NSCLC. Moreover, the overexpression of cyclin B1 and survivin was associated with the clinic stages of NSCLC.

  9. Protein kinase A-mediated phosphorylation of Pah1p phosphatidate phosphatase functions in conjunction with the Pho85p-Pho80p and Cdc28p-cyclin B kinases to regulate lipid synthesis in yeast.

    Science.gov (United States)

    Su, Wen-Min; Han, Gil-Soo; Casciano, Jessica; Carman, George M

    2012-09-28

    Pah1p, which functions as phosphatidate phosphatase (PAP) in the yeast Saccharomyces cerevisiae, plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The diacylglycerol produced by PAP is used for the synthesis of triacylglycerol as well as for the synthesis of phospholipids via the Kennedy pathway. Pah1p is a highly phosphorylated protein in vivo and has been previously shown to be phosphorylated by the protein kinases Pho85p-Pho80p and Cdc28p-cyclin B. In this work, we showed that Pah1p was a bona fide substrate for protein kinase A, and we identified by mass spectrometry and mutagenesis that Ser-10, Ser-677, Ser-773, Ser-774, and Ser-788 were the target sites of phosphorylation. Protein kinase A-mediated phosphorylation of Pah1p inhibited its PAP activity by decreasing catalytic efficiency, and the inhibitory effect was primarily conferred by phosphorylation at Ser-10. Analysis of the S10A and S10D mutations (mimicking dephosphorylation and phosphorylation, respectively), alone or in combination with the seven alanine (7A) mutations of the sites phosphorylated by Pho85p-Pho80p and Cdc28p-cyclin B, indicated that phosphorylation at Ser-10 stabilized Pah1p abundance and inhibited its association with membranes, PAP activity, and triacylglycerol synthesis. The S10A mutation enhanced the physiological effects imparted by the 7A mutations, whereas the S10D mutations attenuated the effects of the 7A mutations. These data indicated that the protein kinase A-mediated phosphorylation of Ser-10 functions in conjunction with the phosphorylations mediated by Pho85p-Pho80p and Cdc28p-cyclin B and that phospho-Ser-10 should be dephosphorylated for proper PAP function.

  10. Spatial reorganization of the endoplasmic reticulum during mitosis relies on mitotic kinase cyclin A in the early Drosophila embryo.

    Science.gov (United States)

    Bergman, Zane J; Mclaurin, Justin D; Eritano, Anthony S; Johnson, Brittany M; Sims, Amanda Q; Riggs, Blake

    2015-01-01

    Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope.

  11. Spatial Reorganization of the Endoplasmic Reticulum during Mitosis Relies on Mitotic Kinase Cyclin A in the Early Drosophila Embryo

    Science.gov (United States)

    Bergman, Zane J.; Mclaurin, Justin D.; Eritano, Anthony S.; Johnson, Brittany M.; Sims, Amanda Q.; Riggs, Blake

    2015-01-01

    Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope. PMID:25689737

  12. Cyclin B1 and p21cipl在急性白血病中的表达及其相关性研究%Expressions of Cyclin B1 and p21cipl in Adult Acute Leukemia and Their Correlation

    Institute of Scientific and Technical Information of China (English)

    马卫东; 徐世荣; 阎蕴力; 郭晓楠; 乔淑凯; 薛芳; 高晓丽

    2005-01-01

    细胞周期蛋白(cyclin)B1作为调节细胞进行有丝分裂的正性调节因子,在细胞增殖中起到重要作用.本研究旨在评价cyclin B1在急性白血病(AL)发生、发展中的作用及其对AL患者预后判断的价值.采用流式细胞术对136例初治AL成人患者、10例持续完全缓解(CCR)患者及17例正常人进行cyclin B1、p21cipl蛋白表达水平检测和细胞周期分布分析;采用RT-PCR测定Cyclin B1、p21 cipl和增殖细胞核抗原(PCNA)mRNA水平.结果显示:136例初治AL患者cyclin B1蛋白表达水平明显高于正常对照组;复发AL患者的cyclin B1表达水平明显高于正常对照组,但低于初治AL患者;缓解AL患者及CCR的AL患者cyclin B1蛋白表达水平与正常对照比较均无显著性差异;所有cyclin B1高表达的AL患者出现非顺序表达(cyclin B1在G1期出现,有些甚至G2期高表达),cyclin B1与p21cipl的蛋白表达呈负相关;cyclin B1蛋白表达与其mRNA水平呈正相关;cyclin B1表达与PCNA及细胞增殖指数(PI)表达水平呈正相关;cyclin B1表达水平高的AL患者有较高的缓解率和生存率,但有较高的复发率.结论:本研究首次发现cyclin B1在成人AL患者有过度表达和异常分布,认为cyclin B1与AL发生、发展有关,是影响AL患者预后的因素之一.%Cyclin B1, a positive regulator, controls mitosis occurrence, plays an important role in cell proliferation.To investigate the clinical significance of cyclin B1, the expression of cyclin B1 in acute leukemia (AL) patients was measured; the expression of cyclin B1 and p21 cipl, and their cell cycle distribution were assayed by flow cytometry in 136 adult patients with newly diagnosed AL, 10 continuous complete remission (CCR) AL and 17 normal controls; the mRNA of cyclin B 1 and p21 cipl, and the proliferation cell nuclear antigen (PCNA) in patients and normal controls were detected with semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The

  13. Cyclin D1 Expression and Its Correlation with Histopathological Differentiation in Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Swati Saawarn

    2012-01-01

    Full Text Available Background. Cyclin D1 regulates the G1 to S transition of cell cycle. Its deregulation or overexpression may lead to disturbance in the normal cell cycle control and tumour formation. Overexpression of cyclin D1 has been reported in various tumors of diverse histogenesis. This case control retrospective study was carried out to study the immunohistochemical reactivity and expression of cyclin D1 and its association with site, clinical staging, and histopathological differentiation of oral squamous cell carcinoma (OSCC. Methods. Forty formalin-fixed paraffin-embedded tissue blocks of biopsy specimens of oral squamous cell carcinoma were immunohistochemically evaluated for expression of cyclin D1. Results. Cyclin D1 expression was seen in 45% cases of OSCC. It did not correlate with site and clinical staging. Highest expression was seen in well-differentiated, followed by moderately differentiated, and poorly differentiated squamous cell carcinomas, with a statistically significant correlation. Conclusion. Cyclin D1 expression significantly increases with increase in differentiation.

  14. Temperature dependent expression of cdc2 and cyclin B1 in spermatogenic cells during spermatogenesis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    p34cdc2 and Cyclin Bi are key components of cell cycle controlling machine and are believed to play a fundamental role in gametogenesis. It is also well known that, in scrotal mammals, spermatogenesis depends greatly on the maintenance of comparatively low temperature in the scrotum. To investigate whether the expression of cdc2 and cyclin B1 in spermatogenic cells during spermatogenesis is actually a temperature dependent event, in situ hybridization, Western blotting and immunohistochemistry analysis were used to study the expression of cdc2 and cyclin B1 in normal and cryptorchid testis. Results showed that the abdominal temperature had no significant influence on the transcription of cdc2 and cyclin B1 in the spermatogonia and pachytene/diplotene primary spermatocytes, but it blocked the translation of them. Due to the deficiency of p34cdc2 and Cyclin B1, the spermatogonia and pachytene/diplotene primary spermatocytes were unable to form MPF, hence, they couldn't undergo karyokinesis. The development of primary spermato cytes was arrested at the G2 to M phase transition. We also found that testosterone could regulate the Cyclin B1 expression in spermatogenic cells. Muscular injection of testosterone could recover spermatogenesis in the unilateral scrotal testis which was influenced by the contralateral cryptorchid testis, but it could not salvage the spermatogenesis block in the cryptorchid testis.

  15. Cyclin G2 Promotes Hypoxia- Driven Local Invasion of Glioblastoma by Orchestrating Cytoskeletal Dynamics

    Directory of Open Access Journals (Sweden)

    Atsushi Fujimura

    2013-11-01

    Full Text Available Microenvironmental conditions such as hypoxia potentiate the local invasion of malignant tumors including glioblastomas by modulating signal transduction and protein modification, yet the mechanism by which hypoxia controls cytoskeletal dynamics to promote the local invasion is not well defined. Here, we show that cyclin G2 plays pivotal roles in the cytoskeletal dynamics in hypoxia-driven invasion by glioblastoma cells. Cyclin G2 is a hypoxia-induced and cytoskeleton-associated protein and is required for glioblastoma expansion. Mechanistically, cyclin G2 recruits cortactin to the juxtamembrane through its SH3 domain-binding motif and consequently promotes the restricted tyrosine phosphorylation of cortactin in concert with src. Moreover, cyclin G2 interacts with filamentous actin to facilitate the formation of membrane ruffles. In primary glioblastoma, cyclin G2 is abundantly expressed in severely hypoxic regions such as pseudopalisades, which consist of actively migrating glioma cells. Furthermore, we show the effectiveness of dasatinib against hypoxia-driven, cyclin G2-involved invasion in vitro and in vivo. Our findings elucidate the mechanism of cytoskeletal regulation by which severe hypoxia promotes the local invasion and may provide a therapeutic target in glioblastoma.

  16. The Expression of Cyclins in Neurons of Rats after Focal Cerebral Ischelma

    Institute of Scientific and Technical Information of China (English)

    Bin CHEN; WANG Wei

    2008-01-01

    The change of the expression of Cyclins in neurons of rats after focal cerebral ischemia was investigated. Ischemia was induced by temporary middle cerebral artery occlusion (MCAO). The experimental rats induced by MCAO were sacrificed on 7th and 14th day after reperfusion. The brain was taken out at 7th and 14th day after injury, and the expression of Cyclin D1>, E, A and B1> in neu- rons of cerebral cortex or hippocampal Cal region was detected by immunofluorescence and confo- cai microscope. The results showed that after MCAO, in the ipsilateral CAI subfield of hippocampus the expression of Cyclin D1, E, A and B1 in neurons was significantly gradually up-regulated at 7th and 14th day after reperfusion (P<0.05) as compared with that in control group. In the ipsilateral cerebral cortex the expression of Cyclin D1 and B1 in neurons was notably gradually down-regulated at 7th and 14th day, and that of Cyclin E and A was significantly up-regulated at 14th day after reper- fusion as compared with that in control group (all P<0.05). It was concluded that there was a differ- ential sensitivity among neurons from different brain regions to ischemic injury. But all of them re-enter into cell cycle after MCAO.

  17. Molecular cloning and chromosomal localization of the human cyclin C (CCNC) and cyclin E (CCNE) genes: Deletion of the CCNC gene in human tumors

    Energy Technology Data Exchange (ETDEWEB)

    Li, Haimin; Lahti, J.M.; Kidd, V.J. [St. Jude Children`s Research Hospital, Memphis, TN (United States)] [and others

    1996-03-01

    The human G1-phase cyclins are important regulators of cell cycle progression that interact with various cyclin-dependent kinases and facilitate entry into S-phase. We have confirmed the localization of the human cyclin C (CCNC) gene to chromosome 6q21 and of human cyclin E (CCNE to 19q12). The CCNC gene structure was also determined, and we have shown that it is deleted in a subset of acute lymphoblastic leukemias, including a patient sample containing a t(2;6)(p21;q15), with no apparent cytogenetic deletion. Single-strand conformational polymorphism analysis of the remaining CCNC allele from patients with a deletion of one allele established that there were no further mutations within the exons or the flanking intronic sequences. These results suggest either that haploinsufficiency of the cyclin C protein is sufficient to promote tumorigenesis or that the important tumor suppressor gene is linked to the CCNC locus. 48 refs., 4 figs., 1 tab.

  18. A jumonji (Jarid2) protein complex represses cyclin D1 expression by methylation of histone H3-K9.

    Science.gov (United States)

    Shirato, Haruki; Ogawa, Satoko; Nakajima, Kuniko; Inagawa, Masayo; Kojima, Mizuyo; Tachibana, Makoto; Shinkai, Yoichi; Takeuchi, Takashi

    2009-01-09

    Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification.

  19. Resibufogenin Induces G1-Phase Arrest through the Proteasomal Degradation of Cyclin D1 in Human Malignant Tumor Cells.

    Directory of Open Access Journals (Sweden)

    Masami Ichikawa

    Full Text Available Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3β (GSK-3β induces the proteasomal degradation of cyclin D1. The addition of GSK-3β inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3β.

  20. Mammalian E-type cyclins control chromosome pairing, telomere stability and CDK2 localization in male meiosis.

    Directory of Open Access Journals (Sweden)

    Laetitia Martinerie

    2014-02-01

    Full Text Available Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis.

  1. 鱼藤素对斑马鱼胚胎中细胞周期蛋白D1基因下调的作用:整胚原位杂交检测%Deguelin down-regulates the expression of cyclin D1 gene in zebrafish embroys through the whole mount in situ hybridization

    Institute of Scientific and Technical Information of China (English)

    海洋; 吴新荣

    2015-01-01

    BACKGROUND:In the early development of zebrafish embryos, cels divide and proliferate rapidly, but low concentration of deguelin can delay the development of zebrafish embryos. OBJECTIVE:To observe the effect of different concentrations of deguelin on cyclin D1 gene expression in zebrafish embryos. METHODS:Though normal fertilization, zebrafish embryos that incubated to the 2-cel stage (about 0.75 hour after fertilization) and shield stage (6 hours after fertilization) were colected and put into 12-wel plates treated with 100, 200, 400 nmol/L deguelin at 28.5℃in an incubator til the shield period and 24 hours after fertilization, respectively. Simultaneously embroys treated with 1% dimethyl sulfoxide solution were as a control group, cultured in the same conditions. Cyclin D1 RNA probes were prepared for the whole mountin situhybridization, observing staining by an upright fluorescent microscope camera to detect the effect of deguelin on cyclin D1 expression in zebrafish embryos. RESULTS AND CONCLUSION:Deguelin showed significant negative regulation on the expression of cyclin D1 gene in zebrafish embryos. Cyclin D1 expressed in outsourcing cels in embryos of shield stage, and a significant reduction in the expression of cyclin D1 came up with the increasing concentrations of deguelin. In the 400 nmol/L deguelin treatment group, there was nearly no expression of cyclin D1. Cyclin D1 expressed in the brain, central nervous system, immature eye, somites, trunk, and tail of embryos at 24 hours after fertilization, and reduced significantly in the 100 nmol/L deguelin treatment group, especialy in the proliferative area. In the 200 and 400 nmol/L treatment groups, the embryonic development slowed down signficantly, and cyclin D1 gene mainly expressed in the dorsal ectoderm cels.%背景:斑马鱼胚胎发育早期,细胞分裂增殖快速,低浓度的鱼藤素可以延缓斑马鱼胚胎发育。目的:观察鱼藤素对斑马鱼胚胎中细胞周期蛋白D1

  2. Advanced Colloids Experiment (ACE-T1)

    Science.gov (United States)

    Meyer, William V.; Sicker, Ron; Brown, Dan; Eustace, John

    2015-01-01

    Increment 45 - 46 Science Symposium presentation of Advanced Colloids Experiment (ACE-T1) to RPO. The purpose of this event is for Principal Investigators to present their science objectives, testing approach, and measurement methods to agency scientists, managers, and other investigators.

  3. Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

    Directory of Open Access Journals (Sweden)

    Magali Roques

    2015-11-01

    Full Text Available Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs. Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites in host liver and red blood cells, and sporogony (producing sporozoites in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei.

  4. Cyclin D and cdk4 Are Required for Normal Development beyond the Blastula Stage in Sea Urchin Embryos

    Science.gov (United States)

    Moore, Jennifer C.; Sumerel, Jan L.; Schnackenberg, Bradley J.; Nichols, Jason A.; Wikramanayake, Athula; Wessel, Gary M.; Marzluff, William F.

    2002-01-01

    cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle. PMID:12052892

  5. Prognostic Value of Expression of Cyclin E in Gastrointestinal Cancer: A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Huang, Lanshan; Ren, Fanghui; Tang, Ruixue; Feng, Zhenbo; Chen, Gang

    2016-02-01

    Cyclin E is a critical regulator in cell cycle and promotes the initiation of DNA replication and centrosome duplication in late G1. The overexpression of cyclin E is common in cancers of the digestive system. However, whether cyclin E represents a prognostic biomarker in gastrointestinal cancer remains controversial. We reviewed the published literatures to clarify the association between cyclin E determined by immunohistochemistry (IHC) and survival in gastrointestinal cancer. Literatures were searched in PubMed and Cochrane Library published up to December 1, 2014. A total of 282 articles were initially identified, and 14 articles were included in this study. Meta-analysis was performed for 10 studies with a total of 1300 patients. Combined hazard risk (HR) and corresponding 95% confidence interval (CI) were calculated by random-effect model due to the heterogeneity. The quality of included studies was assessed by the Newcastle-Ottawa Scale and the Methodological Index for Non-Randomized Studies (MINORS). We found that high level of cyclin E was a predicator of poor prognosis among patients with gastrointestinal cancer (HR = 1.67, 95% CI = 1.06-2.63, P = .028). In summary, overexpression of cyclin E is associated with poor prognosis in gastrointestinal cancer and expression of cyclin E determined by IHC might be a prognostic marker for gastrointestinal cancer in clinical practice.

  6. Cyclin E-induced S phase without activation of the pRb/E2F pathway

    DEFF Research Database (Denmark)

    Lukas, J; Herzinger, T; Hansen, Klaus

    1997-01-01

    In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 and, possibly, cyclin E-dependent Cdk2 positively regulate the G1- to S-phase transition, by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phase genes. Here we...... performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpressing cyclins D1 or E, and human U-2-OS cells, to explore the action of G1 cyclins and the relationship of E2F and cyclin E in S-phase induction. We demonstrate that ectopic expression...... that the cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G1 block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence...

  7. Placental Estrogen Suppresses Cyclin D1 Expression in the Nonhuman Primate Fetal Adrenal Cortex*

    Science.gov (United States)

    Dumitrescu, Adina; Aberdeen, Graham W.; Pepe, Gerald J.

    2014-01-01

    We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27Kip1 and p57Kip2 were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy. PMID:25247468

  8. Placental estrogen suppresses cyclin D1 expression in the nonhuman primate fetal adrenal cortex.

    Science.gov (United States)

    Dumitrescu, Adina; Aberdeen, Graham W; Pepe, Gerald J; Albrecht, Eugene D

    2014-12-01

    We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27(Kip1) and p57(Kip2) were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy.

  9. Cyclin Dependent Kinase 9 Inhibitors for Cancer Therapy.

    Science.gov (United States)

    Sonawane, Yogesh A; Taylor, Margaret A; Napoleon, John Victor; Rana, Sandeep; Contreras, Jacob I; Natarajan, Amarnath

    2016-10-13

    Cyclin dependent kinase (CDK) inhibitors have been the topic of intense research for nearly 2 decades due to their widely varied and critical functions within the cell. Recently CDK9 has emerged as a druggable target for the development of cancer therapeutics. CDK9 plays a crucial role in transcription regulation; specifically, CDK9 mediated transcriptional regulation of short-lived antiapoptotic proteins is critical for the survival of transformed cells. Focused chemical libraries based on a plethora of scaffolds have resulted in mixed success with regard to the development of selective CDK9 inhibitors. Here we review the regulation of CDK9, its cellular functions, and common core structures used to target CDK9, along with their selectivity profile and efficacy in vitro and in vivo.

  10. Cyclin D2 is a critical mediator of exercise-induced cardiac hypertrophy.

    Science.gov (United States)

    Luckey, Stephen W; Haines, Chris D; Konhilas, John P; Luczak, Elizabeth D; Messmer-Kratzsch, Antke; Leinwand, Leslie A

    2017-01-01

    A number of signaling pathways underlying pathological cardiac hypertrophy have been identified. However, few studies have probed the functional significance of these signaling pathways in the context of exercise or physiological pathways. Exercise studies were performed on females from six different genetic mouse models that have been shown to exhibit alterations in pathological cardiac adaptation and hypertrophy. These include mice expressing constitutively active glycogen synthase kinase-3β (GSK-3βS9A), an inhibitor of CaMK II (AC3-I), both GSK-3βS9A and AC3-I (GSK-3βS9A/AC3-I), constitutively active Akt (myrAkt), mice deficient in MAPK/ERK kinase kinase-1 (MEKK1(-/-)), and mice deficient in cyclin D2 (cyclin D2(-/-)). Voluntary wheel running performance was similar to NTG littermates for five of the mouse lines. Exercise induced significant cardiac growth in all mouse models except the cyclin D2(-/-) mice. Cardiac function was not impacted in the cyclin D2(-/-) mice and studies using a phospho-antibody array identified six proteins with increased phosphorylation (greater than 150%) and nine proteins with decreased phosphorylation (greater than 33% decrease) in the hearts of exercised cyclin D2(-/-) mice compared to exercised NTG littermate controls. Our results demonstrate that unlike the other hypertrophic signaling molecules tested here, cyclin D2 is an important regulator of both pathologic and physiological hypertrophy. Impact statement This research is relevant as the hypertrophic signaling pathways tested here have only been characterized for their role in pathological hypertrophy, and not in the context of exercise or physiological hypertrophy. By using the same transgenic mouse lines utilized in previous studies, our findings provide a novel and important understanding for the role of these signaling pathways in physiological hypertrophy. We found that alterations in the signaling pathways tested here had no impact on exercise performance. Exercise

  11. Effects of Serum from Aplastic Anemia patients on the Expression of Cyclin D3 Isoform in Umbilical Cord Blood CD34+ Cells

    Institute of Scientific and Technical Information of China (English)

    孟凡凯; 谭细友; 刘文励; 孙汉英; 周剑锋; 李春蕊; 刘丹; 何莉; 孙岚

    2004-01-01

    Summary: The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34+ cells were isolated from the cord blood with MIDIMACS Semi-solid methylcellulose culture technique was used to measure the formation of CFUGM;The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.

  12. [Clinical significance of cyclin Dl expression in non-small cell lung cancer].

    Science.gov (United States)

    Dworakowska, Dorota

    2005-01-01

    Lung cancer remains interdisciplinary problem. The genetic alterations in non-small cell lung cancer (NSCLC) are related to tumor suppressor genes and proto-oncogenes. CCND1 gene, coding cyclin DI, in correlation with pRb is involved in regulation of cell cycle arrest in G1 phase. Amplification of CCND1 gene and cyclin D1 over-expression was found in several cancers including head and neck cancers or colorectal cancer, where these alterations were correlated with worse prognosis. The literature addressing the clinical significance of CCND1 gene amplification/expression in NSCLC remains poor and prognostic value of these alterations in that cancer is still controversial.

  13. [A cyclin A-like protein is localized in the cells of Physarum polycephalum and functions in the cell cycle].

    Science.gov (United States)

    Li, Xiao-Xue; Li, Gui-Ying; Xing, Miao

    2003-05-01

    The subcellular distribution of a Cyclin A-like protein in the cells of Physarum polycephalum and the function of the protein in the cell cycle were studied by immunoelectron microscope and anti-Cyclin A antibody blocking. After labeled with an anti-Cyclin A monoclonal antibody, the density of gold particles in the labeled specimens was much higher than that in the control, indicating that a Cyclin A-like protein existed in Physarum polycephalum. In the labeled specimen, the gold particles density of the nucleus was higher than that of cytoplasm, which was similar to that of the control, demonstrating that the Cyclin A-like protein was a nuclear protein. The gold particles density of the nuclei varied during the cell cycle. The highest appeared in S phase and the lowest came in metaphase and ana-telophase, which was close to that in the control. From S phase to metaphase, the gold particle densities dropped down gradually. The changes in the gold particle density showed the changes in the content of the Cyclin A-like protein. After treated with the anti-Cyclin A antibody in S phase and G2 phase respectively, the nuclei of Physarum polycephalum were arrested in the phases and the morphology of these nuclei became irregular. When treated with the anti-Cyclin A antibody in prophase, the nuclei appeared abnormal. These results suggested that the Cyclin A-like protein is important in cell cycle regulation of Physarum polycephalum, essentially in S/G2 and G2/M changes.

  14. Cyclin D1 in ASM Cells from Asthmatics Is Insensitive to Corticosteroid Inhibition.

    Science.gov (United States)

    Allen, Jodi C; Seidel, Petra; Schlosser, Tobias; Ramsay, Emma E; Ge, Qi; Ammit, Alaina J

    2012-01-01

    Hyperplasia of airway smooth muscle (ASM) is a feature of the remodelled airway in asthmatics. We examined the antiproliferative effectiveness of the corticosteroid dexamethasone on expression of the key regulator of G(1) cell cycle progression-cyclin D1-in ASM cells from nonasthmatics and asthmatics stimulated with the mitogen platelet-derived growth factor BB. While cyclin D1 mRNA and protein expression were repressed in cells from nonasthmatics in contrast, cyclin D1 expression in asthmatics was resistant to inhibition by dexamethasone. This was independent of a repressive effect on glucocorticoid receptor translocation. Our results corroborate evidence demonstrating that corticosteroids inhibit mitogen-induced proliferation only in ASM cells from subjects without asthma and suggest that there are corticosteroid-insensitive proliferative pathways in asthmatics.

  15. Distinct Effects of Mitogens and the Actin Cytoskeleton on CREB and Pocket Protein Phosphorylation Control the Extent and Timing of Cyclin A Promoter Activity

    Science.gov (United States)

    Bottazzi, Maria Elena; Buzzai, Monica; Zhu, Xiaoyun; Desdouets, Chantal; Bréchot, Christian; Assoian, Richard K.

    2001-01-01

    Soluble mitogens and adhesion-dependent organization of the actin cytoskeleton are required for cells to enter S phase in fibroblasts. The induction of cyclin A is also required for S-phase entry, and we now report that distinct effects of mitogens and the actin cytoskeleton on the phosphorylation of CREB and pocket proteins regulate the extent and timing of cyclin A promoter activity, respectively. First, we show that CREB phosphorylation and binding to the cyclic AMP response element (CRE) determines the extent, but not the timing, of cyclin A promoter activity. Second, we show that pocket protein inactivation regulates the timing, but not the extent, of cyclin A promoter activity. CREB phosphorylation and CRE occupancy are regulated by soluble mitogens alone, while the phosphorylation of pocket proteins requires both mitogens and the organized actin cytoskeleton. Mechanistically, cytoskeletal integrity controls pocket protein phosphorylation by allowing for sustained ERK signaling and, thereby, the expression of cyclin D1. Our results lead to a model of cyclin A gene regulation in which mitogens play a permissive role by stimulating early G1-phase phosphorylation of CREB and a distinct regulatory role by cooperating with the organized actin cytoskeleton to regulate the duration of ERK signaling, the expression of cyclin D1, and the timing of pocket protein phosphorylation. PMID:11604497

  16. Is mortalin a candidate gene for T1DM ?

    Science.gov (United States)

    Johannesen, Jesper; Pie, Angeles; Karlsen, Allan Ertmann; Larsen, Zenia Marian; Jensen, Allan; Vissing, Henrik; Kristiansen, Ole Peter; Pociot, Flemming; Nerup, Jørn

    2004-01-01

    Mortalin has been found to be up-regulated by 2D-protein gel analysis in isolated rodent islets exposed to cytokines. In islets from two rat strains with different sensitivity to the toxic effects of cytokines we observed a significant difference in IL-1beta mediated mortalin expression. Constitutive over-expression of rat mortalin in NIH3T3 cells reduced cellular survival in accordance with mortalin being associated to cellular senescence. Hence we consider the gene encoding for mortalin at chromosome 5q31.1 a putative candidate gene in cytokine induced beta-cell destruction. We scanned the human mortalin gene for polymorphisms and identified three novel polymorphisms. Neither the SNPs individually nor as constructed haplotypes showed disease association tested by (E)TDT in a Danish type 1 diabetes (T1DM) population. Furthermore, we tested the D5S500 microsatelite located close to 5q31.1 without finding linkage to (T1DM). In conclusion, the functional data identifying a difference in mortalin expression in IL-1beta stimulated islets between two rat strains and over-expression of mortalin in NIH3T3 cells associated with decreased viability suggests a functional role for mortalin in cytokine mediated beta cell destruction; however, the identified polymorphisms did not reveal any association in the presence of linkage disequilibrium of mortalin to T1DM in the Danish population.

  17. THE EXPRESSION OF p16 AND CYCLIN D1 IN PROLIFERATIVE ENDOMETRIUM AND ENDOMETRIAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To studythe role of p16 and cyclin in the genesis and development of endometrial car-cinoma. Methods 12 cases of normal endometrium, 22 cases of proliferative endometrium and 41 cases of endome- trial carcinoma were detected for the expression of p16 and cyclin D1 by means of immunohistochemical S-P. Results In normal endometrium p16 was expressed while cyclm D1was almost negative in the proliferative phase, but both of them were negative in the secretory phase. Among the groups of the simple and compound hyperplasia, the atypical hyperplasia and the endometrial carcinoma,the expression of p16 showed a descending tendency, while the expression of cyclin showed an ascending tendency. In endometrial carcinomas the expression of p16 was significantly lower than that of normal endometrium and proliferative endometrium (P<0. 01 ,P<0.05). However, the expression of cy- clin in proliferate endometrium and endometrial carcinoma was significantly higher than that in normal endometri- un (P<0. 05,P<0. 01). The overexpression of cyclin D1 in the atypical hyperplasia group was obviously different from that in the simple and compound hyperplasia group (P<0.01). In endometrial carcinoma,the expression of p16 was decreasing with the descending of cell differentiate degree, on the opposite, the expression of cyclin was in-creased and there existed a negative correlation between them. It was also observed that the overexpression of cyclin was significant different between and ( P <0. 01 ). Conclusion p1 6 is a negative regulating factor of cell cycle in endometrial carcinoma, while cyclin is a positive one. Both of them are important in the genesis and devel-opment of endometrial carcinoma. The Iow expression of p1 6 and the overexpression of cyclin are related with the malicious biological behaviors of endometrial carcinoma and maybe play an important role in the judgement of prog- nosis. Overexpression of cyclin may be an earlier molecular event in the genesis of

  18. p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells.

    Science.gov (United States)

    Deng, Wen; Zhou, Yuan; Tiwari, Agnes F Y; Su, Hang; Yang, Jie; Zhu, Dandan; Lau, Victoria Ming Yi; Hau, Pok Man; Yip, Yim Ling; Cheung, Annie L M; Guan, Xin-Yuan; Tsao, Sai Wah

    2015-03-15

    Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21.

  19. Berberine inhibits growth, induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase 3 and PARP.

    Science.gov (United States)

    Mantena, Sudheer K; Sharma, Som D; Katiyar, Santosh K

    2006-10-01

    Chemotherapeutic approach using non-toxic botanicals may be one of the strategies for the management of the skin cancers. Here we report that in vitro treatment of human epidermoid carcinoma A431 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability (3-77%, P berberine-induced G(1) cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), a simultaneous decrease in Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and enhanced binding of Cdki-Cdk. In additional studies, treatment of A431 cells with berberine (15-75 microM) for 72 h resulted in a significant dose-dependent increase in apoptosis (31-60%, P berberine-treated control (11.7%), which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase. Pretreatment of A431 cells with the pan-caspase inhibitor (z-VAD-fmk) significantly blocked the berberine-induced apoptosis in A431 cells confirmed that berberine-induced apoptosis is mediated through activation of caspase 3-dependent pathway. Together, this study for the first time identified berberine as a chemotherapeutic agent against human epidermoid carcinoma A431 cells in vitro, further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of non-melanoma skin cancers.

  20. HIV-1 expression induces cyclin D1 expression and pRb phosphorylation in infected podocytes: cell-cycle mechanisms contributing to the proliferative phenotype in HIV-associated nephropathy

    Directory of Open Access Journals (Sweden)

    Husain Mohammad

    2002-09-01

    Full Text Available Abstract Background The aberrant cell-cycle progression of HIV-1-infected kidney cells plays a major role in the pathogenesis of HIV-associated nephropathy, however the mechanisms whereby HIV-1 induces infected glomerular podocytes or infected tubular epithelium to exit quiescence are largely unknown. Here, we ask whether the expression of HIV-1 genes in infected podocytes induces cyclin D1 and phospho-pRb (Ser780 expression, hallmarks of cyclin D1-mediated G1 → S phase progression. Results We assessed cyclin D1 and phospho-pRb (Ser780 expression in two well-characterized models of HIV-associated nephropathy pathogenesis: HIV-1 infection of cultured podocytes and HIV-1 transgenic mice (Tg26. Compared to controls, cultured podocytes expressing HIV-1 genes, and podocytes and tubular epithelium from hyperplastic nephrons in Tg26 kidneys, had increased levels of phospho-pRb (Ser780, a target of active cyclin D1/cyclin-dependent kinase-4/6 known to promote G1 → S phase progression. HIV-1-infected podocytes showed markedly elevated cyclin D1 mRNA and cyclin D1 protein, the latter of which did not down-regulate during cell-cell contact or differentiation, suggesting post-transcriptional stabilization of cyclin D1 protein levels by HIV-1. The selective suppression of HIV-1 transcription by the cyclin-dependent kinase inhibitor, flavopiridol, abrogated cyclin D1 expression, underlying the requirement for HIV-1 encoded products to induce cyclin D1. Indeed, HIV-1 virus deleted of nef failed to induce cyclin D1 mRNA to the level of other single gene mutant viruses. Conclusions HIV-1 expression induces cyclin D1 and phospho-pRb (Ser780 expression in infected podocytes, suggesting that HIV-1 activates cyclin D1-dependent cell-cycle mechanisms to promote proliferation of infected renal epithelium.

  1. The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells

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    Pumfery Anne

    2004-04-01

    Full Text Available Abstract Background The human T-cell leukemia virus type 1 (HTLV-1 Tax protein indirectly influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. The function of Tax primarily relies on protein-protein interactions. We have previously shown that Tax upregulates the cell cycle checkpoint proteins p21/waf1 and cyclin D2. Here we describe the consequences of upregulating these G1/S checkpoint regulators in HTLV-1 infected cells. Results To further decipher any physical and functional interactions between cyclin D2 and p21/waf1, we used a series of biochemical assays from HTLV-1 infected and uninfected cells. Immunoprecipitations from HTLV-1 infected cells showed p21/waf1 in a stable complex with cyclin D2/cdk4. This complex is active as it phosphorylates the Rb protein in kinase assays. Confocal fluorescent microscopy indicated that p21/waf1 and cyclin D2 colocalize in HTLV-1 infected, but not in uninfected cells. Furthermore, in vitro kinase assays using purified proteins demonstrated that the addition of p21/waf1 to cyclin D2/cdk4 increased the kinase activity of cdk4. Conclusion These data suggest that the p21/cyclin D2/cdk4 complex is not an inhibitory complex and that p21/waf1 could potentially function as an assembly factor for the cyclin D2/cdk4 complex in HTLV-1 infected cells. A by-product of this assembly with cyclin D2/cdk4 is the sequestration of p21/waf1 away from the cyclin E/cdk2 complex, allowing this active cyclin-cdk complex to phosphorylate Rb pocket proteins efficiently and push cells through the G1/S checkpoint. These two distinct functional and physical activities of p21/waf1 suggest that RNA tumor viruses manipulate the G1/S checkpoint by deregulating cyclin and cdk complexes.

  2. The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells

    Science.gov (United States)

    Kehn, Kylene; Deng, Longwen; de la Fuente, Cynthia; Strouss, Katharine; Wu, Kaili; Maddukuri, Anil; Baylor, Shanese; Rufner, Robyn; Pumfery, Anne; Bottazzi, Maria Elena; Kashanchi, Fatah

    2004-01-01

    Background The human T-cell leukemia virus type 1 (HTLV-1) Tax protein indirectly influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. The function of Tax primarily relies on protein-protein interactions. We have previously shown that Tax upregulates the cell cycle checkpoint proteins p21/waf1 and cyclin D2. Here we describe the consequences of upregulating these G1/S checkpoint regulators in HTLV-1 infected cells. Results To further decipher any physical and functional interactions between cyclin D2 and p21/waf1, we used a series of biochemical assays from HTLV-1 infected and uninfected cells. Immunoprecipitations from HTLV-1 infected cells showed p21/waf1 in a stable complex with cyclin D2/cdk4. This complex is active as it phosphorylates the Rb protein in kinase assays. Confocal fluorescent microscopy indicated that p21/waf1 and cyclin D2 colocalize in HTLV-1 infected, but not in uninfected cells. Furthermore, in vitro kinase assays using purified proteins demonstrated that the addition of p21/waf1 to cyclin D2/cdk4 increased the kinase activity of cdk4. Conclusion These data suggest that the p21/cyclin D2/cdk4 complex is not an inhibitory complex and that p21/waf1 could potentially function as an assembly factor for the cyclin D2/cdk4 complex in HTLV-1 infected cells. A by-product of this assembly with cyclin D2/cdk4 is the sequestration of p21/waf1 away from the cyclin E/cdk2 complex, allowing this active cyclin-cdk complex to phosphorylate Rb pocket proteins efficiently and push cells through the G1/S checkpoint. These two distinct functional and physical activities of p21/waf1 suggest that RNA tumor viruses manipulate the G1/S checkpoint by deregulating cyclin and cdk complexes. PMID:15169570

  3. Molecular evolution of cyclin proteins in animals and fungi

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    Afonnikov Dmitry A

    2011-07-01

    Full Text Available Abstract Background The passage through the cell cycle is controlled by complexes of cyclins, the regulatory units, with cyclin-dependent kinases, the catalytic units. It is also known that cyclins form several families, which differ considerably in primary structure from one eukaryotic organism to another. Despite these lines of evidence, the relationship between the evolution of cyclins and their function is an open issue. Here we present the results of our study on the molecular evolution of A-, B-, D-, E-type cyclin proteins in animals and fungi. Results We constructed phylogenetic trees for these proteins, their ancestral sequences and analyzed patterns of amino acid replacements. The analysis of infrequently fixed atypical amino acid replacements in cyclins evidenced that accelerated evolution proceeded predominantly during paralog duplication or after it in animals and fungi and that it was related to aromorphic changes in animals. It was shown also that evolutionary flexibility of cyclin function may be provided by consequential reorganization of regions on protein surface remote from CDK binding sites in animal and fungal cyclins and by functional differentiation of paralogous cyclins formed in animal evolution. Conclusions The results suggested that changes in the number and/or nature of cyclin-binding proteins may underlie the evolutionary role of the alterations in the molecular structure of cyclins and their involvement in diverse molecular-genetic events.

  4. F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

    Science.gov (United States)

    Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Zou, Chunbin; Miller, Hannah L.; Fenton, Moon; McDyer, John F.; Boyiadzis, Michael

    2012-01-01

    Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G0 phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G0 phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCFFBXL2 in lymphoproliferative malignancies. PMID:22323446

  5. MicroRNA-206 induces G1 arrest in melanoma by inhibition of CDK4 and Cyclin D.

    Science.gov (United States)

    Georgantas, Robert W; Streicher, Katie; Luo, Xiaobing; Greenlees, Lydia; Zhu, Wei; Liu, Zheng; Brohawn, Philip; Morehouse, Christopher; Higgs, Brandon W; Richman, Laura; Jallal, Bahija; Yao, Yihong; Ranade, Koustubh

    2014-03-01

    Expression profiling of microRNAs in melanoma lesional skin biopsies compared with normal donor skin biopsies, as well as melanoma cell lines compared with normal melanocytes, revealed that hsa-miR-206 was down-regulated in melanoma (-75.4-fold, P = 1.7 × 10(-4)). MiR-206 has been implicated in a large number of cancers, including breast, lung, colorectal, ovarian, and prostate cancers; however, its role in tumor development remains largely unknown, its biologic function is poorly characterized, and its targets affecting cancer cells are largely unknown. MiR-206 reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatics identified cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate targets. Western blots and 3'UTR reporter gene assays revealed that miR-206 inhibited translation of CDK4, Cyclin D1, and Cyclin C. Additionally, hsa-miR-206 transfection induced G1 arrest in multiple melanoma cell lines. These observations support hsa-miR-206 as a tumor suppressor in melanoma and identify Cyclin C, Cyclin D1, and CDK4 as miR-206 targets.

  6. Dysregulation of CDK8 and Cyclin C in tumorigenesis %Dysregulation of CDK8 and Cyclin C in tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Wu Xu; Jun-Yuan Ji

    2011-01-01

    Appropriately controlled gene expression is fundamental for normal growth and survival of all living organisms.In eukaryotes,the transcription of protein-coding mRNAs is dependent on RNA polymerase Ⅱ (Pol Ⅱ).The multi-subunit transcription cofactor Mediator complex is proposed to regulate most,if not all,of the Pol Ⅱ-dependent transcription.Here we focus our discussion on two subunits of the Mediator complex,cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC),because they are either mutated or amplified in a variety of human cancers.CDK8 functions as an oncoprotein in melanoma and colorectal cancers,thus there are considerable interests in developing drugs specifically targeting the CDK8 kinase activity.However,to evaluate the feasibility of targeting CDK8 for cancer therapy and to understand how their dysregulation contributes to tumorigenesis,it is essential to elucidate the in vivo function and regulation of CDK8-CycC,which are still poorly understood in multi-cellular organisms.We summarize the evidence linking their dysregulation to various cancers and present our bioinformatics and computational analyses on the structure and evolution of CDK8.We also discuss the implications of these observations in tumorigenesis.Because most of the Mediator subunits,including CDK8 and CycC,are highly conserved during eukaryotic evolution,we expect that investigations using model organisms such as Drosophila will provide important insights into the function and regulation of CDK8 and CycC in different cellular and developmental contexts.

  7. Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Sun, Shiqin [College of Pharmacy, Harbin Medical University-Daqing, Daqing, Heilongjiang 163319 (China); Chen, Xiangmei, E-mail: xm_chen6176@bjmu.edu.cn [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Lu, Fengmin [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China)

    2014-04-25

    Highlights: • The expression of Fbx4 was significantly lower in HCC tissues. • Novel splicing variants of Fbx4 were identified. • These novel variants are much more abundant in human cancer tissues and cells. • The novel Fbx4 isoforms could promote cell proliferation and migration in vitro. • These isoforms showed less capability for cyclin D1 binding and degradation. - Abstract: Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant

  8. Preparation and application of polyclonal antibodiesagainst KSHV v-cyclin.

    Science.gov (United States)

    Xue, Min; Guo, Yuanyuan; Yan, Qin; Qin, Di; Lu, Chun

    2013-09-01

    We prepared rabbit polyclonal antibodies against Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded v-cyclin (ORF 72) and detected the natural viral protein using these polyclonal antibodies. Three antigenic polypeptides of v-cyclin were designed and synthesized. A fragment of the v-cyclin gene was cloned into a eukaryotic expression vector pEF-MCS-Flag-IRES/Puro to construct a recombinant vector, pEF v-cyclin. Then, pEF v-cyclin was transfected into 293T and EA.hy926 cells to obtain v-cyclin-Flag fusion proteins. Six New Zealand white rabbits were immunized with KLH-conjugated peptides to generate polyclonal antibodies against v-cyclin. The polyclonal antibodies were then characterized by ELISA and Western blotting assays. Finally, the polyclonal antibodies against v-cyclin were used to detect natural viral protein expressed in BCBL-1, BC-3, and JSC-1 cells. The results showed that using the Flag antibody, v-cyclin-Flag fusion protein was detected in 293T and EA.hy926 cells transfected with pEF-v-cyclin. Furthermore, ELISA showed that the titer of the induced polyclonal rabbit anti-v-cyclin antibodies was higher than 1:8,000. In Western blotting assays, the antibodies reacted specifically with the v-cyclin-Flag fusion protein as well as the natural viral protein. The recombinant expression vector pEF-v-cyclin was constructed successfully, and the polyclonal antibodies prepared can be used for various biological tests including ELISA and Western blotting assays.

  9. The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain.

    Science.gov (United States)

    Steingruber, Mirjam; Socher, Eileen; Hutterer, Corina; Webel, Rike; Bergbrede, Tim; Lenac, Tihana; Sticht, Heinrich; Marschall, Manfred

    2015-08-11

    Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.

  10. The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain

    Directory of Open Access Journals (Sweden)

    Mirjam Steingruber

    2015-08-01

    Full Text Available Replication of human cytomegalovirus (HCMV is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.

  11. Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells

    DEFF Research Database (Denmark)

    Friedrichsen, Birgitte N; Richter, Henrijette E; Hansen, Johnny A

    2003-01-01

    in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1...... cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and beta-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced......-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate...

  12. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex

    DEFF Research Database (Denmark)

    Lukas, C; Sørensen, Claus Storgaard; Kramer, E

    1999-01-01

    In mammalian somatic-cell cycles, progression through the G1-phase restriction point and initiation of DNA replication are controlled by the ability of the retinoblastoma tumour-suppressor protein (pRb) family to regulate the E2F/DP transcription factors. Continuing transcription of E2F target ge...... in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the post-restriction point period.......In mammalian somatic-cell cycles, progression through the G1-phase restriction point and initiation of DNA replication are controlled by the ability of the retinoblastoma tumour-suppressor protein (pRb) family to regulate the E2F/DP transcription factors. Continuing transcription of E2F target...... genes beyond the G1/S transition is required for coordinating S-phase progression with cell division, a process driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. How E2F-dependent events at G1/S transition are orchestrated with cyclin B and APC activity...

  13. Inotodiol inhabits proliferation and induces apoptosis through modulating expression of cyclinE, p27, bcl-2, and bax in human cervical cancer HeLa cells.

    Science.gov (United States)

    Zhao, Li-Wei; Zhong, Xiu-Hong; Yang, Shu-Yan; Zhang, Yi-Zhong; Yang, Ning-Jiang

    2014-01-01

    Inonotus obliquus is a medicinal mushroom that has been used as an effective agent to treat various diseases such as diabetes, tuberculosis and cancer. Inotodiol, an included triterpenoid shows significant anti-tumor effect. However, the mechanisms have not been well documented. In this study, we aimed to explore the effect of inotodiol on proliferation and apoptosis in human cervical cancer HeLa cells and investigated the underlying molecular mechanisms. HeLa cells were treated with different concentrations of inotodiol. The MTT assay was used to evaluate cell proliferating ability, flow cytometry (FCM) was employed for cell cycle analysis and cell apoptosis, while expression of cyclinE, p27, bcl-2 and bax was detected by immunocytochemistry. Proliferation of HeLa cells was inhibited by inotodiolin a dose-dependent manner at 24h (r=0.9999, pHeLa cells was detected after treatment and the apoptosis rate with the concentration and longer incubation time (r=1.0, pHeLa cells and induced apoptosis in vitro. The mechanisms may be related to promoting apoptosis through increasing the expression of bax and cutting bcl-2 and affecting the cell cycle by down-regulation the expression of cyclin E and up-regulation of p27. The results further indicate the potential value of inotodiol for treatment of human cervical cancer.

  14. RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Hoban PR

    2006-06-01

    Full Text Available Abstract Background Idiopathic Pulmonary Fibrosis (IPF is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. Methods Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. Results Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p Conclusion These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

  15. Cyclin D3 promotes pancreatic β-cell fitness and viability in a cell cycle-independent manner and is targeted in autoimmune diabetes.

    Science.gov (United States)

    Saavedra-Ávila, Noemí Alejandra; Sengupta, Upasana; Sánchez, Begoña; Sala, Ester; Haba, Laura; Stratmann, Thomas; Verdaguer, Joan; Mauricio, Dídac; Mezquita, Belén; Ropero, Ana Belén; Nadal, Ángel; Mora, Conchi

    2014-08-19

    Type 1 diabetes is an autoimmune condition caused by the lymphocyte-mediated destruction of the insulin-producing β cells in pancreatic islets. We aimed to identify final molecular entities targeted by the autoimmune assault on pancreatic β cells that are causally related to β cell viability. Here, we show that cyclin D3 is targeted by the autoimmune attack on pancreatic β cells in vivo. Cyclin D3 is down-regulated in a dose-dependent manner in β cells by leukocyte infiltration into the islets of the nonobese diabetic (NOD) type 1 diabetes-prone mouse model. Furthermore, we established a direct in vivo causal link between cyclin D3 expression levels and β-cell fitness and viability in the NOD mice. We found that changes in cyclin D3 expression levels in vivo altered the β-cell apoptosis rates, β-cell area homeostasis, and β-cell sensitivity to glucose without affecting β-cell proliferation in the NOD mice. Cyclin D3-deficient NOD mice exhibited exacerbated diabetes and impaired glucose responsiveness; conversely, transgenic NOD mice overexpressing cyclin D3 in β cells exhibited mild diabetes and improved glucose responsiveness. Overexpression of cyclin D3 in β cells of cyclin D3-deficient mice rescued them from the exacerbated diabetes observed in transgene-negative littermates. Moreover, cyclin D3 overexpression protected the NOD-derived insulinoma NIT-1 cell line from cytokine-induced apoptosis. Here, for the first time to our knowledge, cyclin D3 is identified as a key molecule targeted by autoimmunity that plays a nonredundant, protective, and cell cycle-independent role in β cells against inflammation-induced apoptosis and confers metabolic fitness to these cells.

  16. Acetylation of cyclin-dependent kinase 5 is mediated by GCN5

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Juhyung; Yun, Nuri; Kim, Chiho [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of); Song, Min-Young; Park, Kang-Sik [Department of Physiology and Biomedical Science Institute, Kyung Hee University School of Medicine, Seoul 130-701 (Korea, Republic of); Oh, Young J., E-mail: yjoh@yonsei.ac.kr [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of)

    2014-04-25

    Highlights: • Cyclin-dependent kinase 5 (CDK5) is present as an acetylated form. • CDK5 is acetylated by GCN5. • CDK5’s acetylation site is mapped at Lys33. • Its acetylation may affect CDK5’s kinase activity. - Abstract: Cyclin-dependent kinase 5 (CDK5), a member of atypical serine/threonine cyclin-dependent kinase family, plays a crucial role in pathophysiology of neurodegenerative disorders. Its kinase activity and substrate specificity are regulated by several independent pathways including binding with its activator, phosphorylation and S-nitrosylation. In the present study, we report that acetylation of CDK5 comprises an additional posttranslational modification within the cells. Among many candidates, we confirmed that its acetylation is enhanced by GCN5, a member of the GCN5-related N-acetyl-transferase family of histone acetyltransferase. Co-immunoprecipitation assay and fluorescent localization study indicated that GCN5 physically interacts with CDK5 and they are co-localized at the specific nuclear foci. Furthermore, liquid chromatography in conjunction with a mass spectrometry indicated that CDK5 is acetylated at Lys33 residue of ATP binding domain. Considering this lysine site is conserved among a wide range of species and other related cyclin-dependent kinases, therefore, we speculate that acetylation may alter the kinase activity of CDK5 via affecting efficacy of ATP coordination.

  17. A Whi7-anchored loop controls the G1 Cdk-cyclin complex at start.

    Science.gov (United States)

    Yahya, Galal; Parisi, Eva; Flores, Alba; Gallego, Carme; Aldea, Martí

    2014-01-09

    Cells commit to a new cell cycle at Start by activation of the G1 Cdk-cyclin complex which, in turn, triggers a genome-wide transcriptional wave that executes the G1/S transition. In budding yeast, the Cdc28-Cln3 complex is regulated by an ER-retention mechanism that is important for proper cell size control. We have isolated small-cell-size CDC28 mutants showing impaired retention at the ER and premature accumulation of the Cln3 cyclin in the nucleus. The differential interactome of a quintuple Cdc28(wee) mutant pinpointed Whi7, a Whi5 paralog targeted by Cdc28 that associates to the ER in a phosphorylation-dependent manner. Our results demonstrate that the Cln3 cyclin and Whi7 act in a positive feedback loop to release the G1 Cdk-cyclin complex and trigger Start once a critical size has been reached, thus uncovering a key nonlinear mechanism at the earliest known events of cell-cycle entry.

  18. Overexpression of Hyaluronan-binding Protein 1 (HABP1/p32/gC1qR) in HepG2 Cells Leads to Increased Hyaluronan Synthesis and Cell Proliferation by Up-regulation of Cyclin D1 in AKT-dependent Pathway*

    Science.gov (United States)

    Kaul, Rachna; Saha, Paramita; Saradhi, Mallampati; Prasad, Ramachandra L. A.; Chatterjee, Soumya; Ghosh, Ilora; Tyagi, Rakesh K.; Datta, Kasturi

    2012-01-01

    Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased “HA pool,” formation of the “HA cable” structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways. PMID:22451658

  19. Loss of PiT-1 results in abnormal endocytosis in the yolk sac visceral endoderm.

    Science.gov (United States)

    Wallingford, Mary C; Giachelli, Cecilia M

    2014-08-01

    PiT-1 protein is a transmembrane sodium-dependent phosphate (Pi) transporter. PiT-1 knock out (KO) embryos die from largely unknown causes by embryonic day (E) 12.5. We tested the hypothesis that PiT-1 is required for endocytosis in the embryonic yolk sac (YS) visceral endoderm (VE). Here we present data supporting that PiT-1 KO results in a YS remodeling defect and decreased endocytosis in the YS VE. The remodeling defect is not due to an upstream cardiomyocyte requirement for PiT-1, as SM22αCre-specific KO of PiT-1 in the developing heart and the YS mesodermal layer (ME) does not recapitulate the PiT-1 global KO phenotype. Furthermore, we find that high levels of PiT-1 protein localize to the YS VE apical membrane. Together these data support that PiT-1 is likely required in YS VE. During normal development maternal immunoglobulin (IgG) is endocytosed into YS VE and accumulates in the apical side of the VE in a specialized lysosome termed the apical vacuole (AV). We have identified a reduction in PiT-1 KO VE cell height and a striking loss of IgG accumulation in the PiT-1 KO VE. The endocytosis genes Tfeb, Lamtor2 and Snx2 are increased at the RNA level. Lysotracker Red staining reveals a loss of distinct AVs, and yolk sacs incubated ex vivo with phRODO Green Dextran for Endocytosis demonstrate a functional loss of endocytosis. As yolk sac endocytosis is controlled in part by microautophagy, but expression of LC3 had not been examined, we investigated LC3 expression during yolk sac development and found stage-specific LC3 RNA expression that is predominantly from the YS VE layer at E9.5. Normalized LC3-II protein levels are decreased in the PiT-1 KO YS, supporting a requirement for PiT-1 in autophagy in the YS. Therefore, we propose the novel idea that PiT-1 is central to the regulation of endocytosis and autophagy in the YS VE.

  20. Activation of the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway in human cholesteatoma epithelium.

    Science.gov (United States)

    Liu, Wei; Yin, Tuanfang; Ren, Jihao; Li, Lihua; Xiao, Zian; Chen, Xing; Xie, Dinghua

    2014-02-01

    Cholesteatoma is a benign keratinizing squamous epithelial lesion characterized by the hyper-proliferation of keratinocytes with abundant production of keratin debris in the middle ear. The epidermal growth factor receptor (EGFR)/Akt/nuclear factor-kappa B (NF-κB)/cyclinD1 signaling pathway is one of the most important pathways in regulating cell survival and proliferation. We hypothesized that the EGFR/Akt/NF-κB/cyclinD1 signaling pathway may be activated and involved in the cellular hyperplasia mechanism in acquired cholesteatoma epithelium. Immunohistochemical staining of phosphorylated EGFR (p-EGFR), phosphorylated Akt (p-Akt), activated NF-κB and cyclinD1 protein was performed in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium. Protein expression of p-EGFR, p-Akt, activated NF-κB and cyclinD1 in cholesteatoma epithelium was significantly increased when compared with normal EAC epithelium (p epithelium, a significant positive association was observed between p-EGFR and p-Akt expression and between the expressions of p-Akt and NF-κB, NF-κB and cyclinD1, respectively (p 0.05). The increased protein expression of p-EGFR, p-Akt, NF-κB and cyclinD1 and their associations in cholesteatoma epithelium suggest that the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway is active and may be involved in the regulatory mechanisms of cellular hyperplasia in cholesteatoma epithelium.

  1. A simple method for NMR t1 noise suppression

    Science.gov (United States)

    Mo, Huaping; Harwood, John S.; Yang, Danzhou; Post, Carol Beth

    2017-03-01

    t1 noise appears as random or semi-random spurious streaks along the indirect t1 (F1) dimension of a 2D or nD NMR spectrum. It can significantly downgrade spectral quality, especially for spectra with strong diagonal signals such as NOESY, because useful and weak cross-peaks can be easily buried under t1 noise. One of the significant contributing factors to t1 noise is unwanted and semi-random F2 signal modulation during t1 acquisition. As such, t1 noise from different acquisitions is unlikely to correlate with each other strongly. In the case of NOESY, co-addition of multiple spectra significantly reduces t1 noise compared with conventional acquisition with the same amount of total acquisition time and resolution.

  2. A Study to investigate the role of p27 and Cyclin E immunoexpression as a prognostic factor in early breast carcinoma

    Directory of Open Access Journals (Sweden)

    Chetty Runjan

    2011-03-01

    Full Text Available Abstract Background Cyclin E and p27 expression is easy to assess in human tissues by standard immunohistochemical techniques. Immunohistochemistry is cost effective, relatively easy to perform and will play more of a role in the future management of cancer. The aim of this study was to investigate the role of p27 and cyclin E immunoexpression as a prognostic factor in early breast carcinoma. Methods Cyclin E and p27 immunohistochemistry was performed on sixty six cases of breast carcinoma submitted over a five year period to the Division of Anatomical Pathology, Groote Schuur hospital; Whittaker and Associates; and PathCare. All tumours included in this study were less than 5 cm in diameter (pT1 and pT2 stage and all the patients had wide local excisions performed. Follow up information was obtained from patient folders in the Department of Radiation Oncology. Results There was no significant association of cyclin E and p27 expression with distant metastasis free survival (MFS for all invasive carcinomas in contrast to grade, lymph node spread and vascular invasion. However, there was a statistically significant direct association of cyclin E with distant metastases in all invasive carcinomas, in the subgroup of infiltrating duct carcinomas (IDC and in the node negative group when cyclin E was stratified as negative and positive (low/high. In this study of early breast carcinoma, only 9/66 cases showed cyclin E expression. Of these, four patients had distant metastases, one patient had a local recurrence and four patients were alive at last follow-up. Furthermore, cyclin E expression was significantly associated with grade, lymph node spread, oestrogen receptor status and histological type. None of the lobular carcinomas showed cyclin E positivity and only one case of lobular carcinoma presented with distant metastases. 59/66 cases were positive (low/high for p27 while seven cases were negative, 22 cases showed low expression and 37 cases

  3. A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    OpenAIRE

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-01-01

    Cyclin dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel...

  4. Phosphorylation of threonine 204 of DEAD-box RNA helicase DDX3 by cyclin B/cdc2 in vitro.

    Science.gov (United States)

    Sekiguchi, Takeshi; Kurihara, Yoshiko; Fukumura, Junko

    2007-05-11

    DDX3 is a DEAD-box RNA helicase involved in human immunodeficiency virus mRNA export and translation. Previously, we reported that DDX3 is required for cyclin A expression. To examine whether DDX3 is regulated at the post-transcriptional level, we determined the phosphorylation sites of hamster DDX3 in vitro. Threonine 204 (Thr204) is a conserved amino acid residue of DDX3 homologues in yeast, frog, hamster, and human that is located within motif Q of DEAD-box RNA helicases. A Thr204 to Glu204 DDX3 mutant protein lost its function, suggesting that phosphorylation at Thr204 affects DDX3 function. Thr204 was phosphorylated by cyclin B/cdc2. Thr323 in motif Ib was also phosphorylated by cyclin B/cdc2 kinase. We propose a novel function of cyclin B/cdc2 kinase in mitosis, which is to cause a loss of DDX3 function to repress cyclin A expression and to decrease ribosome biogenesis and translation during mitosis.

  5. The E3 Ubiquitin Ligase SCF(Cyclin F) Transmits AKT Signaling to the Cell-Cycle Machinery.

    Science.gov (United States)

    Choudhury, Rajarshi; Bonacci, Thomas; Wang, Xianxi; Truong, Andrew; Arceci, Anthony; Zhang, Yanqiong; Mills, Christine A; Kernan, Jennifer L; Liu, Pengda; Emanuele, Michael J

    2017-09-26

    The oncogenic AKT kinase is a key regulator of apoptosis, cell growth, and cell-cycle progression. Despite its important role in proliferation, it remains largely unknown how AKT is mechanistically linked to the cell cycle. We show here that cyclin F, a substrate receptor F-box protein for the SCF (Skp1/Cul1/F-box) family of E3 ubiquitin ligases, is a bona fide AKT substrate. Cyclin F expression oscillates throughout the cell cycle, a rare feature among the 69 human F-box proteins, and all of its known substrates are involved in proliferation. AKT phosphorylation of cyclin F enhances its stability and promotes assembly into productive E3 ligase complexes. Importantly, expression of mutant versions of cyclin F that cannot be phosphorylated by AKT impair cell-cycle entry. Our data suggest that cyclin F transmits mitogen signaling through AKT to the core cell-cycle machinery. This discovery has potential implications for proliferative control in malignancies where AKT is activated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Gene expression of δ1- and δ3- cyclins in root meristem cells of Pisum sativum L. under clinorotation

    Science.gov (United States)

    Artemenko, Olga A.

    2005-08-01

    The data on the influence of altered gravity on plant cell proliferation are ambiguity [1, 3], although a delay of the cell cycle duration in comparison with the control was observed in the most cases. Evidently, the principal regulatory processes in a cell cycle occur in the G1-phase. Cyclins are important regulators in different phases of a cell cycle and have a high homology in plant and mammalian cells. δ-cyclins are specific for plants and control the presynthetic phase events and beginning of S-phase. Therefore, we firstly performed the study the influence of slow horizontal clinorotation on δ3- and δ1-cyclins genes expression in early events of pea root development.

  7. Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic acid: a Potential Role in Cancer Prevention

    Science.gov (United States)

    Dachineni, Rakesh; Ai, Guoqiang; Kumar, D. Ramesh; Sadhu, Satya S.; Tummala, Hemachand; Bhat, G. Jayarama

    2015-01-01

    Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anti-cancer effects are still being elucidated. We hypothesized that aspirin’s chemopreventive actions may involve cell cycle regulation through modulation of the levels or activity of cyclin A2/cyclin dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The down regulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. Firstly, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Secondly, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, pre-incubation of CDK2 with salicylic acid, dose-dependently quenched the fluorescence due to ANS. Thirdly, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid down-regulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention. Implications Biochemical and structural studies indicate that the anti-proliferative actions of aspirin are mediated through cyclin A2/CDK2. PMID:26685215

  8. Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition*

    Science.gov (United States)

    Timofeev, Oleg; Cizmecioglu, Onur; Settele, Florian; Kempf, Tore; Hoffmann, Ingrid

    2010-01-01

    Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr161 by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr14 and Tyr15 phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G2/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr15. In addition, Tyr15-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G2 and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr161 phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G2/M is tightly coupled and regulated by Cdc25 phosphatases. PMID:20360007

  9. Targeting Cyclin-Dependent Kinases in Human Cancers: From Small Molecules to Peptide Inhibitors

    Directory of Open Access Journals (Sweden)

    Marion Peyressatre

    2015-01-01

    Full Text Available Cyclin-dependent kinases (CDK/Cyclins form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported.

  10. Targeting zebrafish and murine pituitary corticotroph tumors with a cyclin-dependent kinase (CDK) inhibitor

    Science.gov (United States)

    Liu, Ning-Ai; Jiang, Hong; Ben-Shlomo, Anat; Wawrowsky, Kolja; Fan, Xue-Mo; Lin, Shuo; Melmed, Shlomo

    2011-01-01

    Cushing disease caused by adrenocorticotropin (ACTH)-secreting pituitary adenomas leads to hypercortisolemia predisposing to diabetes, hypertension, osteoporosis, central obesity, cardiovascular morbidity, and increased mortality. There is no effective pituitary targeted pharmacotherapy for Cushing disease. Here, we generated germline transgenic zebrafish with overexpression of pituitary tumor transforming gene (PTTG/securin) targeted to the adenohypophyseal proopiomelanocortin (POMC) lineage, which recapitulated early features pathognomonic of corticotroph adenomas, including corticotroph expansion and partial glucocorticoid resistance. Adult Tg:Pomc-Pttg fish develop neoplastic coticotrophs and pituitary cyclin E up-regulation, as well as metabolic disturbances mimicking hypercortisolism caused by Cushing disease. Early development of corticotroph pathologies in Tg:Pomc-Pttg embryos facilitated drug testing in vivo. We identified a pharmacologic CDK2/cyclin E inhibitor, R-roscovitine (seliciclib; CYC202), which specifically reversed corticotroph expansion in live Tg:Pomc-Pttg embryos. We further validated that orally administered R-roscovitine suppresses ACTH and corticosterone levels, and also restrained tumor growth in a mouse model of ACTH-secreting pituitary adenomas. Molecular analyses in vitro and in vivo showed that R-roscovitine suppresses ACTH expression, induces corticotroph tumor cell senescence and cell cycle exit by up-regulating p27, p21 and p57, and downregulates cyclin E expression. The results suggest that use of selective CDK inhibitors could effectively target corticotroph tumor growth and hormone secretion. PMID:21536883

  11. Cyclin D3 is selectively required for proliferative expansion of germinal center B cells.

    Science.gov (United States)

    Cato, Matthew H; Chintalapati, Suresh K; Yau, Irene W; Omori, Sidne A; Rickert, Robert C

    2011-01-01

    The generation of robust T-cell-dependent humoral immune responses requires the formation and expansion of germinal center structures within the follicular regions of the secondary lymphoid tissues. B-cell proliferation in the germinal center drives ongoing antigen-dependent selection and the generation of high-affinity class-switched plasma and memory B cells. However, the mechanisms regulating B-cell proliferation within this microenvironment are largely unknown. Here, we report that cyclin D3 is uniquely required for germinal center progression. Ccnd3(-/-) mice exhibit a B-cell-intrinsic defect in germinal center maturation and fail to generate an affinity-matured IgG response. We determined that the defect resulted from failed proliferative expansion of GL7(+) IgD(-) PNA(+) B cells. Mechanistically, sustained expression of cyclin D3 was found to be regulated at the level of protein stability and controlled by glycogen synthase kinase 3 in a cyclic AMP-protein kinase A-dependent manner. The specific defect in proliferative expansion of GL7(+) IgD(-) PNA(+) B cells in Ccnd3(-/-) mice defines an underappreciated step in germinal center progression and solidifies a role for cyclin D3 in the immune response, and as a potential therapeutic target for germinal center-derived B-cell malignancies.

  12. Accelerated Stem Growth Rates and Improved Fiber Properties of Loblolly Pine: Functional Analysis Of CyclinD from Pinus taeda

    Energy Technology Data Exchange (ETDEWEB)

    Dr. John Cairney, School of Biology and Institute of Paper Science and Technology @ Georgia Tech, Georgia Institute of Technology; Dr. Gary Peter, University of Florida; Dr. Ulrika Egertsdotter, Dept. of Forestry, Virgina Tech; Dr. Armin Wagner, New Zealand Forest Research Institute Ltd. (Scion Research.)

    2005-11-30

    A sustained supply of low-cost, high quality raw materials is essential for the future success of the U.S. forest products industry. To maximize stem (trunk) growth, a fundamental understanding of the molecular mechanisms that regulate cell divisions within the cambial meristem is essential. We hypothesize that auxin levels within the cambial meristem regulate cyclin gene expression and this in turn controls cell cycle progression as occurs in all eukaryotic cells. Work with model plant species has shown that ectopic overexpression of cyclins promotes cell division thereby increasing root growth > five times. We intended to test whether ectopic overexpression of cambial cyclins in the cambial zone of loblolly pine also promotes cell division rates that enhance stem growth rates. Results generated in model annual angiosperm systems cannot be reliably extrapolated to perennial gymnosperms, thus while the generation and development of transgenic pine is time consuming, this is the necessary approach for meaningful data. We succeeded in isolating a cyclin D gene and Clustal analysis to the Arabidopsis cyclin D gene family indicates that it is more closely related to cyclin D2 than D1 or D3 Using this gene as a probe we observed a small stimulation of cyclin D expression in somatic embryo culture upon addition of auxin. We hypothesized that trees with more cells in the vascular cambial and expansion zones will have higher cyclin mRNA levels. We demonstrated that in trees under compressive stress where the rates of cambial divisions are increased on the underside of the stem relative to the top or opposite side, there was a 20 fold increase in the level of PtcyclinD1 mRNA on the compressed side of the stem relative to the opposite. This suggests that higher secondary growth rates correlate with PtcyclinD1 expression. We showed that larger diameter trees show more growth during each year and that the increased growth in loblolly pine trees correlates with more cell

  13. Piperine suppresses tumor growth and metastasis in vitro and in vivo in a 4T1 murine breast cancer model

    Institute of Scientific and Technical Information of China (English)

    Li-hua LAI; Qi-hong FU; Yang LIU; Kai JIANG; Qing-ming GUO; Qing-yun CHEN; Bin YAN; Qing-qing WANG; Jian-gen SHEN

    2012-01-01

    Aim:To investigate the effects of piperine,a major pungent alkaloid present in Piper nigrum and Piper Iongum,on the tumor growth and metastasis of mouse 4T1 mammary carcinoma in vitro and in vivo,and elucidate the underlying mechanisms.Methods:Growth of 4T1 cells was assessed using MTT assay.Apoptosis and cell cycle of 4T1 cells were evaluated with flow cytometry,and the related proteins were examined using Western blotting.Real-time quantitative PCR was applied to detect the expression of matrix metalloproteinases (MMPs).A highly malignant,spontaneously metastasizing 4T1 mouse mammary carcinoma model was used to evaluate the in vivo antitumor activity.Piperine was injected into tumors every 3 d for 3 times.Results:Piperine (35-280 μmol/L)inhibited the growth of 4T1 cells in time-and dose-dependent manners (the IC50 values were 105+1.08 and 78.52+1.06 μmol/L,respectively,at 48 and 72 h).Treatment of 4T1 cells with piperine (70-280 μmol/L)dose-dependently induced apoptosis of 4T1 cells,accompanying activation of caspase 3.The cells treated with piperine (140 and 280μmol/L)significantly increased the percentage of cells in G2/M phase with a reduction in the expression of cyclin B1.Piperine (140and 280 μmol/L)significantly decreased the expression of MMP-9 and MMP-13,and inhibited 4T1 cell migration in vitro.Injection of piperine (2.5 and 5 mg/kg)dose-dependently suppressed the primary 4T1 tumor growth and injection of piperine (5 mg/kg)significantly inhibited the lung metastasis.Conclusion:These results demonstrated that piperine is an effective antitumor compound in vitro and in vivo,and has the potential to be developed as a new anticancer drug.

  14. A medical device-grade T1 and ECV phantom for global T1 mapping quality assurance—the T1 Mapping and ECV Standardization in cardiovascular magnetic resonance (T1MES) program

    NARCIS (Netherlands)

    Captur, Gabriella; Gatehouse, Peter; Keenan, Kathryn; Heslinga, Friso Gerben; Bruehl, Ruediger; Prothmann, Marcel; Graves, Martin J.; Eames, Richard J.; Torlasco, Camilla; Benedetti, Giulia; Donovan, Jacqueline; Itterman, Bernd; Boubertakh, Redha; Bathgate, Andrew; Royet, Celine; Pang, Wenjie; Nezafat, Reza; Salerno, Michael; Kellman, Peter; Moon, James C.

    2016-01-01

    Background T1 mapping and extracellular volume (ECV) have the potential to guide patient care and serve as surrogate end-points in clinical trials, but measurements differ between cardiovascular magnetic resonance (CMR) scanners and pulse sequences. To help deliver T1 mapping to global clinical

  15. A medical device-grade T1 and ECV phantom for global T1 mapping quality assurance—the T1 Mapping and ECV Standardization in cardiovascular magnetic resonance (T1MES) program

    NARCIS (Netherlands)

    Captur, Gabriella; Gatehouse, Peter; Keenan, Kathryn; Heslinga, Friso Gerben; Bruehl, Ruediger; Prothmann, Marcel; Graves, Martin J.; Eames, Richard J.; Torlasco, Camilla; Benedetti, Giulia; Donovan, Jacqueline; Itterman, Bernd; Boubertakh, Redha; Bathgate, Andrew; Royet, Celine; Pang, Wenjie; Nezafat, Reza; Salerno, Michael; Kellman, Peter; Moon, James C.

    2016-01-01

    Background T1 mapping and extracellular volume (ECV) have the potential to guide patient care and serve as surrogate end-points in clinical trials, but measurements differ between cardiovascular magnetic resonance (CMR) scanners and pulse sequences. To help deliver T1 mapping to global clinical care

  16. Nucleophosmin phosphorylation by v-cyclin-CDK6 controls KSHV latency.

    Directory of Open Access Journals (Sweden)

    Grzegorz Sarek

    2010-03-01

    Full Text Available Nucleophosmin (NPM is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL and multicentric Castleman disease (MCD. In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199 in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.

  17. Epstein-Barr virus-encoded latent membrane protein 1 modulates cyclin D1 by c-Jun/Jun B heterodimers

    Institute of Scientific and Technical Information of China (English)

    SONG; Xin; TAO; Yongguang; ZENG; Liang; YANG; Jing; TANG; F

    2005-01-01

    In our recent studies, we found that LMP1 encoded by Epstein-Barr virus could accelerate the formation of active c-Jun/Jun B heterodimer. We studied the regulation of cyclinD1 by c-Jun/Jun B heterodimers by laser scanning confocal influorescence microscopy, Western blot, luciferase activity assay, super-EMSA and flow cytometry in the Tet-on-LMP1 HNE2 cell line, in which LMP1 expression was regulated by Tet-on system. c-Jun/Jun B heterodimers induced by LMP1 could up regulate cyclin D1 promoter activity and expression. Overexpression of cyclinD1 accelerated the progression of cell cycle.

  18. Three-dimensional ultrashort echo time cones T1ρ (3D UTE-cones-T1ρ ) imaging.

    Science.gov (United States)

    Ma, Ya-Jun; Carl, Michael; Shao, Hongda; Tadros, Anthony S; Chang, Eric Y; Du, Jiang

    2017-03-20

    We report a novel three-dimensional (3D) ultrashort echo time (UTE) sequence employing Cones trajectory and T1ρ preparation (UTE-Cones-T1ρ ) for quantitative T1ρ assessment of short T2 tissues in the musculoskeletal system. A basic 3D UTE-Cones sequence was combined with a spin-locking preparation pulse for T1ρ contrast. A relatively short TR was used to decrease the scan time, which required T1 measurement and compensation using 3D UTE-Cones data acquisitions with variable TRs. Another strategy to reduce the total scan time was to acquire multiple Cones spokes (Nsp ) after each T1ρ preparation and fat saturation. Four spin-locking times (TSL = 0-20 ms) were acquired over 12 min, plus another 7 min for T1 measurement. The 3D UTE-Cones-T1ρ sequence was compared with a two-dimensional (2D) spiral-T1ρ sequence for the imaging of a spherical CuSO4 phantom and ex vivo meniscus and tendon specimens, as well as the knee and ankle joints of healthy volunteers, using a clinical 3-T scanner. The CuSO4 phantom showed a T1ρ value of 76.5 ± 1.6 ms with the 2D spiral-T1ρ sequence, as well as 85.7 ± 3.6 and 89.2 ± 1.4 ms for the 3D UTE-Cones-T1ρ sequences with Nsp of 1 and 5, respectively. The 3D UTE-Cones-T1ρ sequence provided shorter T1ρ values for the bovine meniscus sample relative to the 2D spiral-T1ρ sequence (10-12 ms versus 16 ms, respectively). The cadaveric human Achilles tendon sample could only be imaged with the 3D UTE-Cones-T1ρ sequence (T1ρ  = 4.0 ± 0.9 ms), with the 2D spiral-T1ρ sequence demonstrating near-zero signal intensity. Human studies yielded T1ρ values of 36.1 ± 2.9, 18.3 ± 3.9 and 3.1 ± 0.4 ms for articular cartilage, meniscus and the Achilles tendon, respectively. The 3D UTE-Cones-T1ρ sequence allows volumetric T1ρ measurement of short T2 tissues in vivo.

  19. P53 but not cyclin E acts in a negative regulatory loop to control HER-2 expression in MCF-7 breast carcinoma cell line.

    Directory of Open Access Journals (Sweden)

    Hamed Montazeri

    2013-08-01

    Full Text Available Cyclin E, HER-2 and p53, are considered as major prognostic markers in breast cancer. As they are related in patho-clinical level, we aimed to check if they have any direct interaction on expression of each other. To study the effect of cyclin E on HER-2 expression, cell lines stably overexpressing cyclin E or its low molecular weight (LMW isoforms were generated. To understand the results of p53 silencing either alone or in combination with cyclin E overexpression, we created three different p53 stably knocked down cell lines. Protein expression was analyzed by western blot, HER-2 expression in the established cell lines were determined using SYBR green real time PCR and data analyzed by REST software. Results indicate that HER-2 expression is only downregulated following p53 silencing and none of cyclin E isoforms can alter its expression. The presence of cyclin E isoforms in p53 silenced clones also does not altered HER-2 expression. Given the fact that p53 degradation is increased by HER-2 overexpression, these data can draw a regulatory loop in which a non-mutated functional p53 and HER-2 can bidirectionally regulate the expression of these two genes. This study improves our understandings of these pathways and these proteins can be introduced either as a marker or as a target in cancer treatment.

  20. Canonical and Alternative Pathways in Cyclin-Dependent Kinase 1/Cyclin B Inactivation upon M-Phase Exit in Xenopus laevis Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Jacek Z. Kubiak

    2011-01-01

    Full Text Available Cyclin-Dependent Kinase 1 (CDK1 is the major M-phase kinase known also as the M-phase Promoting Factor or MPF. Studies performed during the last decade have shown many details of how CDK1 is regulated and also how it regulates the cell cycle progression. Xenopus laevis cell-free extracts were widely used to elucidate the details and to obtain a global view of the role of CDK1 in M-phase control. CDK1 inactivation upon M-phase exit is a primordial process leading to the M-phase/interphase transition during the cell cycle. Here we discuss two closely related aspects of CDK1 regulation in Xenopus laevis cell-free extracts: firstly, how CDK1 becomes inactivated and secondly, how other actors, like kinases and phosphatases network and/or specific inhibitors, cooperate with CDK1 inactivation to assure timely exit from the M-phase.

  1. PKCeta associates with cyclin E/Cdk2 complex in serum-starved MCF-7 and NIH-3T3 cells.

    Science.gov (United States)

    Shtutman, Marat; Hershko, Tzippi; Maissel, Adva; Fima, Eyal; Livneh, Etta

    2003-05-15

    Protein kinase C (PKC) encodes a family of enzymes implicated in cellular differentiation, growth control, and tumor promotion. However, very little is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that PKCeta associates with the cyclin E/Cdk2 complex. This is shown for the ectopically overexpressed PKCeta in NIH-3T3 cells, the inducibly expressed PKCeta in MCF-7 cells (under control of the tetracycline-responsive promoter), and the endogenously expressed PKCeta in mouse mammary epithelial HC11 cells. Subcellular cell fractionation experiments revealed that the complex with cyclin E is formed mostly in the nuclear fractions, although in these cells PKCeta is predominantly expressed in the cytosolic fractions. The complex of PKCeta and cyclin E was studied at various phases of the cell cycle, in serum-starved quiescent cells and in cells stimulated with serum to reenter the cell cycle. Interestingly, the interaction between PKCeta and cyclin E was most prominent in serum-starved cells and was disintegrated when cells entered the cells cycle. Immunofluorescence staining demonstrated that in serum-starved cells PKCeta is concentrated at the perinuclear zone, which is also the site of its colocalization with cyclin E. Colocalization of PKCeta and cyclin E in the perinuclear region was observed in serum-starved cells, and less in proliferating cells. These experiments suggest that the interaction between PKCeta and cyclin E is carefully regulated, and is correlated with the inactivated form of the cyclin E/Cdk2 complex. Thus, our studies support an important link between PKC and cell cycle control.

  2. SirT1—A Sensor for Monitoring Self-Renewal and Aging Process in Retinal Stem Cells

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    Chi-Hsien Peng

    2010-06-01

    Full Text Available Retinal stem cells bear potency of proliferation, self-renewal, and differentiation into many retinal cells. Utilizing appropriate sensors one can effectively detect the self-renewal and aging process abilities. Silencing information regulator (SirT1, a member of the sirtuin family, is a NAD-dependent histone deacetylase and an essential mediator for longevity in normal cells by calorie restriction. We firstly investigate the SirT1 mRNA expression in retinal stem cells from rats and 19 human eyes of different ages. Results revealed that SirT1 expression was significantly decreased in in vivo aged eyes, associated with poor self-renewal abilities. Additionally, SirT1 mRNA levels were dose-dependently increased in resveratrol- treated retinal stem cells. The expression of SirT1 on oxidative stress-induced damage was significantly decreased, negatively correlated with the level of intracellular reactive oxygen species production. Treatment with resveratrol could effectively further reduce oxidative stress induced by H2O2 treatment in retinal stem cells. Importantly, the anti-oxidant effects of resveratrol in H2O2-treated retinal stem cells were significantly abolished by knockdown of SirT1 expression (sh-SirT1. SirT1 expression provides a feasible sensor in assessing self-renewal and aging process in retinal stem cells. Resveratrol can prevent reactive oxygen species-induced damages via increased retinal SirT1 expression.

  3. Dynamic Quantitative T1 Mapping in Orthotopic Brain Tumor Xenografts

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    Kelsey Herrmann

    2016-04-01

    Full Text Available Human brain tumors such as glioblastomas are typically detected using conventional, nonquantitative magnetic resonance imaging (MRI techniques, such as T2-weighted and contrast enhanced T1-weighted MRI. In this manuscript, we tested whether dynamic quantitative T1 mapping by MRI can localize orthotopic glioma tumors in an objective manner. Quantitative T1 mapping was performed by MRI over multiple time points using the conventional contrast agent Optimark. We compared signal differences to determine the gadolinium concentration in tissues over time. The T1 parametric maps made it easy to identify the regions of contrast enhancement and thus tumor location. Doubling the typical human dose of contrast agent resulted in a clearer demarcation of these tumors. Therefore, T1 mapping of brain tumors is gadolinium dose dependent and improves detection of tumors by MRI. The use of T1 maps provides a quantitative means to evaluate tumor detection by gadolinium-based contrast agents over time. This dynamic quantitative T1 mapping technique will also enable future quantitative evaluation of various targeted MRI contrast agents.

  4. CyclinE在甲状腺乳头状癌组织中的表达及临床意义%TThe expression and clinical significance of CyclinE in the thyroid gland papilliform tumor organizes

    Institute of Scientific and Technical Information of China (English)

    范晓东; 张鹏霞; 王茉琳; 王伟群

    2014-01-01

    Objective To study the expression and clinical significance of CyclinE in the thyroid gland papilliform tumor .Methods The expression of CyclinE was tested by immunohistochemistry SP law of the thyroid gland papilliform tumor , the thyroid gland adenoma and the lump in the normal thyroid gland .Results The CyclinE expression rate was elevated gradually (P<0.01).CyclinE expression was correlated with stages, peritonsillar tissue infiltration and lymph node shift related of thyroid gland papilliform tumor (P<0.01).Conclusions CyclinE expression level is correlated with the occurrence and development of thyroid gland papilliform tumor .%目的:探讨CyclinE在甲状腺乳头状癌组织中的表达及其与临床病理特征的关系。方法用免疫组化 SP法检测CyclinE在甲状腺乳头状癌、甲状腺腺瘤及瘤旁正常甲状腺组织中的表达情况。结果 CyclinE的阳性表达率在正常甲状腺组织、甲状腺腺瘤、甲状腺乳头状癌组织中逐渐升高(P<0.01)。 CyclinE表达与甲状腺乳头状癌的临床分期、周围组织浸润及淋巴结转移有关(P<0.01)。结论 Cyclin E 的表达水平与甲状腺乳头状癌的发生、发展及预后有关,为甲状腺乳头状癌的治疗提供新的思路。

  5. Nuclear Targeting of Cyclin-Dependent Kinase 2 Reveals Essential Roles of Cyclin-Dependent Kinase 2 Localization and Cyclin E in Vitamin D-Mediated Growth Inhibition

    OpenAIRE

    Flores, Omar; Wang, Zhengying; Knudsen, Karen E; Burnstein, Kerry L.

    2010-01-01

    1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), inhibits proliferation of a variety of cell types including adenocarcinoma of the prostate. We have previously shown that 1,25-(OH)2D3 increases the stability of the cyclin-dependent kinase inhibitor p27KIP1, decreases cyclin-dependent kinase 2 (CDK2) activity, and promotes G1 phase accumulation in human prostate cancer cells. These effects correlate with cytoplasmic relocalization of CDK2. In this study, we investigated the role of CDK2 cytoplasmic re...

  6. Reduced expression of cyclin D2 is associated with poor recurrence-free survival independent of cyclin D1 in stage III non-small cell lung cancer.

    Science.gov (United States)

    Ko, Eunkyung; Kim, Yujin; Park, Seong-Eun; Cho, Eun Yoon; Han, Jungho; Shim, Young Mog; Park, Joobae; Kim, Duk-Hwan

    2012-08-01

    Compared to well-known function of cyclin D1 in lung cancer, the role of cyclin D2 is not clear. This study was aimed at understanding the clinicopathological significance of cyclin D2 in primary non-small cell lung cancer (NSCLC). We retrospectively analyzed expression statuses of cyclin D1, cyclin D2, p16, p21, p27, Ki-67, and phospho-pRb (Ser-807/811) using immunohistochemistry in 626 NSCLCs. Cyclin D2 was expressed in normal lung tissue, and its expression was reduced in 170 (27%) of 626 NSCLCs with a median duration of follow-up of 64 months. Mean phospho-pRb (Ser-807/811) levels were not associated with expression levels of cyclin D2 (P=0.15). The relationship between recurrence and the reduced expression of cyclin D2 was not homogenous by stage (Breslow-Day test for homogeneity, P=0.04). Reduced expression of cyclin D2 was not associated with patient's prognosis in 370 stage I, 112 stage II, and 18 stage IV NSCLCs. However, for 126 stage III NSCLCs, reduced expression of cyclin D2 was adversely associated with recurrence-free survival (RFS) (hazard ratio [HR]=3.71, 95% CI=1.54-13.17; P=0.01), independent of histology and expression of cyclin D1. The reduced expression of cyclin D2 was not associated with the overexpression of cyclin D1 (P=0.65). The present study suggests that reduced expression of cyclin D2 in stage III NSCLC may be associated with poor RFS. And, cyclin D2 may have a distinct role from cyclin D1 in NSCLC. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  7. Effects of Oridonin on proliferation and apoptosis of PC-3 cells%冬凌草甲素通过改变CyclinD2、CyclinE、P27的表达对PC-3细胞抑制增殖和凋亡诱导效应

    Institute of Scientific and Technical Information of China (English)

    李进; 杨罗艳; 吴洪涛

    2012-01-01

    Objective To investigate the effect of Oridonin on apoptosis of human prostate cancer cell lines (PC-3 cells) and their molecular mechanism. Methods The PC-3 cells were intervened by Oridonin in different concentration. The vitality of the PC-3 cells was detected by MTT assay. The change of cell cycle was analyzed by the flow cytometry; and the changes of expressions of CyclinD2, CyclinE, P27 in PC-3 cells were detected by the real-time fluorescent quantitative determination. Results (1) Oridonin increased the percentage of the G0/G1 phase and decreased the S phase of PC-3 cells; (2) Oridonin down regulated the expression of CyclinD2 and CyclinE, and up regulated the expression of P27 in a concentration-dependent way in PC-3 cells. Conclusion Oridonin can inhibit proliferation of PC-3 cells and induce their apoptosis through regulating the cell cycle protein, blocking the "checkpoint" of the G1/S phase, down-regulating CyclinD2 and CyclinE as well as up-regulating P27.%目的 探索冬凌草甲素对人雄激素非依赖性前列腺癌细胞株——PC-3细胞的诱导凋亡作用及其分子机制.方法 用不同浓度的冬凌草甲素干预PC-3细胞,MTT试验分析观察其对PC-3细胞活力的影响;流式细胞仪检测细胞周期变化;实时荧光定量PCR方法检测PC-3细胞CyclinD2、CyclinE、p27蛋白表达的变化.结果 (1)冬凌草甲素增加G0/G1期PC-3细胞百分率,降低S期PC-3细胞百分率;(2)冬凌草甲素以浓度依赖性方式抑制PC-3细胞的CyclinD2、CyclinE蛋白表达,而P27蛋白表达上调;结论 冬凌草甲素通过影响细胞周期调节蛋白、阻断细胞周期G1/S期“稽查点”;抑制CyclinD2、CyclinE,上调P27等途径抑制PC-3细胞增殖及诱导PC-3细胞凋亡.

  8. Sp1 phosphorylation by cyclin-dependent kinase 1/cyclin B1 represses its DNA-binding activity during mitosis in cancer cells.

    Science.gov (United States)

    Chuang, J-Y; Wang, S-A; Yang, W-B; Yang, H-C; Hung, C-Y; Su, T-P; Chang, W-C; Hung, J-J

    2012-11-22

    Sp1 is important for the transcription of many genes. Our previous studies have shown that Sp1 is degraded in normal cell, but it is preserved in cancer cells during mitosis and exists a priori in the daughter cells, ready to engage in gene transcription and thereby contributes to the proliferation and survival of cancer cells. The mechanism by which Sp1 is preserved in cancer cells during mitosis remains unknown. In this study, we observed that Sp1 strongly colocalized with cyclin-dependent kinase 1 (CDK1)/cyclin B1 during mitosis. Moreover, we showed that Sp1 is a novel mitotic substrate of CDK1/cyclin B1 and is phosphorylated by it at Thr 739 before the onset of mitosis. Phospho-Sp1 reduced its DNA-binding ability and facilitated the chromatin condensation process during mitosis. Mutation of Thr739 to alanine resulted in Sp1 remaining in the chromosomes, delayed cell-cycle progression, and eventually led to apoptosis. Screening of Sp1-associated proteins during mitosis by using liquid chromatography/mass spectrometry indicated the tethering of Sp1 to myosin/F-actin. Furthermore, phospho-Sp1 and myosin/F-actin appeared to exist as a congregated ring at the periphery of the chromosome. However, at the end of mitosis and the beginning of interphase, Sp1 was dephosphorylated by PP2A and returned to the chromatin. These results indicate that cancer cells use CDK1 and PP2A to regulate the movement of Sp1 in and out of the chromosomes during cell-cycle progression, which may benefit cancer-cell proliferation.

  9. T1 theorem for Besov and Triebel-Lizorkin spaces

    Institute of Scientific and Technical Information of China (English)

    DENG Donggao; HAN Yongsheng

    2005-01-01

    Using the discrete Calderon type reproducing formula and the PlancherelPolya characterization for the Besov and Triebel-Lizorkin spaces, the T1 theorem for the Besov and Triebel-Lizorkin spaces was proved.

  10. SCYL1-BP1 affects cell cycle arrest in human hepatocellular carcinoma cells via Cyclin F and RRM2.

    Science.gov (United States)

    Wang, Yang; Zhi, Qiaoming; Ye, Qin; Zhou, Chengyuan; Zhang, Lei; Yan, Wei; Wu, Qun; Zhang, Di; Li, Pu; Huo, Keke

    2016-01-01

    The cell cycle is regulated via important biological mechanisms. Controlled expression of cell cycle regulatory proteins is crucial to maintain cell cycle progression. However, unbalanced protein expression leads to many diseases, such as cancer. Previous research suggests that SCYL1-BP1 function might be related to cell cycle progression and SCYL1-BP1 dysfunction to diseases through undefined mechanisms. In this research, an unbiased yeast two-hybrid screen was used to find protein(s) with potential biological relevance to SCYL1-BP1 function, and a novel interaction was recognized between SCYL1-BP1 and Cyclin F. This interaction was chosen as a paradigm to study SCYL1-BP1 function in cell cycle progression and its possible role in tumorigenesis. We found that SCYL1-BP1 binds to Cyclin F both in vivo and in vitro. SCYL1-BP1 overexpression promoted expression of the CCNF gene and simultaneously delayed Cyclin F protein degradation. SCYL1-BP1 knockdown reduced the expression of endogenous Cyclin F. It was also demonstrated in functional assays that SCYL1-BP1 overexpression induces G2/M arrest in cultured liver cells. Furthermore, SCYL1-BP1 sustained RRM2 protein expression by reducing its ubiquitination. Thus, we propose that SCYL1- BP1 affects the cell cycle through increasing steady state levels of Cyclin F and RRM2 proteins, thus constituting a dual regulatory circuit. This study provides a possible mechanism for SCYL1-BP1-mediated cell cycle regulation and related diseases.

  11. The LIM-only protein FHL2 mediates ras-induced transformation through cyclin D1 and p53 pathways.

    Directory of Open Access Journals (Sweden)

    Charlotte Labalette

    Full Text Available BACKGROUND: Four and a half LIM-only protein 2 (FHL2 has been implicated in multiple signaling pathways that regulate cell growth and tissue homeostasis. We reported previously that FHL2 regulates cyclin D1 expression and that immortalized FHL2-null mouse embryo fibroblasts (MEFs display reduced levels of cyclin D1 and low proliferative activity. METHODOLOGY/PRINCIPAL FINDINGS: Here we address the contribution of FHL2 in cell transformation by investigating the effects of oncogenic Ras in FHL2-null context. We show that H-RasV12 provokes cell cycle arrest accompanied by accumulation of p53 and p16(INK4a in immortalized FHL2(-/- MEFs. These features contrast sharply with Ras transforming activity in wild type cell lines. We further show that establishment of FHL2-null cell lines differs from conventional immortalization scheme by retaining functional p19(ARF/p53 checkpoint that is required for cell cycle arrest imposed by Ras. However, after serial passages of Ras-expressing FHL2(-/- cells, dramatic increase in the levels of D-type cyclins and Rb phosphorylation correlates with the onset of cell proliferation and transformation without disrupting the p19(ARF/p53 pathway. Interestingly, primary FHL2-null cells overexpressing cyclin D1 undergo a classical immortalization process leading to loss of the p19(ARF/p53 checkpoint and susceptibility to Ras transformation. CONCLUSIONS/SIGNIFICANCE: Our findings uncover a novel aspect of cellular responses to mitogenic stimulation and illustrate a critical role of FHL2 in the signalling network that implicates Ras, cyclin D1 and p53.

  12. Sustained spindle-assembly checkpoint response requires de novo transcription and translation of cyclin B1.

    Directory of Open Access Journals (Sweden)

    Ana Lúcia Mena

    Full Text Available BACKGROUND: Microtubule-targeting drugs induce mitotic delay at pro-metaphase by preventing the spindle assembly checkpoint to be satisfied. However, especially after prolonged treatments, cells can escape this arrest in a process called mitotic slippage. The mechanisms underlying the spindle assembly checkpoint and slippage are not fully understood. It has been generally accepted that during mitosis there is a temporary shutdown of high-energy-consuming processes, such as transcription and translation. However, the synthesis of specific proteins is maintained or up-regulated since protein synthesis is necessary for entry into and progression through mitosis. METHODOLOGY/PRINCIPAL FINDINGS: In this work we investigated whether the mitotic arrest caused by the mitotic checkpoint is independent of transcription and translation. By using immunofluorescent microscopy and western blotting, we demonstrate that inhibition of either of these processes induces a shortening of the mitotic arrest caused by the nocodazole treatment, and ultimately leads to mitotic slippage. Our western blotting and RTQ-PCR results show that inhibition of transcription during mitotic arrest does not affect the expression of the spindle checkpoint proteins, whereas it induces a significant decrease in the mRNA and protein levels of Cyclin B1. The exogenous expression of Cyclin B1 substantially rescued the mitotic phenotype in nocodazole cells treated with the inhibitors of transcription and translation. CONCLUSIONS/SIGNIFICANCE: This work emphasizes the importance of transcription and translation for the maintenance of the spindle assembly checkpoint, suggesting the existence of a mechanism dependent on cyclin B1 gene regulation during mitosis. We propose that continuous transcription of mitotic regulators is required to sustain the activation of the spindle assembly checkpoint.

  13. leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3.

    Science.gov (United States)

    Saxena, Neeraj K; Vertino, Paula M; Anania, Frank A; Sharma, Dipali

    2007-05-01

    Numerous epidemiological studies documented that obesity is a risk factor for breast cancer development in postmenopausal women. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease state. In this study, we analyzed the role of leptin and the molecular mechanism(s) underlying its action in breast cancer cells that express both short and long isoforms of leptin receptor. Leptin increased MCF7 cell population in the S-phase of the cell cycle along with a robust increase in CYCLIN D1 expression. Also, leptin induced Stat3-phosphorylation-dependent proliferation of MCF7 cells as blocking Stat3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation. Using deletion constructs of CYCLIN D1 promoter and chromatin immunoprecipitation assay, we show that leptin induced increase in CYCLIN D1 promoter activity is mediated through binding of activated Stat3 at the Stat binding sites and changes in histone acetylation and methylation. We also show specific involvement of coactivator molecules, histone acetyltransferase SRC1, and mediator complex in leptin-mediated regulation of CYCLIN D1 promoter. Importantly, silencing of SRC1 and Med1 abolished the leptin induced increase in CYCLIN D1 expression and MCF7 cell proliferation. Intriguingly, recruitment of both SRC1 and Med1 was dependent on phosphorylated Stat3 as AG490 treatment inhibited leptin-induced recruitment of these coactivators to CYCLIN D1 promoter. Our data suggest that CYCLIN D1 may be a target gene for leptin mediated growth stimulation of breast cancer cells and molecular mechanisms involve activated Stat3-mediated recruitment of distinct coactivator complexes.

  14. APC/C and SCF(cyclin F) Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry.

    Science.gov (United States)

    Choudhury, Rajarshi; Bonacci, Thomas; Arceci, Anthony; Lahiri, Debojyoti; Mills, Christine A; Kernan, Jennifer L; Branigan, Timothy B; DeCaprio, James A; Burke, Daniel J; Emanuele, Michael J

    2016-09-20

    The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems.

  15. Cyclin D2 in the basal process of neural progenitors is linked to non-equivalent cell fates

    Science.gov (United States)

    Tsunekawa, Yuji; Britto, Joanne M; Takahashi, Masanori; Polleux, Franck; Tan, Seong-Seng; Osumi, Noriko

    2012-01-01

    Asymmetric cell division plays an indispensable role during corticogenesis for producing new neurons while maintaining a self-renewing pool of apical progenitors. The cellular and molecular determinants favouring asymmetric division are not completely understood. Here, we identify a novel mechanism for generating cellular asymmetry through the active transportation and local translation of Cyclin D2 mRNA in the basal process. This process is regulated by a unique cis-regulatory sequence found in the 3′ untranslated region (3′UTR) of the mRNA. Unequal inheritance of Cyclin D2 protein to the basally positioned daughter cell with the basal process confers renewal of the apical progenitor after asymmetric division. Conversely, depletion of Cyclin D2 in the apically positioned daughter cell results in terminal neuronal differentiation. We demonstrate that Cyclin D2 is also expressed in the developing human cortex within similar domains, thus indicating that its role as a fate determinant is ancient and conserved. PMID:22395070

  16. Hyperpolarized (129)Xe T (1) in oxygenated and deoxygenated blood

    Science.gov (United States)

    Albert, M. S.; Balamore, D.; Kacher, D. F.; Venkatesh, A. K.; Jolesz, F. A.

    2000-01-01

    The viability of the new technique of hyperpolarized (129)Xe MRI (HypX-MRI) for imaging organs other than the lungs depends on whether the spin-lattice relaxation time, T(1), of (129)Xe is sufficiently long in the blood. In previous experiments by the authors, the T(1) was found to be strongly dependent upon the oxygenation of the blood, with T(1) increasing from about 3 s in deoxygenated samples to about 10 s in oxygenated samples. Contrarily, Tseng et al. (J. Magn. Reson. 1997; 126: 79-86) reported extremely long T(1) values deduced from an indirect experiment in which hyperpolarized (129)Xe was used to create a 'blood-foam'. They found that oxygenation decreased T(1). Pivotal to their experiment is the continual and rapid exchange of hyperpolarized (129)Xe between the gas phase (within blood-foam bubbles) and the dissolved phase (in the skin of the bubbles); this necessitated a complicated analysis to extract the T(1) of (129)Xe in blood. In the present study, the experimental design minimizes gas exchange after the initial bolus of hyperpolarized (129)Xe has been bubbled through the sample. This study confirms that oxygenation increases the T(1) of (129)Xe in blood, from about 4 s in freshly drawn venous blood, to about 13 s in blood oxygenated to arterial levels, and also shifts the red blood cell resonance to higher frequency. Copyright 2000 John Wiley & Sons, Ltd. Abbreviations used BOLD blood oxygen level dependent NOE nuclear overhouses effect PO(2) oxygen partial pressure RBC red blood cells RF radio frequency SNR signal-to-noise ratio.

  17. Systematic T1 improvement for hyperpolarized 129xenon.

    Science.gov (United States)

    Repetto, Maricel; Babcock, Earl; Blümler, Peter; Heil, Werner; Karpuk, Sergei; Tullney, Kathlynne

    2015-03-01

    The spin-lattice relaxation time T1 of hyperpolarized (HP)-(129)Xe was improved at typical storage conditions (i.e. low and homogeneous magnetic fields). Very long wall relaxation times T(1)(wall) of about 18 h were observed in uncoated, spherical GE180 glass cells of ∅=10 cm which were free of rubidium and not permanently sealed but attached to a standard glass stopcock. An "aging" process of the wall relaxation was identified by repeating measurements on the same cell. This effect could be easily removed by repeating the initial cleaning procedure. In this way, a constant wall relaxation was ensured. The Xe nuclear spin-relaxation rate 1/T1(Xe-Xe) due to van der Waals molecules was investigated too, by admixing three different buffer gases (N(2), SF(6) and CO(2)). Especially CO(2) exhibited an unexpected high efficiency (r) in shortening the lifetime of the Xe-Xe dimers and hence prolonging the total T1 relaxation even further. These measurements also yielded an improved accuracy for the van der Waals relaxation for pure Xe (with 85% (129)Xe) of T(1)(Xe-Xe)=(4.6±0.1)h. Repeating the measurements with HP (129)Xe in natural abundance in mixtures with SF6, a strong dependence of T(1)(Xe-Xe) and r on the isotopic enrichment was observed, uncovering a shorter T(1)(Xe-Xe) relaxation for the (129)Xe in natural composition as compared to the 85% isotopically enriched gas.

  18. Virtual mutagenesis of the yeast cyclins genetic network reveals complex dynamics of transcriptional control networks.

    Directory of Open Access Journals (Sweden)

    Eliska Vohradska

    Full Text Available Study of genetic networks has moved from qualitative description of interactions between regulators and regulated genes to the analysis of the interaction dynamics. This paper focuses on the analysis of dynamics of one particular network--the yeast cyclins network. Using a dedicated mathematical model of gene expression and a procedure for computation of the parameters of the model from experimental data, a complete numerical model of the dynamics of the cyclins genetic network was attained. The model allowed for performing virtual experiments on the network and observing their influence on the expression dynamics of the genes downstream in the regulatory cascade. Results show that when the network structure is more complicated, and the regulatory interactions are indirect, results of gene deletion are highly unpredictable. As a consequence of quantitative behavior of the genes and their connections within the network, causal relationship between a regulator and target gene may not be discovered by gene deletion. Without including the dynamics of the system into the network, its functional properties cannot be studied and interpreted correctly.

  19. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation.

    Science.gov (United States)

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-04-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  20. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    Institute of Scientific and Technical Information of China (English)

    Hai Jiang; Jianchun Wu; Chen He; Wending Yang; Honglin Li

    2009-01-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdkl activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chkl and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  1. Evolutionarily conserved transcription factor Apontic controls the G1/S progression by inducing cyclin e during eye development

    KAUST Repository

    Liu, Qingxin

    2014-06-16

    During Drosophila eye development, differentiation initiates in the posterior region of the eye disk and progresses anteriorly as a wave marked by the morphogenetic furrow (MF), which demarcates the boundary between anterior undifferentiated cells and posterior differentiated photoreceptors. However, the mechanism underlying the regulation of gene expression immediately before the onset of differentiation remains unclear. Here, we show that Apontic (Apt), which is an evolutionarily conserved transcription factor, is expressed in the differentiating cells posterior to the MF. Moreover, it directly induces the expression of cyclin E and is also required for the G1-to-S phase transition, which is known to be essential for the initiation of cell differentiation at the MF. These observations identify a pathway crucial for eye development, governed by a mechanism in which Cyclin E promotes the G1-to-S phase transition when regulated by Apt.

  2. Monoclonal antibody raised against human mitotic cyclin B1, identifies cyclin B-like mitotic proteins in synchronized onion (Allium cepa L.) root meristem.

    Science.gov (United States)

    Chaudhuri, S K; Ghosh, S

    1997-03-01

    Cyclin B-like mitotic proteins have been detected in synchronized Allium cepa L. root tip cells by using mouse monoclonal anti-cyclin B1 antibody raised against human cyclin B1. Immunoblot shows two closely placed isoforms of cyclin B-like proteins having an apparent molecular weight around 54 kDa. In vivo [35S]-methionine labelling followed by immunoprecipitation and autoradiography indicates that cyclin B-like proteins are mainly synthesized in the G2 phase of the cell cycle and destroyed in late mitosis. Immunoblotting data depict that the level of cyclin B-like proteins reaches the maximum at the late G2 to early M phase; and it becomes degraded in the late hours of mitosis. Moreover, the cyclin B isoforms are stabilized in colchicine-arrested metaphase cells as already reported in animal cells.

  3. RhoA promotes epidermal stem cell proliferation via PKN1-cyclin D1 signaling

    Science.gov (United States)

    Wang, Fan; Zhan, Rixing; Chen, Liang; Dai, Xia; Wang, Wenping; Guo, Rui; Li, Xiaoge; Li, Zhe; Wang, Liang; Huang, Shupeng; Shen, Jie

    2017-01-01

    Objective Epidermal stem cells (ESCs) play a critical role in wound healing, but the mechanism underlying ESC proliferation is not well defined. Here, we explore the effects of RhoA on ESC proliferation and the possible underlying mechanism. Methods Human ESCs were enriched by rapid adhesion to collagen IV. RhoA(+/+)(G14V), RhoA(-/-)(T19N) and pGFP control plasmids were transfected into human ESCs. The effect of RhoA on cell proliferation was detected by cell proliferation and DNA synthesis assays. Induction of PKN1 activity by RhoA was determined by immunoblot analysis, and the effects of PKN1 on RhoA in terms of inducing cell proliferation and cyclin D1 expression were detected using specific siRNA targeting PKN1. The effects of U-46619 (a RhoA agonist) and C3 transferase (a RhoA antagonist) on ESC proliferation were observed in vivo. Results RhoA had a positive effect on ESC proliferation, and PKN1 activity was up-regulated by the active RhoA mutant (G14V) and suppressed by RhoA T19N. Moreover, the ability of RhoA to promote ESC proliferation and DNA synthesis was interrupted by PKN1 siRNA. Additionally, cyclin D1 protein and mRNA expression levels were up-regulated by RhoA G14V, and these effects were inhibited by siRNA-mediated knock-down of PKN1. RhoA also promoted ESC proliferation via PKN in vivo. Conclusion This study shows that the effect of RhoA on ESC proliferation is mediated by activation of the PKN1-cyclin D1 pathway in vitro, suggesting that RhoA may serve as a new therapeutic target for wound healing. PMID:28222172

  4. Overexpression of cyclin Y in non-small cell lung cancer is associated with cancer cell proliferation

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Cyclin Y (CCNY) is a key cell cycle regulator that acts as a growth factor sensor to integrate extracellular signals with the cell cycle machinery. The expression status of CCNY in lung cancer and its clinical significance remain unknown. The data indicates that CCNY may be deregulated in non-small cell lung cancer, where it may act to promote cell proliferation. These studies suggest that CCNY may be a candidate biomarker of NSCLC and a possible therapeutic target for lung cancer treatment.

  5. [Early Detection of Manganese Intoxication Based on Occupational History and T1-weighted MRI].

    Science.gov (United States)

    Fukutake, Toshio; Yano, Hajime; Kushida, Ryutaro; Sunada, Yoshihide

    2016-02-01

    Manganese regulates many enzymes and is essential for normal cell function. Chronic manganese intoxication has an insidious and progressive course terminating to atypical parkinsonism with little therapeutic efficacy. For subjects with chronic manganese exposure such as welders, manganese intoxication can be detected early based on the presence of hyperintensity in the globus pallidus on T(1)-weighted MRI and abnormally high urinary excretion of manganese with a chelating agent even in cases of normal serum/urine level of manganese.

  6. Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells Inverse relationship between TCTP/RhoA and p53/ /cyclin A/actin expression in ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Malgorzata Kloc

    2012-10-01

    Full Text Available The translationally controlled tumor protein (TCTP plays a role in cell growth, cell cycle and cancer
    progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the
    cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased
    migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,
    a key regulator of cell cycle, controls actin organization and negatively regulates cell motility via regulation of RhoA
    expression. We studied the organization of actin and cytokeratin cytoskeleton and the expression of TCTP, p53,
    cyclin A, RhoA and actin in HIO180 non-transformed ovarian epithelial cells, and OVCAR3 and SKOV3 (expressing
    low level of inducible p53 ovarian epithelial cancer cells with different metastatic potential. Immunostaining
    and ultrastructural analyses illustrated a dramatic difference in the organization of the cytokeratin and actin
    filaments in non-transformed versus cancer cell lines. We also determined that there is an inverse relationship between
    the level of TCTP/RhoA and actin/p53/cyclin A expression in ovarian cancer cell lines. This previously unidentified
    negative relationship between TCTP/RhoA and actin/p53/cyclin A may suggest that this interaction is linked
    with the high aggressiveness of ovarian cancers.The translationally controlled tumor protein (TCTP plays a role in cell growth, cell cycle and cancer
    progression. TCTP controls negatively the stability of the p53 tumor suppressor protein and interacts with the
    cellular cytoskeleton. The deregulation of the actin and cytokeratin cytoskeleton is responsible for the increased
    migratory activity of tumor cells and is linked with poor patient outcome. Recent studies indicate that cyclin A,
    a key regulator of cell cycle, controls actin organization

  7. Berberine inhibits cyclin D1 expression via suppressed binding of AP-1 transcription factors to CCND1 AP-1 motif

    Institute of Scientific and Technical Information of China (English)

    Ye LUO; Yu HAO; Tai-ping SHI; Wei-wei DENG; Na LI

    2008-01-01

    Aim: To verify the suppressive effect of berberine on the proliferation of the human pulmonary giant cell carcinoma cell line PG and to demonstrate the mecha-nisms behind the antitumoral effects of berberine. Methods: The proliferative effects of PG cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetry. The cell cycle was examined by flow cytometry. The expression level of cyclin D1 was detected by RT-PCR. The activities of the activating protein-1 (AP-1) and NF-κB signaling pathways related to cyclin D1 were examined by luciferase assay. The cytoplasmic level of c-Jun was detected by Western blot analysis. An electrophoretic mobility shift assay was used to examiae the binding of transcription factors to the cyclin D1 gene (CCNDl) AP-1 motif. Results: The results showed that the proliferation of PG cells treated with different concentrations (10, 20, and 40 μg/mL) of berberine for 24 and 48 h was suppressed significantly compared to the control group. After treatment with berberine, the proportion of PG cells at the G0/G1 phase increased, while cells at the S and G2/M phases decreased. Berberine could inhibit the expression of cyclin D1 in PG cells. Berberine inhibited the activity of the AP-1 signaling pathway, but had no significant effect on the NF-κB signaling pathway. Berberine suppressed the expression of c-Jun and decreased the binding of tran-scription factors to the CCND1 AP-1 motif. Conclusion: Berberine suppresses the activity of the AP-1 signaling pathway and decreases the binding of transcrip-tion factors to the CCND1 AP-1 motif. This is one of the important mechanisms behind the antitumoral effects of berberine as a regulator of cyclin D1.

  8. CDK-1 and two B-type cyclins promote PAR-6 stabilization during polarization of the early C. elegans embryo.

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    Alexia Rabilotta

    Full Text Available In the C. elegans embryo, formation of an antero-posterior axis of polarity relies on signaling by the conserved PAR proteins, which localize asymmetrically in two mutually exclusive groups at the embryonic cortex. Depletion of any PAR protein causes a loss of polarity and embryonic lethality. A genome-wide RNAi screen previously identified two B-type cyclins, cyb-2.1 and cyb-2.2, as suppressors of par-2(it5ts lethality. We found that the loss of cyb-2.1 or cyb-2.2 suppressed the lethality and polarity defects of par-2(it5ts mutants and that these cyclins act in cell polarity with their cyclin-dependent kinase partner, CDK-1. Interestingly, cyb-2.1; cyb-2.2 double mutants did not show defects in cell cycle progression or timing of polarity establishment, suggesting that they regulate polarity independently of their typical role in cell cycle progression. Loss of both cyclin genes or of cdk-1 resulted in a decrease in PAR-6 levels in the embryo. Furthermore, the activity of the cullin CUL-2 was required to achieve suppression of par-2 lethality when both cyclins were absent. Our results support a model in which CYB-2.1/2/CDK-1 antagonize CUL-2 activity to promote stabilization of PAR-6 levels during polarization of the early C. elegans embryo. They also suggest that CYB-2.1 and CYB-2.2 contribute to the coupling of cell cycle progression and asymmetric segregation of cell fate determinants.

  9. Improvement of Thermal Stability via Outer-Loop Ion Pair Interaction of Mutated T1 Lipase from Geobacillus zalihae Strain T1

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    Mahiran Basri

    2012-01-01

    Full Text Available Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The Tm for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher Tm as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.

  10. The role of cyclin E1 in hepatocellular carcinoma

    OpenAIRE

    Chan, Yan-yan; 陳茵茵

    2014-01-01

    Hepatocellular carcinoma (HCC) accounts for 70-85% of liver cancer, which is the sixth most common cancer in the world. Prognosis of HCC is dismal with little chance of complete recovery after diagnosis. It is of essence to discover the key molecules involved in the tumor progression. This could help earlier detection of HCC and establish targeted molecular therapies. Cyclin E1 (CCNE1) is a cyclin molecule responsible for the transition from G1 to S phase of the cell cycle and is often dysreg...

  11. A novel enterocin T1 with anti-Pseudomonas activity produced by Enterococcus faecium T1 from Chinese Tibet cheese.

    Science.gov (United States)

    Liu, Hui; Zhang, Lanwei; Yi, Huaxi; Han, Xue; Gao, Wei; Chi, Chunliang; Song, Wei; Li, Haiying; Liu, Chunguang

    2016-02-01

    An enterocin-producing Enterococcus faecium T1 was isolated from Chinese Tibet cheese. The enterocin was purified by SP-Sepharose and reversed phase HPLC. It was identified as unique from other reported bacteriocins based on molecular weight (4629 Da) and amino acid compositions; therefore it was subsequently named enterocin T1. Enterocin T1 was stable at 80-100 °C and over a wide pH range, pH 3.0-10.0. Protease sensitivity was observed to trypsin, pepsin, papain, proteinase K, and pronase E. Importantly, enterocin T1 was observed to inhibit the growth of numerous Gram-negative and Gram-positive bacteria including Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Escherichia coli, Salmonella typhimurium, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Listeria monocytogenes. Take together, these results suggest that enterocin T1 is a novel bacteriocin with the potential to be used as a bio-preservative to control Pseudomonas spp. in food.

  12. MyT1 Counteracts the Neural Progenitor Program to Promote Vertebrate Neurogenesis

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    Francisca F. Vasconcelos

    2016-10-01

    Full Text Available The generation of neurons from neural stem cells requires large-scale changes in gene expression that are controlled to a large extent by proneural transcription factors, such as Ascl1. While recent studies have characterized the differentiation genes activated by proneural factors, less is known on the mechanisms that suppress progenitor cell identity. Here, we show that Ascl1 induces the transcription factor MyT1 while promoting neuronal differentiation. We combined functional studies of MyT1 during neurogenesis with the characterization of its transcriptional program. MyT1 binding is associated with repression of gene transcription in neural progenitor cells. It promotes neuronal differentiation by counteracting the inhibitory activity of Notch signaling at multiple levels, targeting the Notch1 receptor and many of its downstream targets. These include regulators of the neural progenitor program, such as Hes1, Sox2, Id3, and Olig1. Thus, Ascl1 suppresses Notch signaling cell-autonomously via MyT1, coupling neuronal differentiation with repression of the progenitor fate.

  13. Cell type-specific translational repression of Cyclin B during meiosis in males.

    Science.gov (United States)

    Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T

    2015-10-01

    The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I. © 2015. Published by The Company of Biologists Ltd.

  14. Characterization of cyclin E expression in multiple myeloma and its functional role in seliciclib-induced apoptotic cell death.

    Directory of Open Access Journals (Sweden)

    Liat Josefsberg Ben-Yehoshua

    Full Text Available Multiple Myeloma (MM is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator--cyclin dependent kinase (CDK. Genomic instability was reported to be affected by over expression of another CDK regulator--cyclin E (CCNE. This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs. Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion-mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy.

  15. Characterization of cyclin E expression in multiple myeloma and its functional role in seliciclib-induced apoptotic cell death.

    Science.gov (United States)

    Josefsberg Ben-Yehoshua, Liat; Beider, Katia; Shimoni, Avichai; Ostrovsky, Olga; Samookh, Michal; Peled, Amnon; Nagler, Arnon

    2012-01-01

    Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator--cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator--cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion-mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy.

  16. Free-breathing MOLLI: application to myocardial T(1) mapping.

    Science.gov (United States)

    Tsai, Jyun-Ming; Huang, Teng-Yi; Tseng, Yu-Shen; Lin, Yi-Ru

    2012-12-01

    Of the myocardial T(1) mapping techniques, the modified Look-Locker inversion recovery (MOLLI) sequence is accurate and highly reproducible. The MOLLI sequence requires patients to hold their breath for 17 heartbeats during the scanning process to minimize respiratory motion-related artifacts. However, some patients are unable to hold their breath because of illness or limited breath-hold capacity. This study, therefore, aimed to develop a robust myocardial T(1) mapping method based on the MOLLI sequence for patients unable to perform voluntary breath-holds. This study presents a free-breathing MOLLI (FB-MOLLI) sequence and an optimized reconstruction method to allow myocardial T(1) mapping in vivo without breath-hold. Nine healthy volunteers participated in this study after providing institutionally approved consent. The FB-MOLLI sequence acquires 20 images within 29 heartbeats. The reconstruction program employs a two-step automatic image registration technique and an image selection method inspired by the self-gating cardiac imaging method. Results indicate that the proposed reconstruction method increases the accuracy and reproducibility of free-breathing T(1) measurements significantly (p mapping, and could greatly facilitate acquisition procedures during routine examinations.

  17. Caenorhabditis elegans cyclin B3 is required for multiple mitotic processes including alleviation of a spindle checkpoint-dependent block in anaphase chromosome segregation.

    Directory of Open Access Journals (Sweden)

    Gary M R Deyter

    2010-11-01

    Full Text Available The master regulators of the cell cycle are cyclin-dependent kinases (Cdks, which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3-dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.

  18. Early B-cell-specific inactivation of ATM synergizes with ectopic CyclinD1 expression to promote pre-germinal center B-cell lymphomas in mice.

    Science.gov (United States)

    Yamamoto, K; Lee, B J; Li, C; Dubois, R L; Hobeika, E; Bhagat, G; Zha, S

    2015-06-01

    Ataxia telangiectasia-mutated (ATM) kinase is a master regulator of the DNA damage response. ATM is frequently inactivated in human B-cell non-Hodgkin lymphomas, including ~50% of mantle cell lymphomas (MCLs) characterized by ectopic expression of CyclinD1. Here we report that early and robust deletion of ATM in precursor/progenitor B cells causes cell autonomous, clonal mature B-cell lymphomas of both pre- and post-germinal center (GC) origins. Unexpectedly, naive B-cell-specific deletion of ATM is not sufficient to induce lymphomas in mice, highlighting the important tumor suppressor function of ATM in immature B cells. Although EμCyclinD1 is not sufficient to induce lymphomas, EμCyclinD1 accelerates the kinetics and increases the incidence of clonal lymphomas in ATM-deficient B-cells and skews the lymphomas toward pre-GC-derived small lymphocytic neoplasms, sharing morphological features of human MCL. This is in part due to CyclinD1-driven expansion of ATM-deficient naive B cells with genomic instability, which promotes the deletions of additional tumor suppressor genes (i.e. Trp53, Mll2, Rb1 and Cdkn2a). Together these findings define a synergistic function of ATM and CyclinD1 in pre-GC B-cell proliferation and lymphomagenesis and provide a prototypic animal model to study the pathogenesis of human MCL.

  19. Early B-cell Specific Inactivation of ATM Synergizes with Ectopic CyclinD1 Expression to Promote Pre-germinal center B-cell Lymphomas in Mice

    Science.gov (United States)

    Yamamoto, Kenta; Lee, Brian J.; Li, Chen; Dubois, Richard L.; Hobeika, Elias; Bhagat, Govind; Zha, Shan

    2017-01-01

    Ataxia Telangiectasia Mutated (ATM) kinase is a master regulator of the DNA damage response. ATM is frequently inactivated in human B-cell non-Hodgkin Lymphomas (B-NHL), including ~50% of mantle cell lymphomas (MCLs) characterized by ectopic expression of CyclinD1. Here we report that early and robust deletion of ATM in precursor/progenitor B-cells causes cell-autonomous, clonal mature B cell lymphomas of both pre- and post-germinal center (GC) origins. Unexpectedly naïve B cell specific deletion of ATM is not sufficient to induce lymphomas in mice, highlighting the important tumor suppressor function of ATM in immature B cells. While EμCyclinD1 is not sufficient to induce lymphomas, EμCyclinD1 accelerates the kinetics and increased the incidence of clonal lymphomas in ATM-deficient B-cells and skews the lymphomas towards pre-GC derived small lymphocytic neoplasms sharing morphological features of human MCL. This is in part due to CyclinD1-driven expansion of ATM-deficient naïve B cells with genomic instability, which promotes the deletions of additional tumor suppressor genes (i.g. Trp53, Mll2, Rb1 and Cdkn2a). Together these findings define a synergistic function of ATM and CyclinD1 in pre-germinal center B-cell proliferation and lymphomagenesis and provide a prototypic animal model to study the pathogenesis of human MCL. PMID:25676421

  20. Peroxisome proliferator-activated receptor gamma agonists induce proteasome-dependent degradation of cyclin D1 and estrogen receptor alpha in MCF-7 breast cancer cells.

    Science.gov (United States)

    Qin, Chunhua; Burghardt, Robert; Smith, Roger; Wormke, Mark; Stewart, Jessica; Safe, Stephen

    2003-03-01

    Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) gamma agonists ciglitazone or 15-deoxy-Delta 12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) alpha proteins, and this was accompanied by decreased cell proliferation and G(1)-G(0)-->S-phase progression. Down-regulation of cyclin D1 and ER alpha by PPARgamma agonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-Delta 12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ER alpha antibodies, there was enhanced formation of ubiquitinated cyclin D1 and ER alpha bands. Thus, PPARgamma-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER alpha), and this pathway may be important for other cancer cell lines.

  1. Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation.

    Science.gov (United States)

    Wang, Chenguang; Pattabiraman, Nagarajan; Zhou, Jian Nian; Fu, Maofu; Sakamaki, Toshiyuki; Albanese, Chris; Li, Zhiping; Wu, Kongming; Hulit, James; Neumeister, Peter; Novikoff, Phyllis M; Brownlee, Michael; Scherer, Philipp E; Jones, Joan G; Whitney, Kathleen D; Donehower, Lawrence A; Harris, Emily L; Rohan, Thomas; Johns, David C; Pestell, Richard G

    2003-09-01

    The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPAR gamma induces hepatic steatosis, and liganded PPAR gamma promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPAR gamma function, transactivation, expression, and promoter activity. PPAR gamma transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPAR gamma ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPAR gamma-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1(-/-) fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPAR gamma ligands of PPAR gamma and PPAR gamma-responsive genes, and cyclin D1(-/-) mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPAR gamma in vivo. The inhibition of PPAR gamma function by cyclin D1 is a new mechanism of signal transduction cross talk between PPAR gamma ligands and mitogenic signals that induce cyclin D1.

  2. Cyclin CYB-3 controls both S-phase and mitosis and is asymmetrically distributed in the early C. elegans embryo.

    Science.gov (United States)

    Michael, W Matthew

    2016-09-01

    In early C. elegans embryos the timing of cell division is both invariant and developmentally regulated, yet how the cell cycle is controlled in the embryo and how cell cycle timing impacts early development remain important, unanswered questions. Here, I focus on the cyclin B3 ortholog CYB-3, and show that this cyclin has the unusual property of controlling both the timely progression through S-phase and mitotic entry, suggesting that CYB-3 is both an S-phase-promoting and mitosis-promoting factor. Furthermore, I find that CYB-3 is asymmetrically distributed in the two-cell embryo, such that the somatic precursor AB cell contains ∼2.5-fold more CYB-3 than its sister cell, the germline progenitor P1 CYB-3 is not only physically limited in P1 but also functionally limited, and this asymmetry is controlled by the par polarity network. These findings highlight the importance of the CYB-3 B3-type cyclin in cell cycle regulation in the early embryo and suggest that CYB-3 asymmetry helps establish the well-documented cell cycle asynchrony that occurs during cell division within the P-lineage.

  3. Activation of P-TEFb by Androgen Receptor-Regulated Enhancer RNAs in Castration-Resistant Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Yu Zhao

    2016-04-01

    Full Text Available The androgen receptor (AR is required for castration-resistant prostate cancer (CRPC progression, but the function and disease relevance of AR-bound enhancers remain unclear. Here, we identify a group of AR-regulated enhancer RNAs (e.g., PSA eRNA that are upregulated in CRPC cells, patient-derived xenografts (PDXs, and patient tissues. PSA eRNA binds to CYCLIN T1, activates P-TEFb, and promotes cis and trans target gene transcription by increasing serine-2 phosphorylation of RNA polymerase II (Pol II-Ser2p. We define an HIV-1 TAR RNA-like (TAR-L motif in PSA eRNA that is required for CYCLIN T1 binding. Using TALEN-mediated gene editing we further demonstrate that this motif is essential for increased Pol II-Ser2p occupancy levels and CRPC cell growth. We have uncovered a P-TEFb activation mechanism and reveal altered eRNA expression that is related to abnormal AR function and may potentially be a therapeutic target in CRPC.

  4. PAC exhibits potent anti-colon cancer properties through targeting cyclin D1 and suppressing epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Al-Qasem, Abeer; Al-Howail, Huda A; Al-Swailem, Mashael; Al-Mazrou, Amer; Al-Otaibi, Basem; Al-Jammaz, Ibrahim; Al-Khalaf, Huda H; Aboussekhra, Abdelilah

    2016-03-01

    Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide. Although response rates and overall survival have been improved in recent years, resistance to multiple drug combinations is inevitable. Therefore, the development of more efficient drugs, with fewer side effects is urgently needed. To this end, we have investigated in the present report the effect of PAC, a novel cucumin analogue, on CRC cells both in vitro and in vivo. We have shown that PAC induces apoptosis, mainly via the internal mitochondrial route, and inhibits cell proliferation through delaying the cell cycle at G2/M phase. Interestingly, the pro-apoptotic effect was mediated through STAT3-dependent down-regulation of cyclin D1 and its downstream target survivin. Indeed, change in the expression level of cyclin D1 modulated the expression of survivin and the response of CRC cells to PAC. Furthermore, using the ChIP assay, we have shown PAC-dependent reduction in the binding of STAT3 to the cyclin D1 promoter in vivo. Additionally, PAC suppressed the epithelial-to-mesenchymal process through down-regulating the mesenchymal markers (N-cadherin, vimentin and Twist1) and inhibiting the invasion/migration abilities of the CRC cells via repressing the pro-migration/invasion protein kinases AKT and ERK1/2. In addition, PAC inhibited tumor growth and repressed the JAK2/STAT3, AKT/mTOR and MEK/ERK pathways as well as their common downstream effectors cyclin D1 and survivin in humanized CRC xenografts. Collectively, these results indicate that PAC has potent anti-CRC effects, and therefore could constitute an effective alternative chemotherapeutic agent, which may consolidate the adjuvant treatment of colon cancer.

  5. The transcription factor NFAT5 is required for cyclin expression and cell cycle progression in cells exposed to hypertonic stress.

    Directory of Open Access Journals (Sweden)

    Katherine Drews-Elger

    Full Text Available BACKGROUND: Hypertonicity can perturb cellular functions, induce DNA damage-like responses and inhibit proliferation. The transcription factor NFAT5 induces osmoprotective gene products that allow cells to adapt to sustained hypertonic conditions. Although it is known that NFAT5-deficient lymphocytes and renal medullary cells have reduced proliferative capacity and viability under hypertonic stress, less is understood about the contribution of this factor to DNA damage responses and cell cycle regulation. METHODOLOGY/PRINCIPAL FINDINGS: We have generated conditional knockout mice to obtain NFAT5(-/- T lymphocytes, which we used as a model of proliferating cells to study NFAT5-dependent responses. We show that hypertonicity triggered an early, NFAT5-independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins, and cell cycle arrest. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced an osmoprotective gene expression program, downregulated stress markers, resumed cyclin expression and proliferation, and displayed enhanced NFAT5 transcriptional activity in S and G2/M. In contrast, NFAT5(-/- cells failed to induce osmoprotective genes and exhibited poorer viability. Although surviving NFAT5(-/- cells downregulated genotoxic stress markers, they underwent cell cycle arrest in G1/S and G2/M, which was associated with reduced expression of cyclins E1, A2 and B1. We also show that pathologic hypertonicity levels, as occurring in plasma of patients and animal models of osmoregulatory disorders, inhibited the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in fresh NFAT5(-/- lymphocytes. CONCLUSIONS/SIGNIFICANCE: We conclude that NFAT5 facilitates cell proliferation under hypertonic conditions by inducing an osmoadaptive response that enables cells to express fundamental regulators needed for cell cycle progression.

  6. The tight junction protein ZO-2 blocks cell cycle progression and inhibits cyclin D1 expression.

    Science.gov (United States)

    Gonzalez-Mariscal, Lorenza; Tapia, Rocio; Huerta, Miriam; Lopez-Bayghen, Esther

    2009-05-01

    ZO-2 is an adaptor protein of the tight junction that belongs to the MAGUK protein family. ZO-2 is a dual localization protein that in sparse cultures is present at the cell borders and the nuclei, whereas in confluent cultures it is concentrated at the cell boundaries. Here we have studied whether ZO-2 is able to regulate the expression of cyclin D1 (CD1) and cell proliferation. We have demonstrated that ZO-2 negatively regulates CD1 transcription by interacting with c-Myc at an E box present in CD1 promoter. We have further found that ZO-2 transfection into epithelial MDCK cells triggers a diminished expression of CD1 protein and decreases the rate of cell proliferation in a wound-healing assay.

  7. Cyclin-dependent kinase inhibitors in yeast, animals, and plants: a functional comparison.

    Science.gov (United States)

    De Clercq, Annelies; Inzé, Dirk

    2006-01-01

    The cell cycle is remarkably conserved in yeast, animals, and plants and is controlled by cyclin-dependent kinases (CDKs). CDK activity can be inhibited by binding of CDK inhibitory proteins, designated CKIs. Numerous studies show that CKIs are essential in orchestrating eukaryotic cell proliferation and differentiation. In yeast, animals, and plants, CKIs act as regulators of the G1 checkpoint in response to environmental and developmental cues and assist during mitotic cell cycles by inhibiting CDK activity required to arrest mitosis. Furthermore, CKIs play an important role in regulating cell cycle exit that precedes differentiation and in promoting differentiation in cooperation with transcription factors. Moreover, CKIs are essential to control CDK activity in endocycling cells. So, in yeast, animals, and plants, CKIs share many functional similarities, but their functions are adapted toward the specific needs of the eukaryote.

  8. [Management of T1a vocal fold carcinoma].

    Science.gov (United States)

    Reiter, R; Brosch, S; Smith, E; Pickhard, A

    2013-12-01

    About 2/3 of the larynx carcinomas affect the vocal chords. The main risk factor is smoking. Carcinomas in this localisation often arise from leukoplakias with dysplasia. A typical symptom is dysphonia. Arrest of vibration in microlaryngostroboscopy is a hint that a carcinoma could be present. Transoral laser cordectomy or radiotherapy show equivalent oncological results and results in quality of voice in the treatment of vocal fold carcinoma (T1a). As lymph node and distant metastasis are very rare, follow-up can concentrate on microlaryngoscopy. In case of a suspicious area on the vocal fold, biopsy of the affected tissue is needed to plan correct treatment. The prognosis of the T1 vocal chord carcinoma is quite good with a 5-year survival rate of almost 100%.

  9. Towards T1-limited magnetic resonance imaging using Rabi beats

    CERN Document Server

    Fedder, H; Rempp, F; Wolf, T; Hemmer, P; Jelezko, F; Wrachtrup, J

    2010-01-01

    Two proof-of-principle experiments towards T1-limited magnetic resonance imaging with NV centers in diamond are demonstrated. First, a large number of Rabi oscillations is measured and it is demonstrated that the hyperfine interaction due to the NV's 14N can be extracted from the beating oscillations. Second, the Rabi beats under V-type microwave excitation of the three hyperfine manifolds is studied experimentally and described theoretically.

  10. Towards T 1-limited magnetic resonance imaging using Rabi beats

    Science.gov (United States)

    Fedder, H.; Dolde, F.; Rempp, F.; Wolf, T.; Hemmer, P.; Jelezko, F.; Wrachtrup, J.

    2011-03-01

    Two proof-of-principle experiments toward T 1-limited magnetic resonance imaging with NV centers in diamond are demonstrated. First, a large number of Rabi oscillations is measured and it is demonstrated that the hyperfine interaction due to the NV's 14N can be extracted from the beating oscillations. Second, the Rabi beats under V-type microwave excitation of the three hyperfine manifolds is studied experimentally and described theoretically.

  11. 电动T1 大众Bulli

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    新Bulli由电动机提供动力,可容纳六名乘员,并配有iPad控制的多媒体信息娱乐系统。新车比T1车身更长、更高,但宽度比较窄,以2.62m的轴距充分利用了车身整体长度。

  12. Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells

    OpenAIRE

    Ning Wang; Xuanbin Wang; Hor-Yue Tan; Sha Li; Chi Man Tsang; Sai-Wah Tsao; Yibin Feng

    2016-01-01

    The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degrada...

  13. Heme oxygenase-1 regulates cyclin-dependent kinase inhibitor p18INK4C in mice with cisplatin-induced acute kidney injury%周期素依赖性激酶抑制剂p18INK4C在急性肾损伤中的表达受血红素加氧酶1调控

    Institute of Scientific and Technical Information of China (English)

    张懿; 谌卫; 付莉莉; 胡惠民; 梅长林

    2011-01-01

    目的 初步探讨周期素依赖性激酶抑制剂p18INK4C(p18)在急性肾损伤(AKI)中的肾保护作用机制.方法 利用血红素加氧酶(HO)-1的化学诱导剂(hemin)和抑制剂(锌原卟啉,ZnPP)观察在顺铂诱导的小鼠肾脏上皮细胞(TCMK-1)损伤中HO-1与p18之间的关系.利用p18基因敲除鼠,观察p18基因缺失对于顺铂诱导的HO-1表达的影响.结果 在顺铂诱导的AKI小鼠肾组织及损伤的TCMK-1细胞中,p18、HO-1的mRNA和蛋白表达均明显上调(P值均<0.05),HO-1的诱导表达峰值先于p18上调峰值.在无血清培养的TCMK-1细胞中,HO-1诱导剂hemin与细胞共同孵育后明显上调TCMK-1细胞中HO-1和p18蛋白的表达(P值均<0.05).相反,HO-1抑制剂ZnPP与顺铂共同孵育TCMK-1后的第8和16小时,ZnPP不仅抑制了顺铂诱导的HO-1蛋白表达的上调,而且使p18的表达受到明显抑制.p18-/-与p18+/+小鼠肾组织中HO-1蛋白表达基线值的差异无统计学意义(P>0.05),顺铂注射后第3天,p18-/-与p18+/+小鼠肾组织中HO-1蛋白的诱导表达量的差异亦无统计学意义(P>0.05).顺铂注射后第3天,p18+/+(WT)小鼠肾组织中p18蛋白表达上调,而p18-/-小鼠肾脏中p18蛋白表达缺失.结论 在顺铂诱导的AKI中,HO-1调控p18的表达,p18部分介导了HO-1在AKI中的保护作用.%Objective To determine the protective role of cyclin-dependent kinase inhibitor p18INMO(p18) in cisplatin-induced acute kidney injury (AKI) by using p18 gene knockout mice and investigate the renoprotective mechanism of p18. Methods Chemical inducer (hemin) and inhibitor of HO-1 (ZnPP) were used to study the relationship between p18 and HO-1 in cisplatin-induced mouse kidney epithelial cells (TCMK-1). Further, p18 gene knockout mice were used to demonstrate the effect of p18 on the inducible expression of HO-1 in cisplatin-induced AKI. Results Expressions of p18 and HO-1 were both upregulated at mRNA and protein levels in cisplatin-induced kidneys and

  14. CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    Directory of Open Access Journals (Sweden)

    Breuer Denitra

    2012-11-01

    Full Text Available Abstract Background HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity. Results The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94 that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation. Conclusion Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for

  15. T1 and susceptibility contrast at high fields

    Science.gov (United States)

    Neelavalli, Jaladhar

    Clinical imaging at high magnetic field strengths (≥ 3Tesla) is sought after primarily due to the increased signal strength available at these fields. This increased SNR can be used to perform: (a) high resolution imaging in the same time as at lower field strengths; (b) the same resolution imaging with much faster acquisition; and (c) functional MR imaging (fMRI), dynamic perfusion and diffusion imaging with increased sensitivity. However they are also associated with increased power deposition (SAR) due to increase in imaging frequency and longer T1 relaxation times. Longer T1s mean longer imaging times for generating good T1 contrast images. On the other hand for faster imaging, at high fields fast spin echo or magnetization prepared sequences are conventionally proposed which are, however, associated with high SAR values. Imaging with low SAR is more and more important as we move towards high fields and particularly for patients with metallic implants like pacemakers or deep brain stimulator. The SAR limit acceptable for these patients is much less than the limit acceptable for normal subjects. A new method is proposed for imaging at high fields with good contrast with simultaneous reduction in power deposition. Further, T1 based contrast optimization problem in FLASH imaging is considered for tissues with different T1s but same spin densities. The solution providing optimal imaging parameters is simplified for quick and easy computation in a clinical setting. The efficacy of the simplification is evaluated and practical limits under which the simplification can be applied are worked out. The phase difference due to variation in magnetic susceptibility property among biological tissues is another unique source of contrast which is different from the conventional T1, T2 and T2* contrast. This susceptibility based phase contrast has become more and more important at high fields, partly due to contrast generation issues due to longer T 1s and shorter T2s and

  16. EXPRESSION OF CYCLIN D1 AND CDK4 IN OSTEOSARCOMA OF THE JAWS

    Institute of Scientific and Technical Information of China (English)

    司晓辉; 刘正

    2001-01-01

    Objective: To analyze cyclin D1 and cyclin- dependent kinase 4 (CDK4) expression and their significance in osteosarcoma of the jaws. Methods: Immunohistochemical ABC method was used to detect the expression of cyclin D1 and CDK4 in 20 cases of osteosarcoma and 8 cases of osteochondroma of the jaws. Results: The positive rates of cyclin D1 and CDK4 were 65% (13/20) and 60% (12/20), respectively. There was significant positive correlation between cyclin D1 and CDK4 expression (gs=0.48, P<0.05). Both cyclin D1 and CDK4 were present in 1/8 (12.5%) osteochondroma. The positive rate was remarkably different between osteosarcoma and osteochondroma (P<0.05). Conclusion: Cyclin D1 and CDK4 are overexpressed in osteosarcoma of the jaws and closely related to its occurrence and development.

  17. Immunohistochemical analysis of p53, cyclinD1, RB1, c-fos and N-ras gene expression in hepatocellular carcinoma in Iran

    Institute of Scientific and Technical Information of China (English)

    SJ Moghaddam; EN Haghighi; S Samiee; N Shahid; AR Keramati; S Dadgar; MR Zali

    2007-01-01

    AIM: To study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma.METHODS: Paraffin-embedded tissue samples of 25 patients (18 males and 7 females) with hepatocellular carcinoma were collected from 22 pathology centers in Tehran during 2000-2001, and stained using immunohistochemistry method (avidin-biotin-peroxidase)for detection of p53, cyclinD1, RB1, c-fos and N-ras proteins.RESULTS: Six (24%), 5 (20%), 12 (48%) and 2 samples (8%) were positive for p53, cyclinD1, C-fos and N-ras expression, respectively. Twenty-two (88%) samples had alterations in the G1 cell-cycle checkpoint protein expression (RB1 or cyclinD1). P53 positive samples showed a higher (9 times) risk of being positive for RB1 protein than p53 negative samples. Loss of expression of RB1 in association with p53 over-expression was observed in 4 (66.7%) of 6 samples. Loss of expression of RB1 was seen in all cyclinD1 positive, 20 (90.9%) N-ras negative, and 11 (50%) C-fos positive samples,respectively. CyclinD1 positive samples showed a higher (2.85 and 4.75 times) risk of being positive for c-fos and N-ras expression than cyclinD1 negative samples.CONCLUSION: The expression of p53, RB1 and c-fos genes appears to have a key role in the pathogenesis of hepatocellular carcinoma in Iran. Simultaneous overexpression of these genes is significantly associated with their loss of expression during development of hepatocellular carcinoma.

  18. Local IGF-1 isoform protects cardiomyocytes from hypertrophic and oxidative stresses via SirT1 activity.

    Science.gov (United States)

    Vinciguerra, Manlio; Santini, Maria Paola; Claycomb, William C; Ladurner, Andreas G; Rosenthal, Nadia

    2009-12-10

    Oxidative and hypertrophic stresses contribute to the pathogenesis of heart failure. Insulin-like growth factor-1 (IGF-1) is a peptide hormone with a complex post-transcriptional regulation, generating distinct isoforms. Locally acting IGF-1 isoform (mIGF-1) helps the heart to recover from toxic injury and from infarct. In the murine heart, moderate overexpression of the NAD(+)-dependent deacetylase SirT1 was reported to mitigate oxidative stress. SirT1 is known to promote lifespan extension and to protect from metabolic challenges. Circulating IGF-1 and SirT1 play antagonizing biological roles and share molecular targets in the heart, in turn affecting cardiomyocyte physiology. However, how different IGF-1 isoforms may impact SirT1 and affect cardiomyocyte function is unknown. Here we show that locally acting mIGF-1 increases SirT1 expression/activity, whereas circulating IGF-1 isoform does not affect it, in cultured HL-1 and neonatal cardiomyocytes. mIGF-1-induced SirT1 activity exerts protection against angiotensin II (Ang II)-triggered hypertrophy and against paraquat (PQ) and Ang II-induced oxidative stress. Conversely, circulating IGF-1 triggered itself oxidative stress and cardiomyocyte hypertrophy. Interestingly, potent cardio-protective genes (adiponectin, UCP-1 and MT-2) were increased specifically in mIGF-1-overexpressing cardiomyocytes, in a SirT1-dependent fashion. Thus, mIGF-1 protects cardiomyocytes from oxidative and hypertrophic stresses via SirT1 activity, and may represent a promising cardiac therapeutic.

  19. DNA damage leads to a Cyclin A-dependent delay in metaphase-anaphase transition in the Drosophila gastrula.

    Science.gov (United States)

    Su, T T; Jaklevic, B

    2001-01-09

    In response to DNA damage, fission yeast, mammalian cells, and cells of the Drosophila gastrula inhibit Cdk1 to delay the entry into mitosis. In contrast, budding yeast delays metaphase-anaphase transition by stabilization of an anaphase inhibitor, Pds1p. A variation of the second response is seen in Drosophila cleavage embryos; when nuclei enter mitosis with damaged DNA, centrosomes lose gamma-tubulin, spindles lose astral microtubules, chromosomes fail to reach a metaphase configuration, and interphase resumes without an intervening anaphase. The resulting polyploid nuclei are eliminated. The cells of the Drosophila gastrula can also delay metaphase-anaphase transition in response to DNA damage. This delay accompanies the stabilization of Cyclin A, a known inhibitor of sister chromosome separation in Drosophila. Unlike in cleavage embryos, gamma-tubulin remains at the spindle poles, and anaphase always occurs after the delay. Cyclin A mutants fail to delay metaphase-anaphase transition after irradiation and show an increased frequency of chromosome breakage in the subsequent anaphase. DNA damage delays metaphase-anaphase transition in Drosophila by stabilizing Cyclin A. This delay may normally serve to preserve chromosomal integrity during segregation. To our knowledge this is the first report of a metazoan metaphase-anaphase transition being delayed in response to DNA damage. Though mitotic progression is modulated in response to DNA damage in both cleaving and gastruating embryos of Drosophila, different mechanisms operate. These differences are discussed in the context of differential cell cycle regulation in cleavage and gastrula stages.

  20. T1w dark blood imaging improves detection of contrast enhancing lesions in multiple sclerosis.

    Science.gov (United States)

    Thaler, Christian; Schneider, Tanja; Sedlacik, Jan; Kutzner, Daniel; Stellmann, Jan-Patrick; Heesen, Christoph; Fiehler, Jens; Siemonsen, Susanne

    2017-01-01

    In multiple sclerosis (MS) the sensitivity for detection of contrast enhancing lesions (CEL) in T1-weighted scans is essential for diagnostics and therapy decisions. The purpose of our study was to evaluate the sensitivity of T1w MPRAGE scans in comparison to T1w dark blood technique (T1-DB) for CEL in MS. 3T MR imaging was performed in 37 MS patients, including T2-weighted imaging, T1w MPRAGE before and after gadolinium injection (unenhanced-T1 and T1-CE) and T1-DB imaging. After gadolinium application, the T1-DB scan was performed prior to T1-CE. From unenhanced-T1 and T1-CE scans, subtraction images (T1-SUB) were calculated. The number of CEL was determined separately on T1-CE and T1-DB by two raters independently. Lesions only detected on T1-DB scans then were verified on T1-SUB. Only lesions detected by both raters were included in further analysis. In 16 patients, at least one CEL was detected by both rater, either on T1-CE or T1-DB. All lesions that were detected on T1-CE were also detected on T1-DB images. The total number of contrast enhancing lesions detected on T1-DB images (n = 54) by both raters was significantly higher than the corresponding number of lesions identified on T1-CE (n = 27) (p = 0.01); all of these lesions could be verified on SUB images. In 21 patients, no CEL was detected in any of the sequences. The application of T1-DB technique increases the sensitivity for CEL in MS, especially for those lesions that show only subtle increase in intensity after Gadolinium application but remain hypo- or iso-intense to surrounding tissue.

  1. The electronics system of the TOTEM T1 telescope

    Energy Technology Data Exchange (ETDEWEB)

    Minutoli, S., E-mail: minutoli@ge.infn.it [Istituto Nazionale di Fisica Nucleare, Genova (Italy); Bozzo, M. [Istituto Nazionale di Fisica Nucleare, Genova (Italy); Dipartimento di Fisica, Genova (Italy); Ferro, F. [Istituto Nazionale di Fisica Nucleare, Genova (Italy); Lo Vetere, M. [Istituto Nazionale di Fisica Nucleare, Genova (Italy); Dipartimento di Fisica, Genova (Italy); Robutti, E. [Istituto Nazionale di Fisica Nucleare, Genova (Italy)

    2013-08-01

    The T1 detector of the TOTEM experiment is devoted to the measurement of the inelastic rate of proton–proton interactions at the LHC. It is made of Cathode Strip Chambers. The complete electronic chains of front-end, readout and trigger are presented here. The electronics system has been developed keeping into account the hostile environment from the point of view of both radiation and magnetic field. Dedicated VLSI circuits have been extensively used in order to optimize space and power consumption.

  2. The TOTEM T1 read out card motherboard

    CERN Document Server

    Minutoli, S; Robutti, E

    2010-01-01

    This article describes the Read Out Card (ROC) motherboard, which is the main component of the T1 forward telescope front-end electronic system. The ROC main objectives are to acquire tracking data and trigger information from the detector. It performs data conversion from electrical to optical format and transfers the data streams to the next level of the system and it implements Slow Control modules which are able to receive, decode and distribute the LHC machine low jitter clock and fast command. The ROC also provides a spy mezzanine connection based on programmable FPGA and USB2.0 for laboratory and portable DAQ debugging system

  3. The TOTEM T1 read out card motherboard

    Science.gov (United States)

    Minutoli, S.; Lo Vetere, M.; Robutti, E.

    2010-12-01

    This article describes the Read Out Card (ROC) motherboard, which is the main component of the T1 forward telescope front-end electronic system. The ROC main objectives are to acquire tracking data and trigger information from the detector. It performs data conversion from electrical to optical format and transfers the data streams to the next level of the system and it implements Slow Control modules which are able to receive, decode and distribute the LHC machine low jitter clock and fast command. The ROC also provides a spy mezzanine connection based on programmable FPGA and USB2.0 for laboratory and portable DAQ debugging system.

  4. Chk1 is activated at the midblastula transition in Xenopus laevis embryos independently of DNA content and the cyclin E/Cdk2 developmental timer.

    Science.gov (United States)

    Adjerid, Nassiba; Wroble, Brian N; Sible, Jill C

    2008-04-15

    Cell cycle checkpoints that are engaged in response to damaged and unreplicated DNA may serve additional, constitutive functions. In the developing Xenopus laevis embryo, the checkpoint kinase Chk1 is transiently activated at the midblastula transition (MBT), a period of extensive cell cycle remodeling including the acquisition of cell cycle checkpoints. The timing of many cell cycle remodeling events at the MBT, such as the lengthening of cell cycles, depends upon a critical nucleocytoplasmic (N/C) ratio. However, other events, including the degradation of maternal cyclin E, do not depend upon the N/C ratio, and are regulated by an autonomous developmental timer. To better understand what regulates Chk1 activation at the MBT, embryos were treated with aphidicolin, at different developmental times and for different lengths of time, to reduce the DNA content at the MBT. Chk1 was activated at the MBT in these embryos establishing that Chk1 activation occurs independently of the N/C ratio. Cdc25A is normally phosphorylated by Chk1 at the MBT and then degraded. The degradation of Cdc25A demonstrated partial dependence on DNA content, suggesting that factors other than Chk1 regulate its degradation. When the cyclin E developmental timer was disrupted with the Cdk2 inhibitor delta34-Xic1, Chk1 was still activated at the MBT, indicating that activation of Chk1 at the MBT was not directly linked to the cyclin E timer. Conversely, unreplicated or damaged DNA, delayed the degradation of cyclin E at the MBT, indicating that the cyclin E/Cdk2 timer is sensitive to engagement of cell cycle checkpoints.

  5. Expression of pRb, p53, p16 and cyclin D1 and their clinical implications in urothelial carcinoma.

    Science.gov (United States)

    Lee, Kyungji; Jung, Eun Sun; Choi, Young-Jin; Lee, Kyo Young; Lee, Ahwon

    2010-10-01

    The aim of this study was to assess immunohistochemical expression of p53, pRb, p16, and cyclin D1, alone or in combination, as prognostic indicators and to investigate their correlation with clinocopathologic features of urothelial carcinoma. Immunohistochemical staining for p53, pRb, p16, and cyclin D1 was performed on a tissue microarray from 103 patients with urothelial carcinoma who underwent radical cystectomy. Of the patient samples analyzed, 36 (35%), 61 (59%), 47 (46%) and 30 (29%) had altered expression of p53, pRb, p16, and cyclin D1, respectively. Abnormal expression of p53 and pRb correlated with depth of invasion (P=0.040 and P=0.044, respectively). Cyclin D1 expression was associated with tumor stage and recurrence (P=0.017 and P=0.036, respectively). Altered pRb was significantly correlated with overall survival (P=0.040). According to the expression pattern of pRb and p53, p53/pRb (altered/normal) had worse survival than p53/pRb (normal/altered) (P=0.022). Alteration of all markers had worse survival than all normal (P=0.029). As determined by multivariate analysis, tumor stage, lymph node metastasis and the combined expression of p53 and pRb are independent prognostic factors. In conclusion, immunohistochemical evaluation of cell cycle regulators, especially the p53/pRb combination, might be useful in planning appropriate treatment strategies.

  6. Dietary fat increases solid tumor growth and metastasis of 4T1 murine mammary carcinoma cells and mortality in obesity-resistant BALB/c mice.

    Science.gov (United States)

    Kim, Eun Ji; Choi, Mi-Ran; Park, Heesook; Kim, Minhee; Hong, Ji Eun; Lee, Jae-Yong; Chun, Hyang Sook; Lee, Ki Won; Yoon Park, Jung Han

    2011-08-11

    High-fat diets (HFDs) are known to cause obesity and are associated with breast cancer progression and metastasis. Because obesity is associated with breast cancer progression, it is important to determine whether dietary fat per se stimulates breast cancer progression in the absence of obesity. This study investigated whether an HFD increases breast cancer growth and metastasis, as well as mortality, in obesity-resistant BALB/c mice. The 4-week-old, female BALB/c mice were fed HFD (60% kcal fat) or control diet (CD, 10% kcal fat) for 16 weeks. Subsequently, 4T1 mammary carcinoma cells were injected into the inguinal mammary fat pads of mice fed continuously on their respective diets. Cell-cycle progression, angiogenesis, and immune cells in tumor tissues, proteases and adhesion molecules in the lungs, and serum cytokine levels were analyzed with immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). In vitro studies were also conducted to evaluate the effects of cytokines on 4T1 cell viability, migration, and adhesion. Spleen and gonadal fat-pad weights, tumor weight, the number and volume of tumor nodules in the lung and liver, and tumor-associated mortality were increased in the HFD group, with only slight increases in energy intake and body weight. HF feeding increased macrophage infiltration into adipose tissues, the number of lipid vacuoles and the expression of cyclin-dependent kinase (CDK)2, cyclin D1, cyclin A, Ki67, CD31, CD45, and CD68 in the tumor tissues, and elevated serum levels of complement fragment 5a (C5a), interleukin (IL)-16, macrophage colony-stimulating factor (M-CSF), soluble intercellular adhesion molecule (sICAM)-1, tissue inhibitors of metalloproteinase (TIMP)-1, leptin, and triggering receptor expressed on myeloid cells (TREM)-1. Protein levels of the urokinase-type plasminogen activator, ICAM-1, and vascular cell adhesion molecule-1 were increased, but plasminogen activator inhibitor-1 levels were

  7. The regulation of ER export and Golgi retention of ST3Gal5 (GM3/GM4 synthase) and B4GalNAcT1 (GM2/GD2/GA2 synthase) by arginine/lysine-based motif adjacent to the transmembrane domain.

    Science.gov (United States)

    Uemura, Satoshi; Shishido, Fumi; Kashimura, Madoka; Inokuchi, Jin-ichi

    2015-12-01

    In the Golgi maturation model, the Golgi cisternae dynamically mature along a secretory pathway. In this dynamic process, glycosyltransferases are transported from the endoplasmic reticulum (ER) to the Golgi apparatus where they remain and function. The precise mechanism behind this maturation process remains unclear. We investigated two glycosyltransferases, ST3Gal5 (ST3G5) and B4GalNAcT1 (B4GN1), involved in ganglioside synthesis and examined their signal sequences for ER export and Golgi retention. Reports have suggested that the [R/K](X)[R/K] motif functions as an ER exporting signal; however, this signal sequence is insufficient in stably expressed, full-length ST3G5. Through further analysis, we have clarified that the (2)R(3)R(X)(5) (9)K(X)(3) (13)K sequence in ST3G5 is essential for ER export. We have named the sequence the R/K-based motif. On the other hand, for ER export of B4GN1, the homodimer formation in addition to the R/K-based motif is required for ER export suggesting the importance of unidentified lumenal side interaction. We found that ST3G5 R2A/R3A and K9A/K13A mutants localized not only in Golgi apparatus but also in endosomes. Furthermore, the amounts of mature type asparagine-linked (N)-glycans in ST3G5 R2A/R3A and K9A/K13A mutants were decreased compared with those in wild-type proteins, and the stability of the mutants was lower. These results suggest that the R/K-based motif is necessary for the Golgi retention of ST3G5 and that the retention is involved in the maturation of N-glycans and in stability. Thus, several basic amino acids located on the cytoplasmic tail of ST3G5 play important roles in both ER export and Golgi retention.

  8. New T1-based superconductor T1PbSrRCuO without Ca with Tc above 100 K

    Science.gov (United States)

    Sheng, Z. Z.; Xin, Y.; Meason, J. M.

    1991-04-01

    Ca-free T1PbSrRCuO samples (R=rare earths) with Tc up to above 100 K were prepared, and studied by resistance and ac susceptibility measurements and by powder x-ray diffraction analyses. A 1212-type phase (Tl1-xPbx)Sr2(Sr1-yRy)Cu2Oz is responsible for the observed supercondcutivity. A rare-earth is required for the formation of the 1212 phase. Pb-dopping is necessary to increase the Tc of the 1212 phase from 90 K to above 100 K.

  9. The impact of reliable pre-bolus T1 measurements or a fixed T1 value in the assessment of glioma patients with Dynamic Contrast Enhancing MRI

    DEFF Research Database (Denmark)

    Tietze, Anna; Mouridsen, Kim; Mikkelsen, Irene Klærke

    2015-01-01

    Purpose: Accurate quantification of hemodynamic parameters using Dynamic Contrast Enhanced MRI (DCE) requires a measurement of tissue T1 prior to contrast injection (T1). We evaluate (i) T1 estimation using the variable flip angle (VFA) and the saturation recovery (SR) techniques and (ii) investi...

  10. Synaptic roles of cyclin-dependent kinase 5 & its implications in epilepsy

    Directory of Open Access Journals (Sweden)

    Aparna Banerjee Dixit

    2017-01-01

    Full Text Available There is an urgent need to understand the molecular mechanisms underlying epilepsy to find novel prognostic/diagnostic biomarkers to prevent epilepsy patients at risk. Cyclin-dependent kinase 5 (CDK5 is involved in multiple neuronal functions and plays a crucial role in maintaining homeostatic synaptic plasticity by regulating intracellular signalling cascades at synapses. CDK5 deregulation is shown to be associated with various neurodegenerative diseases such as Alzheimer's disease. The association between chronic loss of CDK5 and seizures has been reported in animal models of epilepsy. Genetic expression of CDK5 at transcriptome level has been shown to be abnormal in intractable epilepsy. In this review various possible mechanisms by which deregulated CDK5 may alter synaptic transmission and possibly lead to epileptogenesis have been discussed. Further, CDK5 has been proposed as a potential biomarker as well as a pharmacological target for developing treatments for epilepsy.

  11. Construction of CyclinD1 Gene Overexpression Lentivirus Vector and Its Effect on the Proliferation of Neural Stem Cells%CyclinD1基因过表达慢病毒载体的构建和对神经干细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    马俊芳; 崔博; 沈东超; 崔丽英

    2015-01-01

    Objective:In order to study the effect of cyclinD1 on neural stem cell proliferation, the cyclinD1 gene overexpression lentiviral vector was constructed and the expression of cyclinD1 in neural stem cells in mice after transfection was tested. Methods: Nestin promoter-Ccnd1 gene was inserted into plenti6 lentiviral expression vec-tors by DNA recombination technique. Lentiviral vector plenti-D1 was established after recombination. Recombi-nant lentiviral vector was detected by DNA sequencing. The plenti-D1 was transfected into 293T cell line. The viruses yielded by 293T cell were transfected into mouse embryonic neural stem cells. The expression of cyclinD1 was detected by real-time PCR and Western Blot analysis after transfection. We studied the effects of different MOI values of plenti-D1 on neural stem cell proliferation by MTT assay. Results: It was confirmed by DNA sequencing that the cyclinD1 gene sequencing was correctly inserted into the vector, and that the cyclinD1 gene overexpression lentiviral vector was successfully constructed. After extracting the infected cells, the real-time PCR showed cy-clinD1 mRNA overexpression was significantly higher than the control groups; Western Blot analysis was used to detect expression of cyclinD1 protein in neural stem cells. The cyclinD1 gene overexpression lentiviral vector was significantly up-regulated in mRNA level or in protein level in neural stem cells. When the MOI was 10, 20, and 50, the viruses significantly promoted the proliferation of neural stem cells. Conclusion: The cyclinD1 gene overex-pression lentiviral vector was successfully constructed and it efficiently up-regulated the expression of cyclinD1 in mouse embryonic neural stem cells. CyclinD1 overexpression can promote the proliferation of neural stem cells.%目的:针对小鼠 cyclinD1基因构建质粒并进行慢病毒包装,转染小鼠神经干细胞,检测其表达水平。方法:根据 cyclinD1基因信息,采用 DNA

  12. p16 Cyclin D1在妊娠滋养细胞疾病组织中 的表达及意义%Expression of pi6 and Cyclin D1 in the tissues of gestational trophoblastic diseases and its significance

    Institute of Scientific and Technical Information of China (English)

    梁劲荃; 赵或明; 梁睿; 主改霞; 孙润琴

    2001-01-01

    Objective To detect the significance of p16 and CyclinD1 in the tumorigenesis of trophoblastic tumors.Methods Immunohistochemical SP method was used to detect the p16 and CyclinD1 gene expression in the tissues of 49 cases of trophoblastic diseases ( the primary curretage tissues of moles 32 cases, invasive moles 12 cases and choriocarcinoma 5 cases ) and 4 cases of normal chorion of early gestation. Results The expression of p16 and CyclinD1 was respectively all positive and all negative in the chorion of early gestation. When the moles that didn′t transform to malignancy, the moles that did transform and the trophoblasfic tumors were detected,a descending tendency of p16 expression was found ,while the expression of CyclinD1 showed an ascending tendency. The positive rate of p16 expression was significantly different between the following groups: the moles transforming malignantly later and the moles that didnt transform,the moles that didnt transform and the invasive moles( P 0.05). Conclusions The absent expression of the cell cycle regulating gene pi6 and the excessive expression of cyclinD1 may play a key role in the malignant transformation of trophocyte and in the development of invasive moles. Detection of p16 and Cyclin D1 expression in the primary curretage tissues of moles may be of great value in assessing the prognosis of moles.%目的探讨p16、CyclinD1基因在滋养细胞肿瘤发生方面的意义。方法用免疫组化SP法,对49例滋养细胞疾病组织(葡萄胎首次清宫标本32例,侵蚀性葡萄胎及绒癌子宫标本分别为12例和5例)和4例早孕正常绒毛组织中p16和CyclinD1进行检测。结果在早孕绒毛组织中,pi6和CyclinD1表达分别为全部阳性和全部阴性。在不恶变葡萄胎、以后发生恶变的葡萄胎和滋养细胞肿瘤中p16表达呈下降趋势,CyclinD1表达呈上升趋势。p16和CyclinD1表达阳性率在不恶变葡萄胎与以后发生恶变的葡萄胎组间、在不恶变

  13. Contrast-enhanced 3T MR Perfusion of Musculoskeletal Tumours: T1 Value Heterogeneity Assessment and Evaluation of the Influence of T1 Estimation Methods on Quantitative Parameters.

    Science.gov (United States)

    Gondim Teixeira, Pedro Augusto; Leplat, Christophe; Chen, Bailiang; De Verbizier, Jacques; Beaumont, Marine; Badr, Sammy; Cotten, Anne; Blum, Alain

    2017-06-14

    To evaluate intra-tumour and striated muscle T1 value heterogeneity and the influence of different methods of T1 estimation on the variability of quantitative perfusion parameters. Eighty-two patients with a histologically confirmed musculoskeletal tumour were prospectively included in this study and, with ethics committee approval, underwent contrast-enhanced MR perfusion and T1 mapping. T1 value variations in viable tumour areas and in normal-appearing striated muscle were assessed. In 20 cases, normal muscle perfusion parameters were calculated using three different methods: signal based and gadolinium concentration based on fixed and variable T1 values. Tumour and normal muscle T1 values were significantly different (p = 0.0008). T1 value heterogeneity was higher in tumours than in normal muscle (variation of 19.8% versus 13%). The T1 estimation method had a considerable influence on the variability of perfusion parameters. Fixed T1 values yielded higher coefficients of variation than variable T1 values (mean 109.6 ± 41.8% and 58.3 ± 14.1% respectively). Area under the curve was the least variable parameter (36%). T1 values in musculoskeletal tumours are significantly different and more heterogeneous than normal muscle. Patient-specific T1 estimation is needed for direct inter-patient comparison of perfusion parameters. • T1 value variation in musculoskeletal tumours is considerable. • T1 values in muscle and tumours are significantly different. • Patient-specific T1 estimation is needed for comparison of inter-patient perfusion parameters. • Technical variation is higher in permeability than semiquantitative perfusion parameters.

  14. Identifying allosteric fluctuation transitions between different protein conformational states as applied to Cyclin Dependent Kinase 2

    Directory of Open Access Journals (Sweden)

    Gu Jenny

    2007-02-01

    Full Text Available Abstract Background The mechanisms underlying protein function and associated conformational change are dominated by a series of local entropy fluctuations affecting the global structure yet are mediated by only a few key residues. Transitional Dynamic Analysis (TDA is a new method to detect these changes in local protein flexibility between different conformations arising from, for example, ligand binding. Additionally, Positional Impact Vertex for Entropy Transfer (PIVET uses TDA to identify important residue contact changes that have a large impact on global fluctuation. We demonstrate the utility of these methods for Cyclin-dependent kinase 2 (CDK2, a system with crystal structures of this protein in multiple functionally relevant conformations and experimental data revealing the importance of local fluctuation changes for protein function. Results TDA and PIVET successfully identified select residues that are responsible for conformation specific regional fluctuation in the activation cycle of Cyclin Dependent Kinase 2 (CDK2. The detected local changes in protein flexibility have been experimentally confirmed to be essential for the regulation and function of the kinase. The methodologies also highlighted possible errors in previous molecular dynamic simulations that need to be resolved in order to understand this key player in cell cycle regulation. Finally, the use of entropy compensation as a possible allosteric mechanism for protein function is reported for CDK2. Conclusion The methodologies embodied in TDA and PIVET provide a quick approach to identify local fluctuation change important for protein function and residue contacts that contributes to these changes. Further, these approaches can be used to check for possible errors in protein dynamic simulations and have the potential to facilitate a better understanding of the contribution of entropy to protein allostery and function.

  15. Phospholipase C-delta1 expression is linked to proliferation, DNA synthesis, and cyclin E levels.

    Science.gov (United States)

    Stallings, Jonathan D; Zeng, Yue X; Narvaez, Francisco; Rebecchi, Mario J

    2008-05-16

    We previously reported that phospholipase C-delta1 (PLC-delta1) accumulates in the nucleus at the G1/S transition, which is largely dependent on its binding to phosphatidylinositol 4,5-bisphosphate ( Stallings, J. D., Tall, E. G., Pentyala, S., and Rebecchi, M. J. (2005) J. Biol. Chem. 280, 22060-22069 ). Here, using small interfering RNA (siRNA) that specifically targets rat PLC-delta1, we investigated whether this enzyme plays a role in cell cycle control. Inhibiting expression of PLC-delta1 significantly decreased proliferation of rat C6 glioma cells and altered S phase progression. [3H]Thymidine labeling and fluorescence-activated cell sorting analysis indicated that the rates of G1/S transition and DNA synthesis were enhanced. On the other hand, knockdown cultures released from the G1/S boundary were slower to reach full G2/M DNA content, consistent with a delay in S phase. The levels of cyclin E, a key regulator of the G1/S transition and DNA synthesis, were elevated in asynchronous cultures as well as those blocked at the G1/S boundary. Epifluorescence imaging showed that transient expression of human phospholipase C-delta1, resistant to these siRNA, suppressed expression of cyclin E at the G1/S boundary despite treatment of cultures with rat-specific siRNA. Although whole cell levels of phosphatidylinositol 4,5-bisphosphate were unchanged, suppression of PLC-delta1 led to a significant rise in the nuclear levels of this phospholipid at the G1/S boundary. These results support a role for PLC-delta1 and nuclear phospholipid metabolism in regulating cell cycle progression.

  16. Identification of a T1D Susceptibility Gene

    Directory of Open Access Journals (Sweden)

    Grant Morahan

    2001-01-01

    Full Text Available It is not known what causes type 1 diabetes (T1D, which affects over 1 million people in the U.S. alone. Each year, 30,000 young people in the U.S. develop this disease and depend on insulin injections thereafter. Because of the huge cost to the individual, the family, and to society in increased health care costs, it is important to find what makes these people susceptible. The disease process itself is clear: the individual’s immune system — T lymphocytes in particular — attack and destroy the body’s insulin-producing cells. But how and why this autoimmune process starts or proceeds unregulated is still not known.

  17. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  18. Limited prognostic value of tissue protein expression levels of cyclin E in Danish ovarian cancer patients

    DEFF Research Database (Denmark)

    Heeran, Mel C; Høgdall, Claus K; Kjaer, Susanne K

    2012-01-01

    The primary objective of this study was to assess the expression of cyclin E in tumour tissues from 661 patients with epithelial ovarian tumours. The second was to evaluate whether cyclin E tissue expression levels correlate with clinico-pathological parameters and prognosis of the disease. Using...... tissue arrays (TA), we analysed the cyclin E expression levels in tissues from 168 women with borderline ovarian tumours (BOT) (147 stage I, 4 stage II, 17 stage III) and 493 Ovarian cancer (OC) patients (127 stage I, 45 stage II, 276 stage III, 45 stage IV). Using a 10% cut-off level for cyclin E......-off value showed that cyclin E had no independent prognostic value. In conclusion, we found cyclin E expression in tumour tissue to be of limited prognostic value to Danish OC patients....

  19. Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Christopher C.; Bloodworth, Jeffrey C. [Division of Pharmacology, Columbus, OH 43210 (United States); Mythreye, Karthikeyan [Duke University, Department of Medicine, Durham, NC 27708 (United States); Lee, Nam Y., E-mail: lee.5064@osu.edu [Division of Pharmacology, Columbus, OH 43210 (United States); Davis Heart and Lung Research Institute, Columbus, OH 43210 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Endoglin inhibits ERK activation in endothelial cells. Black-Right-Pointing-Pointer Endoglin is a regulator of c-Myc and cyclin D1 expression. Black-Right-Pointing-Pointer {beta}-arrestin2 interaction with endoglin is required for ERK/c-Myc repression. Black-Right-Pointing-Pointer Endoglin impedes cellular proliferation by targeting ERK-induced mitogenic signaling. -- Abstract: Endoglin is an endothelial-specific transforming growth factor beta (TGF-{beta}) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-{beta} signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previously identified {beta}-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and {beta}-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-{beta}-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/{beta}-arrestin2 interaction is disrupted. Given that TGF-{beta}-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.

  20. Prognostic significance of cyclinD1 amplification and the co-alteration of cyclinD1/pRb/ppRb in patients with esophageal squamous cell carcinoma.

    Science.gov (United States)

    Wang, M-T; Chen, G; An, S-J; Chen, Z-H; Huang, Z-M; Xiao, P; Ben, X-S; Xie, Z; Chen, S-L; Luo, D-L; Tang, J-M; Lin, J-Y; Zhang, X-C; Wu, Y-L

    2012-01-01

    CyclinD1/pRb/ppRb is one of the most important pathways regulating the cell cycle, and related with the development of many cancers. However, the co-alteration of CyclinD1/pRb/ppRb in esophageal squamous cell carcinomas is less understood. This study aims to analyze the combined prognostic significance of cyclinD1 (CCND1) DNA amplification and the co-alteration of CCND1/pRb/ppRB in patients with esophageal squamous cell carcinoma. CCND1 DNA amplification and the protein expression of CCND1, pRb, and ppRb on 100 tumor specimens and 11 normal tissues were detected using real-time quantitative reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Their prognosis significance was analyzed by Kaplan-Meier method. We found that 41% of the patients had CCND1 DNA amplification, which had a short survival time compared with the patients without CCND1 amplification (25.63 months vs. not reached, P=0.007). The patients with the co-alternation of CCND1(+) /pRb(-) /ppRb(+) protein expression levels have a poorer overall survival than the others (11.4 vs. 43.4 months, P=0.001). Cox regression analysis showed that the co-alternation of CCND1/pRb/ppRb and CyclinD1 amplification were the two most independent prognosis factors of patients with esophageal cancer. These findings suggested that CCND1 amplification and co-alternation of CCND1(+) /pRb(-) /ppRb(+) may play a crucial role in the prognostic evaluation of patients with esophageal cancer, and the patients with CCND1(+) /pRb(-) /ppRb(+) have the worst prognosis in all the patients. The results also indicated that the patients with CCND1 amplification or co-alternation of CyclinD1(+) /pRb(-) /ppRb(+) might be the preponderant people for therapy targeting the CCND1/pRb/ppRb pathway in the future.

  1. A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

    Directory of Open Access Journals (Sweden)

    Pinhero Reena

    2004-01-01

    Full Text Available We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His6-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni2+-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004-14. Our protocol provides a novel systematic approach for the purification of these three (and possibly other recombinant CDKs.

  2. Targeting cyclin B1 inhibits proliferation and sensitizes breast cancer cells to taxol

    Directory of Open Access Journals (Sweden)

    Strebhardt Klaus

    2008-12-01

    Full Text Available Abstract Background Cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1, is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers. Methods In order to explore cyclin B1 as a potential target for gynecological cancer therapy, we studied the effect of small interfering RNA (siRNA on different gynecological cancer cell lines by monitoring their proliferation rate, cell cycle profile, protein expression and activity, apoptosis induction and colony formation. Tumor formation in vivo was examined using mouse xenograft models. Results Downregulation of cyclin B1 inhibited proliferation of several breast and cervical cancer cell lines including MCF-7, BT-474, SK-BR-3, MDA-MB-231 and HeLa. After combining cyclin B1 siRNA with taxol, we observed an increased apoptotic rate accompanied by an enhanced antiproliferative effect in breast cancer cells. Furthermore, control HeLa cells were progressively growing, whereas the tumor growth of HeLa cells pre-treated with cyclin B1 siRNA was strongly inhibited in nude mice, indicating that cyclin B1 is indispensable for tumor growth in vivo. Conclusion Our data support the notion of cyclin B1 being essential for survival and proliferation of gynecological cancer cells. Concordantly, knockdown of cyclin B1 inhibits proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy.

  3. Expression of Cyclin E and Its Relationship with the Prognosis of Patients with Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    HE Wenshan; HUANG Tao; WANG Haijiu

    2006-01-01

    Objective: To investigate the expression of cyclin E in breast cancer tissues and its relationship with prognosis of the patients with breast cancer. Methods: The expression of cyclin E, HER-2/neu,nm23-H1 and actin was detected in 80 breast cancer tissues and 18 benign breast tumor tissues by immunohistochemical methods. The relationship between cyclin E and the remaining genes or the clinical data of the patients with breast cancer was analyzed. Results: The over expression rate of cyclin E in malignant tissues was obviously higher than that in benign tumor tissues (P<0.01). The over expression of cyclin E in later stage of disease was higher than that in early stage of disease (P<0.05). The expression of cyclin E in ER positive tissues was lower than that in ER negative tissues (P<0.05). The expression of cyclin E in PR positive tissues and PR negative tissues had no significant difference (P>0.05). The expression of cyclin E in HER-2/neu positive tissues was higher than that in HER-2/neu negative tissues (P<0.05). And the expression of cyclin E in ER, PR and HER-2/neu all positive tissues was much higher (P<0.01). There was no significant difference in the expression of cyclin E between nm23-H1 positive tissues and nm23-H1 negative tissues (P>0.05). The expression of cyclin E in actin positive and continuous distribution tissues was lower than that in actin negative or discontinuous distribution tissues (P<0.05). Conclusion: The expression of cyclin E has a strong correlation to the prognosis of the patients with breast cancer.

  4. Critical Role Played by Cyclin D3 in the MyoD-Mediated Arrest of Cell Cycle during Myoblast Differentiation

    OpenAIRE

    1999-01-01

    During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and wit...

  5. Cyclin-dependent kinase inhibitor p20 controls circadian cell-cycle timing.

    Science.gov (United States)

    Laranjeiro, Ricardo; Tamai, T Katherine; Peyric, Elodie; Krusche, Peter; Ott, Sascha; Whitmore, David

    2013-04-23

    Specific stages of the cell cycle are often restricted to particular times of day because of regulation by the circadian clock. In zebrafish, both mitosis (M phase) and DNA synthesis (S phase) are clock-controlled in cell lines and during embryo development. Despite the ubiquitousness of this phenomenon, relatively little is known about the underlying mechanism linking the clock to the cell cycle. In this study, we describe an evolutionarily conserved cell-cycle regulator, cyclin-dependent kinase inhibitor 1d (20 kDa protein, p20), which along with p21, is a strongly rhythmic gene and directly clock-controlled. Both p20 and p21 regulate the G1/S transition of the cell cycle. However, their expression patterns differ, with p20 predominant in developing brain and peak expression occurring 6 h earlier than p21. p20 expression is also p53-independent in contrast to p21 regulation. Such differences provide a unique mechanism whereby S phase is set to different times of day in a tissue-specific manner, depending on the balance of these two inhibitors.

  6. Cyclin C stimulates β-cell proliferation in rat and human pancreatic β-cells

    OpenAIRE

    2015-01-01

    Activation of pancreatic β-cell proliferation has been proposed as an approach to replace reduced functional β-cell mass in diabetes. Quiescent fibroblasts exit from G0 (quiescence) to G1 through pRb phosphorylation mediated by cyclin C/cdk3 complexes. Overexpression of cyclin D1, D2, D3, or cyclin E induces pancreatic β-cell proliferation. We hypothesized that cyclin C overexpression would induce β-cell proliferation through G0 exit, thus being a potential therapeutic target to recover funct...

  7. A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    Science.gov (United States)

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-01-01

    Cyclin dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexrepsssion. Intriguingly, we found that C53 interacts with checkpoint kinase 1 (Chk1) and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and DNA damage response. PMID:19223857

  8. By using tissue chip technique to study the expression of p27 and cyclinE in breast carcinoma%采用组织芯片技术研究乳腺癌中p27和cyclinE的表达

    Institute of Scientific and Technical Information of China (English)

    戴艳枝; 刘勇; 袁晟; 邓宇

    2003-01-01

    To study the expressions of p27 ,cyclinE and analysis its meaning in breast carcinoma. Methods By using tissue chip technique creating 80 cases breast carcinoma tissue chip. at the same time using the S-P immunohis-tochemical methods, the expressions of p27 and cyclinE in breast carcinoma tissue chip was studied.Results The positive rates of p27 and cyclinE were 38.8% and 52.5% in 80 eases breast carcinoma respectively. A significant correlation was found between the lowexpression of p27, overexpression of cyclinE and metastasis of lymph node in breast carcinoma. A simaificant correlation was found be-tween the lowexpression of p27 and overexpression of cyclinE in breast carcinoma. Conclusion The expression of p27 and cyclinE might be considered to be a reference indicator for prognosis of breast carcinoma. The lowexpression of p27 has an ability to effect the overexpression of cyclinE in breast carcinoma. It is feasible to utilize tissue chip for a rapid, economic and accurate screening of clinical tissue specimens on a large scale.

  9. T1 Colorectal Cancer with Synchronous Liver Metastasis

    Directory of Open Access Journals (Sweden)

    Kiichi Sugimoto

    2013-06-01

    Full Text Available The patient was a 68-year-old man who was admitted to our hospital with a liver tumor. Abdominal ultrasonography and computed tomography revealed a liver tumor 30 mm in diameter. On colonoscopy, a pedunculated tumor with a central depression (20 mm in diameter was observed in the ascending colon, and this tumor was considered to be invading deeply into the submucosal layer. Right hemicolectomy with D3 lymphadenectomy and partial hepatectomy were performed simultaneously. On histopathological examination of the resected specimen, the tumor was a well-differentiated tubular adenocarcinoma with 3,000 μm invasion of the submucosal layer. The liver tumor showed histological findings similar to those of the primary colorectal carcinoma. The pathological stage according to the 7th edition of the TNM classification was stage IV (T1N0M1. Nine months after the operation, computed tomography revealed hepatic hilar lymph node metastases and a great deal of ascites. The patient ultimately died 14 months after the operation.

  10. Transit timing analysis of CoRoT-1b

    CERN Document Server

    Csizmadia, Sz; Barge, P; Agol, E; Aigrain, S; Alonso, R; Almenara, J M; Bonomo, A S; Borde, P; Bouchy, F; Cabrera, J; Deeg, H J; De la Reza, R; Deleuil, M; Dvorak, R; Erikson, A; Guenther, E W; Fridlund, M; Gondoin, P; Guillot, T; Hatzes, A; Jorda, L; Lammer, H; Lázaro, C; Leger, A; Llebaria, A; Magain, P; Moutou, C; Ollivier, M; Paetzold, M; Queloz, D; Rauer, H; Rouan, D; Schneider, J; Wuchterl, G; Gandolfi, D

    2009-01-01

    CoRoT, the pioneer space-based transit search, steadily provides thousands of high-precision light curves with continuous time sampling over periods of up to 5 months. The transits of a planet perturbed by an additional object are not strictly periodic. By studying the transit timing variations (TTVs), additional objects can be detected in the system. A transit timing analysis of CoRoT-1b is carried out to constrain the existence of additional planets in the system. We used data obtained by an improved version of the CoRoT data pipeline (version 2.0). Individual transits were fitted to determine the mid-transit times, and we analyzed the derived $O-C$ diagram. N-body integrations were used to place limits on secondary planets. No periodic timing variations with a period shorter than the observational window (55 days) are found. The presence of an Earth-mass Trojan is not likely. A planet of mass greater than $\\sim 1$ Earth mass can be ruled out by the present data if the object is in a 2:1 (exterior) mean mot...

  11. Flowing Foam: T1 events and solid-liquid transitions.

    Science.gov (United States)

    Dennin, Michael

    2005-11-01

    Flowing aqueous foam is found in many applications ranging from oil recovery, to fire fighting, to spreading shaving cream. Aqueous foam consists of gas bubbles with liquid walls. One of the striking features of foam is that despite being composed entirely of fluids, its mechanical properties are either those of a solid (elastic response) or fluid (viscous flow), depending on the nature of the applied stress and strains. We study the transition between these two regimes using a model foam system: bubble rafts. Bubble rafts are a single layer of bubbles floating on the air-water surface. This allows us to track the motion of all the bubbles during flow. In this talk, we will present two main results. First, we will discuss the observation of the coexistence between a solid-like and fluid-like state during flow. Second, we will discuss the role played by nonlinear, topological rearrangements, known as T1 events, in determining the mechanical response of the system.

  12. Main-Belt Comet P/2012 T1 (PANSTARRS)

    CERN Document Server

    Hsieh, Henry H; Novakovic, Bojan; Yang, Bin; Haghighipour, Nader; Micheli, Marco; Denneau, Larry; Fitzsimmons, Alan; Jedicke, Robert; Kleyna, Jan; Veres, Peter; Wainscoat, Richard J; Ansdell, Megan; Elliott, Garrett T; Keane, Jacqueline V; Meech, Karen J; Moskovitz, Nicholas A; Riesen, Timm E; Sheppard, Scott S; Sonnett, Sarah; Tholen, David J; Urban, Laurie; Kaiser, Nick; Chambers, K C; Burgett, William S; Magnier, Eugene A; Morgan, Jeffrey S; Price, Paul A

    2013-01-01

    We present initial results from observations and numerical analyses aimed at characterizing main-belt comet P/2012 T1 (PANSTARRS). Optical monitoring observations were made between October 2012 and February 2013 using the University of Hawaii 2.2 m telescope, the Keck I telescope, the Baade and Clay Magellan telescopes, Faulkes Telescope South, the Perkins Telescope at Lowell Observatory, and the Southern Astrophysical Research (SOAR) telescope. The object's intrinsic brightness approximately doubles from the time of its discovery in early October until mid-November and then decreases by ~60% between late December and early February, similar to photometric behavior exhibited by several other main-belt comets and unlike that exhibited by disrupted asteroid (596) Scheila. We also used Keck to conduct spectroscopic searches for CN emission as well as absorption at 0.7 microns that could indicate the presence of hydrated minerals, finding an upper limit CN production rate of QCN100 Myr and is unlikely to be a rec...

  13. The secondary eclipse of CoRoT-1b

    CERN Document Server

    Alonso, R; Aigrain, S; Auvergne, M; Baglin, A; Barbieri, M; Barge, P; Bonomo, A S; Borde, P; Bouchy, F; Chaintreuil, S; De la Reza, R; Deeg, H J; Deleuil, M; Dvorak, R; Erikson, A; Fridlund, M; Fialho, F; Gondoin, P; Guillot, T; Hatzes, A; Jorda, L; Lammer, H; Léger, A; Llebaria, A; Magain, P; Mazeh, T; Moutou, C; Ollivier, M; Patzold, M; Pont, F; Queloz, D; Rauer, H; Rouan, D; Schneider, J; Wuchterl, G

    2009-01-01

    The transiting planet CoRoT-1b is thought to belong to the pM-class of planets, in which the thermal emission dominates in the optical wavelengths. We present a detection of its secondary eclipse in the CoRoT white channel data, whose response function goes from ~400 to ~1000 nm. We used two different filtering approaches, and several methods to evaluate the significance of a detection of the secondary eclipse. We detect a secondary eclipse centered within 20 min at the expected times for a circular orbit, with a depth of 0.016+/-0.006%. The center of the eclipse is translated in a 1-sigma upper limit to the planet's eccentricity of ecosomega<0.014. Under the assumption of a zero Bond Albedo and blackbody emission from the planet, it corresponds to a T_{CoRoT}=2330 +120-140 K. We provide the equilibrium temperatures of the planet as a function of the amount of reflected light. If the planet is in thermal equilibrium with the incident flux from the star, our results imply an inefficient transport mechanism ...

  14. HLA and autoimmune diseases: Type 1 diabetes (T1D) as an example.

    Science.gov (United States)

    Gorodezky, Clara; Alaez, Carmen; Murguía, Andrea; Rodríguez, Araceli; Balladares, Sandra; Vazquez, Miriam; Flores, Hilario; Robles, Carlos

    2006-03-01

    Autoimmune diseases need to be considered at a genetic and mechanistic level. T1D is an autoimmune, chronic, multifactorial and polygenic disease characterized by the destruction of the pancreatic beta-cells associated with long term dysfunction of several organs and tissues. Mechanisms of susceptibility include epi-genetic and post-transcriptional effects that regulate transmission and expression of the inherited genes. The HLA complex, constitutes the most relevant region contributing 50% of the inherited risk for T1D. An additional 17 genes with variable but small effects have been described. In non-Caucasians, the presence of E-DRbeta1-74 and/or D-DRbeta1-57 are relevant in predisposition. The "Diabetogenic haplotypes" in Mexicans were DRB1*0301-DQA1*0501-DQB1*0201 (OR = 21.4); DRB1*0405-DQA1-*0301-DQB1*0302 (OR = 44.5) and the same DQA1/DQB1 with DRB1*0404/*0401 conferring lower risk, increasing (OR = 61.3) with an early age at onset and a heterozygote DR3/DR4 genotype. In most populations, the absence of D-57 and the presence of R-52 are important to the susceptibility, but in Hispanics, all DR4s (including the protective DRB1*0403/*0407/*0411) are in linkage disequilibrium with DQA1/DQB1 susceptibility alleles. Thus, susceptibility alleles in Latin American Mestizos are of Mediterranean ancestry whereas protective alleles are of Amerindian origin. In this review, we discuss the complexity of T1D and some aspects of prevention/intervention based on immunogenetics.

  15. Comparison of T1-weighted fast spin-echo and T1-weighted fluid-attenuated inversion recovery images of the lumbar spine at 3.0 Tesla

    Energy Technology Data Exchange (ETDEWEB)

    Lavdas, Eleftherios; Vlychou, Marianna; Arikidis, Nikos; Kapsalaki, Eftychia; Roka, Violetta; Fezoulidis, Ioannis V. (Dept. of Radiology, Univ. Hospital of Larissa, Medical School of Thessaly, Mezourlo (Greece)), e-mail: mvlychou@med.uth.gr

    2010-04-15

    Background: T1-weighted fluid-attenuated inversion recovery (FLAIR) sequence has been reported to provide improved contrast between lesions and normal anatomical structures compared to T1-weighted fast spin-echo (FSE) imaging at 1.5T regarding imaging of the lumbar spine. Purpose: To compare T1-weighted FSE and fast T1-weighted FLAIR imaging in normal anatomic structures and degenerative and metastatic lesions of the lumbar spine at 3.0T. Material and Methods: Thirty-two consecutive patients (19 females, 13 males; mean age 44 years, range 30-67 years) with lesions of the lumbar spine were prospectively evaluated. Sagittal images of the lumbar spine were obtained using T1-weighted FSE and fast T1-weighted FLAIR sequences. Both qualitative and quantitative analyses measuring the signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and relative contrast (ReCon) between degenerative and metastatic lesions and normal anatomic structures were conducted, comparing these sequences. Results: On quantitative evaluation, SNRs of cerebrospinal fluid (CSF), nerve root, and fat around the root of fast T1-weighted FLAIR imaging were significantly lower than those of T1-weighted FSE images (P<0.001). CNRs of normal spinal cord/CSF and disc herniation/ CSF for fast T1-weighted FLAIR images were significantly higher than those for T1-weighted FSE images (P<0.001). ReCon of normal spinal cord/CSF, disc herniation/CSF, and vertebral lesions/CSF for fast T1-weighted FLAIR images were significantly higher than those for T1-weighted FSE images (P<0.001). On qualitative evaluation, it was found that CSF nulling and contrast at the spinal cord (cauda equina)/CSF interface for T1-weighted FLAIR images were significantly superior compared to those for T1-weighted FSE images (P<0.001), and the disc/spinal cord (cauda equina) interface was better for T1-weighted FLAIR images (P<0.05). Conclusion: The T1-weighted FLAIR sequence may be considered as the preferred lumbar spine imaging

  16. Functional lung MRI in chronic obstructive pulmonary disease: comparison of T1 mapping, oxygen-enhanced T1 mapping and dynamic contrast enhanced perfusion.

    Directory of Open Access Journals (Sweden)

    Bertram J Jobst

    Full Text Available Monitoring of regional lung function in interventional COPD trials requires alternative endpoints beyond global parameters such as FEV1. T1 relaxation times of the lung might allow to draw conclusions on tissue composition, blood volume and oxygen fraction. The aim of this study was to evaluate the potential value of lung Magnetic resonance imaging (MRI with native and oxygen-enhanced T1 mapping for the assessment of COPD patients in comparison with contrast enhanced perfusion MRI.20 COPD patients (GOLD I-IV underwent a coronal 2-dimensional inversion recovery snapshot flash sequence (8 slices/lung at room air and during inhalation of pure oxygen, as well as dynamic contrast-enhanced first-pass perfusion imaging. Regional distribution of T1 at room air (T1, oxygen-induced T1 shortening (ΔT1 and peak enhancement were rated by 2 chest radiologists in consensus using a semi-quantitative 3-point scale in a zone-based approach.Abnormal T1 and ΔT1 were highly prevalent in the patient cohort. T1 and ΔT1 correlated positively with perfusion abnormalities (r = 0.81 and r = 0.80; p&0.001, and with each other (r = 0.80; p<0.001. In GOLD stages I and II ΔT1 was normal in 16/29 lung zones with mildly abnormal perfusion (15/16 with abnormal T1. The extent of T1 (r = 0.45; p<0.05, ΔT1 (r = 0.52; p<0.05 and perfusion abnormalities (r = 0.52; p<0.05 showed a moderate correlation with GOLD stage.Native and oxygen-enhanced T1 mapping correlated with lung perfusion deficits and severity of COPD. Under the assumption that T1 at room air correlates with the regional pulmonary blood pool and that oxygen-enhanced T1 reflects lung ventilation, both techniques in combination are principally suitable to characterize ventilation-perfusion imbalance. This appears valuable for the assessment of regional lung characteristics in COPD trials without administration of i.v. contrast.

  17. Activation of cyclin D1 by estradiol and spermine in MCF-7 breast cancer cells: a mechanism involving the p38 MAP kinase and phosphorylation of ATF-2.

    Science.gov (United States)

    Lewis, Joan S; Vijayanathan, Veena; Thomas, T J; Pestell, Richard G; Albanese, Chris; Gallo, Michael A; Thomas, Thresia

    2005-01-01

    Estradiol (E2) and the naturally occurring polyamines (putrescine, spermidine, and spermine) play important roles in breast cancer cell growth and differentiation. We examined the effects of E2 and spermine on the phosphorylation and DNA binding of activating transcription factor-2 (ATF-2) in MCF-7 breast cancer cells. ATF-2 is a transcription factor involved in estrogenic regulation of cyclin D1 gene, and thereby cell cycle progression. DNA affinity immunoblot assays showed a six- to eightfold increase in the binding of ATF-2 to a 74-mer ATF/CRE oligonucleotide (ODN1) from cyclin D1 promoter in the presence of 4 nM E2 and 0.5 mM spermine, compared to untreated control. Individual treatments with E2 or spermine caused a twofold or lower increase in ATF-2 binding to ODN1. Immunoblotting with phospho-ATF-2 antibody showed that increased DNA binding of ATF-2 was associated with its phosphorylation. A p38 MAP kinase inhibitor, PD169316, inhibited ATF-2 phosphorylation. In contrast, the MEK-ERK1/2 inhibitor, PD98059, or the JNK inhibitor, SP600125, had no significant effect on DNA binding of ATF-2. Cyclin D1 promoter (-1745CD1) activity increased by approximately 12-fold (above control) in the presence of E2 and spermine, compared to a sixfold increase in the presence of E2 alone and a twofold increase with spermine. Cells transfected with a dominant negative mutant of ATF-2 showed decreased transactivation of cyclin D1 promoter in response to E2 and spermine. These results indicate that spermine can enhance E2-induced cell signaling and cyclin D1 transcription by activation of the p38 MAP kinase and phosphorylation of ATF-2, contributing to breast cancer cell proliferation.

  18. Cyclin-dependent kinase 2 is an ideal target for ovary tumors with elevated cyclin E1 expression

    OpenAIRE

    Yang, Liu; Fang, Dongdong; Chen, Huijun; Lu, Yiyu; Dong, Zheng; Ding, Han-Fei; Jing, Qing; Su, Shi-bing; Huang, Shuang

    2015-01-01

    CCNE1 gene amplification is present in 15-20% ovary tumor specimens. Here, we showed that Cyclin E1 (CCNE1) was overexpressed in 30% of established ovarian cancer cell lines. We also showed that CCNE1 was stained positive in over 40% of primary ovary tumor specimens regardless of their histological types while CCNE1 staining was either negative or low in normal ovary and benign ovary tumor tissues. However, the status of CCNE1 overexpression was not associated with the tumorigenic potential o...

  19. Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Ya-Hsin, E-mail: yhcheng@mail.cmu.edu.tw [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China); Li, Lih-Ann; Lin, Pinpin; Cheng, Li-Chuan [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan, ROC (China); Hung, Chein-Hui [Graduate Institute of Clinical Medicine Sciences, Chang Gung University, Puizi City, Chiayi 613, Taiwan, ROC (China); Chang, Nai Wen [Department of Biochemistry, School of Medicine, China Medical University, Taichung, Taiwan, ROC (China); Lin, Chingju [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China)

    2012-09-15

    Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phase and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3β, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation. -- Highlights: ► Baicalein causes the G1 phase arrest by decreasing Rb phosphorylation. ► Baicalein modulates AhR-mediated cell proliferation. ► Both AhR activation and cyclin D1 degradation results in hypophosphorylation of Rb. ► Baicalein facilitates cyclin D1 degradation by signalling the GSK-3β pathway.

  20. Quantification of the host response proteome after mammalian reovirus T1L infection.

    Directory of Open Access Journals (Sweden)

    Alicia R Berard

    Full Text Available All viruses are dependent upon host cells for replication. Infection can induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays to measure the cellular "transcriptome." We used SILAC (stable isotope labeling by amino acids in cell culture, combined with high-throughput 2-D HPLC/mass spectrometry, to determine relative quantitative differences in host proteins at 6 and 24 hours after infecting HEK293 cells with reovirus serotype 1 Lang (T1L. 3,076 host proteins were detected at 6 hpi, of which 132 and 68 proteins were significantly up or down regulated, respectively. 2,992 cellular proteins, of which 104 and 49 were up or down regulated, respectively, were identified at 24 hpi. IPA and DAVID analyses indicated proteins involved in cell death, cell growth factors, oxygen transport, cell structure organization and inflammatory defense response to virus were up-regulated, whereas proteins involved in apoptosis, isomerase activity, and metabolism were down-regulated. These proteins and pathways may be suitable targets for intervention to either attenuate virus infection or enhance oncolytic potential.

  1. Sulforaphane, a Dietary Isothiocyanate, Induces G2/M Arrest in Cervical Cancer Cells through CyclinB1 Downregulation and GADD45β/CDC2 Association

    Science.gov (United States)

    Cheng, Ya-Min; Tsai, Ching-Chou; Hsu, Yi-Chiang

    2016-01-01

    Globally, cervical cancer is the most common malignancy affecting women. The main treatment methods for this type of cancer include conization or hysterectomy procedures. Sulforaphane (SFN) is a natural, compound-based drug derived from dietary isothiocyanates which has previously been shown to possess potent anti-tumor and chemopreventive effects against several types of cancer. The present study investigated the effects of SFN on anti-proliferation and G2/M phase cell cycle arrest in cervical cancer cell lines (Cx, CxWJ, and HeLa). We found that cytotoxicity is associated with an accumulation of cells in the G2/M phases of the cell-cycle. Treatment with SFN led to cell cycle arrest as well as the down-regulation of Cyclin B1 expression, but not of CDC2 expression. In addition, the effects of GADD45β gene activation in cell cycle arrest increase proportionally with the dose of SFN; however, mitotic delay and the inhibition of proliferation both depend on the dosage of SFN used to treat cancer cells. These results indicate that SFN may delay the development of cancer by arresting cell growth in the G2/M phase via down-regulation of Cyclin B1 gene expression, dissociation of the cyclin B1/CDC2 complex, and up-regulation of GADD45β proteins. PMID:27626412

  2. Sulforaphane, a Dietary Isothiocyanate, Induces G2/M Arrest in Cervical Cancer Cells through CyclinB1 Downregulation and GADD45β/CDC2 Association

    Directory of Open Access Journals (Sweden)

    Ya-Min Cheng

    2016-09-01

    Full Text Available Globally, cervical cancer is the most common malignancy affecting women. The main treatment methods for this type of cancer include conization or hysterectomy procedures. Sulforaphane (SFN is a natural, compound-based drug derived from dietary isothiocyanates which has previously been shown to possess potent anti-tumor and chemopreventive effects against several types of cancer. The present study investigated the effects of SFN on anti-proliferation and G2/M phase cell cycle arrest in cervical cancer cell lines (Cx, CxWJ, and HeLa. We found that cytotoxicity is associated with an accumulation of cells in the G2/M phases of the cell-cycle. Treatment with SFN led to cell cycle arrest as well as the down-regulation of Cyclin B1 expression, but not of CDC2 expression. In addition, the effects of GADD45β gene activation in cell cycle arrest increase proportionally with the dose of SFN; however, mitotic delay and the inhibition of proliferation both depend on the dosage of SFN used to treat cancer cells. These results indicate that SFN may delay the development of cancer by arresting cell growth in the G2/M phase via down-regulation of Cyclin B1 gene expression, dissociation of the cyclin B1/CDC2 complex, and up-regulation of GADD45β proteins.

  3. CKbeta8-1 alters expression of cyclin E in colony forming units-granulocyte macrophage (CFU-GM) lineage from human cord blood CD34+ cells.

    Science.gov (United States)

    Noh, Eui Kyu; Ra, Jae Sun; Lee, Seong Ae; Kwon, Byoung S; Han, In Seob

    2005-12-31

    A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34+ cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34+ cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34+ cells were compared with those in untreated CD34+ cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34+ cells. Here we describe an in vitro system in which CB CD34+ cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.

  4. Tetrandrine Suppresses Cancer Angiogenesis and Metastasis in 4T1 Tumor Bearing Mice

    Directory of Open Access Journals (Sweden)

    Jian-Li Gao

    2013-01-01

    Full Text Available Metastasis remains the most deadly aspect of cancer and still evades direct treatment. Thus, there is a great need to develop new treatment regimens to suppress tumor cells that have escaped surgical removal or that may have already disseminated. We have found that tetrandrine (TET exhibits anticolon cancer activity. Here, we investigate the inhibition effect of TET to breast cancer metastasis, angiogenesis and its molecular basis underlying TET’s anticancer activity. We compare TET with chemotherapy drug doxorubicin in 4T1 tumor bearing BALB/c mice model and find that TET exhibits an anticancer metastatic and antiangiogenic activities better than those of doxorubicin. The lung metastatic sites were decreased by TET, which is confirmed by bioluminescence imaging in vivo. On the other hand, laser doppler perfusion imaging (LDI was used for measuring the blood flow of tumor in 4T1-tumor bearing mice. As a result, the local blood perfusion of tumor was markedly decreased by TET after 3 weeks. Mechanistically, TET treatment leads to a decrease in p-ERK level and an increase in NF-κB levels in HUVECs. TET also regulated metastatic and angiogenic related proteins, including vascular endothelial growth factor, hypoxia-inducible factor-1α, integrin β5, endothelial cell specific molecule-1, and intercellular adhesion molecule-1 in vivo.

  5. Antitumor activity of Papua’s Myrmecodia pendans in human oral tongue squamous cell carcinoma cell line through induction of cyclin-dependent kinase inhibitor p27Kip1 and suppression of cyclin E

    Directory of Open Access Journals (Sweden)

    Supriatno DRG

    2014-03-01

    Full Text Available Oral tongue squamous cell carcinoma (OTSCC is one of the most common cancers encountered in Indonesia, due to the prevalent habits of tobacco chewing, alcohol drinking and smoking. Oral tongue cancer is characterized by a high degree of local invasion and a high rate of metastasis to the cervical lymph nodes. Interestingly, treatment options for this cancer are limited. The aim of this study was to examine the antitumor activity of Papua’s Myrmecodia pendans (ant nest plant in a human oral tongue squamous cell carcinoma cell line (B88 and to explore the possible mechanism in it. In the present study, B88 cells were treated with various concentration of ethanol extract of Papua’s M. pendans. The results revealed that B88 cells treated with Papua’s M. pendans were remarkable suppressed in cell growth and cell invasion, and had a significant induction of apoptosis characterized by an increase in activation of caspase-3 and -9. Furthermore, up-regulation of p27Kip1 and down-regulation of cyclin E protein was detected in B88 cells treated with Papua’s M. pendans. These results indicated that Papua’s M. pendans exhibited a high potential antitumor activity in human oral tongue squamous cell carcinoma through induction of p27Kip1 and suppression of cycline E.

  6. Loss of cyclin-dependent kinase 5 from parvalbumin interneurons leads to hyperinhibition, decreased anxiety, and memory impairment.

    Science.gov (United States)

    Rudenko, Andrii; Seo, Jinsoo; Hu, Ji; Su, Susan C; de Anda, Froylan Calderon; Durak, Omer; Ericsson, Maria; Carlén, Marie; Tsai, Li-Huei

    2015-02-11

    Perturbations in fast-spiking parvalbumin (PV) interneurons are hypothesized to be a major component of various neuropsychiatric disorders; however, the mechanisms regulating PV interneurons remain mostly unknown. Recently, cyclin-dependent kinase 5 (Cdk5) has been shown to function as a major regulator of synaptic plasticity. Here, we demonstrate that genetic ablation of Cdk5 in PV interneurons in mouse brain leads to an increase in GABAergic neurotransmission and impaired synaptic plasticity. PVCre;fCdk5 mice display a range of behavioral abnormalities, including decreased anxiety and memory impairment. Our results reveal a central role of Cdk5 expressed in PV interneurons in gating inhibitory neurotransmission and underscore the importance of such regulation during behavioral tasks. Our findings suggest that Cdk5 can be considered a promising therapeutic target in a variety of conditions attributed to inhibitory interneuronal dysfunction, such as epilepsy, anxiety disorders, and schizophrenia.

  7. Comparison of Trifecta and Pentafecta Outcomes between T1a and T1b Renal Masses following Robot-Assisted Partial Nephrectomy (RAPN with Minimum One Year Follow Up: Can RAPN for T1b Renal Masses Be Feasible?

    Directory of Open Access Journals (Sweden)

    Dae Keun Kim

    Full Text Available To investigate the feasibility of RAPN on T1b renal mass by assessment of Trifecta and Pentafecta rate between T1a and T1b renal mass.We retrospectively reviewed the medical records of 277 cases of RPN performed from 2006 to 2015. Sixty patients with clinically T1b renal masses (> 4 cm and ≤ 7 cm were identified, and from 180 patients with clinically T1a renal mass, 60 patients were matched with T1b renal mass by propensity score. Tumor complexity was investigated according to R.E.N.A.L nephrometry score. "Pentafecta" was defined as achievement of Trifecta (negative surgical margin, no postoperative complications and warm ischemia time of ≤ 25 minutes with addition of over 90% estimated GFR preservation and no chronic kidney disease stage upgrading at 1 year postoperative period. Propensity score matching was performed by OneToManyMTCH. Logistic regression models were used to identify the variables which predict the Trifecta, and Pentafecta ac.Preoperative variables (age, sex, body mass index, ASA score were similar between T1a and T1b after propensity score matching. The median R.E.N.A.L. nephrometry score was 8 vs 9 for T1a and T1b respectively (p<0.001. The median warm ischemia time was 20.1 min vs 26.2 min (p<0.001. Positive surgical margin rate was 5% vs 6.6% (p = 0.729 and overall complication rate of 13.3%. vs 15% (p = 0.793. The rate of achievement of Trifecta rate were 65.3% vs 43.3% (p = 0.017 and Pentafecta rate were 38.3% vs 26.7% (p = 0.172. For achievement of Pentafecta, R.E.N.A.L nephrometry score (HR 0.80; 95% CI (0.67-0.97; p = 0.031 was significant predictor of achieving Pentafecta. Subanalyis to assess the component of R.E.N.A.L nephrometry score, L component (location relative to the polar lines, HR 0.63; 95% CI (0.38-1.03; P = 0.064 was relatively important component for Pentafecta achievement.The rate of Pentafecta after RAPN was comparable between T1a and T1b renal masses. RAPN is a feasible modality with excellent

  8. Immunohistochemical comparison of cyclin D1 and P16 in odontogenic keratocyst and unicystic ameloblastoma

    Directory of Open Access Journals (Sweden)

    Seyed Mohammad Razavi

    2013-01-01

    Conclusion: Cyclin D1 did show a higher staining intensity in UAs compared to the keratocysts, although the expression of P16 was similar in the studied groups. The invasive growth of OKC might be related to the state of expression of cyclin D1 and P16 in the epithelium of this cyst.

  9. Cyclin A-Cdk2 Phosphorylates BH3 only Protein Bad in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    HE Kan; CHEN Yue; LI Jing-hua; ZHAN Zhuo; WU Yong-ge; KONG Wei; JIN Ying-hua

    2007-01-01

    Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli. To determine a specific substrate of Cyclin A-Cdk2 for apoptosis, in this study, we carried out anin vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source, and recombinant protein GST-Bad as a substrate. Our study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in vitro. To examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo, we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn, a dominant negative version of Cdk2, in Hela cells and determined the phosphorylation status of protein Bad. The test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells,but not in Cdk2-dn or mock transfectent. Moreover, etoposide also caused the phosphorylation of endogenetic Bad. In conclusion, here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.

  10. Elevated cyclin E level in human clear cell renal cell carcinoma: possible causes and consequences.

    Science.gov (United States)

    Nauman, Alicja; Turowska, Olga; Poplawski, Piotr; Master, Adam; Tanski, Zbigniew; Puzianowska-Kuznicka, Monika

    2007-01-01

    The expression of cyclin E gene (CCNE) in relation to the expression of its major regulatory protein, E2F1, was examined in clear cell renal cell carcinomas (ccRCC). We show that the overexpression of E2F1 is accompanied by the significant increase of the mean amounts of cyclin E mRNA, as well as of total cyclin E protein and its low molecular weight forms in cancer tissues as compared to peritumoral controls. A significant increase of the mean amount of total cyclin E was found in peritumoral tissues compared to cancer-free kidneys, suggesting that cancer cells might secrete factors having a profound influence on the metabolism of neighbouring tissues. A significant, positive correlations between E2F1 protein and total cyclin E mRNA, as well as between E2F1 protein and full length cyclin E protein were found in cancer-free kidneys and in peritumoral tissues, but not in ccRCCs. The overexpression of cyclin E positively correlated with the decreasing degree of tumor differentiation, implicating a role for cyclin E in the promotion of tumorigenesis.

  11. Evaluation of bone marrow by opposed phase T1-weighted images and enhanced MR imaging

    Energy Technology Data Exchange (ETDEWEB)

    Amano, Yasuo; Tanabe, Yoshihiro; Miyashita, Tsuguhiro; Hayashi, Hiromitsu; Horiuchi, Junichi; Nomura, Takeo; Kumazaki, Tatsuo (Nippon Medical School, Tokyo (Japan))

    1994-09-01

    We investigated bone marrow in a control group, cases of aplastic anemia and post-irradiation patients by examining T1-weighted (T1W1), short T1 inversion recovery (STIR), opposed phase T1W1 (op-T1W1) and Gd-DTPA enhanced op-T1W1 images obtained by 0.5 T MRI. Bone marrow was classified into four types based on MR findings. Normal marrow showed low intensity on op-T1W1 and STIR images without enhancement (I). Fatty marrow, which showed high intensity on T1W1 and op-T1W1 images was observed in aplastic anemia and post-irradiation patients (II). Hematopoietic marrow (III) showed low intensity on op-T1W1 and enhanced, while active hematopoietic marrow (IV) revealed high intensity on both STIR and op-T1W1 images and was enhanced following Gd-DTPA infusion. Aplastic anemia of moderate grade included types II, III and IV. Enhanced MR was needed to differentiate between types I and III since both types showed low intensity on op-T1W1 images. Furthermore, type IV was considered as hyperplastic compared with type III. Enhanced MR and op-T1W1 images were useful in evaluating hematopoiesis of bone marrow. (author).

  12. SUMO modification of Stra13 is required for repression of cyclin D1 expression and cellular growth arrest.

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    Yaju Wang

    Full Text Available Stra13, a basic helix-loop-helix (bHLH transcription factor is involved in myriad biological functions including cellular growth arrest, differentiation and senescence. However, the mechanisms by which its transcriptional activity and function are regulated remain unclear. In this study, we provide evidence that post-translational modification of Stra13 by Small Ubiquitin-like Modifier (SUMO dramatically potentiates its ability to transcriptionally repress cyclin D1 and mediate G(1 cell cycle arrest in fibroblast cells. Mutation of SUMO acceptor lysines 159 and 279 located in the C-terminal repression domain has no impact on nuclear localization; however, it abrogates association with the co-repressor histone deacetylase 1 (HDAC1, attenuates repression of cyclin D1, and prevents Stra13-mediated growth suppression. HDAC1, which promotes cellular proliferation and cell cycle progression, antagonizes Stra13 sumoylation-dependent growth arrest. Our results uncover an unidentified regulatory axis between Stra13 and HDAC1 in progression through the G(1/S phase of the cell cycle, and provide new mechanistic insights into regulation of Stra13-mediated transcriptional repression by sumoylation.

  13. A plant-specific cyclin-dependent kinase is involved in the control of G2/M progression in plants.

    Science.gov (United States)

    Porceddu, A; Stals, H; Reichheld, J P; Segers, G; De Veylder, L; Barroco, R P; Casteels, P; Van Montagu, M; Inzé, D; Mironov, V

    2001-09-28

    Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G(2)/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G(2)/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G(2)/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G(2)/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G(2)/M progression in plants.

  14. Knocking-down of CREPT prohibits the progression of oral squamous cell carcinoma and suppresses cyclin D1 and c-Myc expression.

    Science.gov (United States)

    Ma, Juntao; Ren, Yipeng; Zhang, Lei; Kong, Xiangpan; Wang, Tong; Shi, Yueyi; Bu, Rongfa

    2017-01-01

    As a regulator essential for many cell cycle-related proteins, the robust expression of Cell cycle-Related and Expression-elevated Protein in Tumor (CREPT) implicates a poor diagnosis of endoderm and mesoderm-derived tumors. Whether CREPT plays the same role in the tumorigenesis derived from ectodermal tissues remains elusive. To explore the role of CREPT in ectoderm-derived tumors, cells from 7oral squamous cell carcinoma (OSCC) lines and 84clinical OSCC samples were exploited in this study. Quantitative PCR, Western blot assay and immunohistochemistry were applied in the evaluation of CREPT, cyclin D1 and c-Myc expression. Knocking-down of CREPT was performed by lentivirus delivering specific shRNA of CREPT. The effects of CREPT on OSCC were examined by cell proliferation, colony formation, apoptosis, cell migration and xenograft implantation experiments. Compared with human normal oral keratinocytes, OSCC cell lines showed a significantly elevated expression of CREPT in both mRNA and protein levels. Consistently, samples from OSCC patients also exhibited a noticeably stronger CREPT expression than the noncancerous samples. In contrast, knocking down of CREPT in OSCC cell lines significantly reduced proliferation, colony formation and migration as well as the expression of cyclin D1 and c-Myc, but promoted apoptosis. Statistical analysis also suggested that CREPT expression was significantly correlated with the T and N classification of OSCC. Furthermore, CAL27 mouse xenograft model confirmed that down-regulation of CREPT prohibited cyclin D1 and c-Myc expression, through which decreased the in vivo tumor growth, but increased the survival ratio of hosts. In OSCC cell lines, up-regulated CREPT expression enhanced cell proliferation, migration and cell cycle as well as promoted cyclin D1 and c-Myc expression as it did in endoderm and mesoderm-origin tumors. Our study strongly suggests that CREPT could be used as a marker for the OSCC prognosis and might work as a

  15. Cigarette smoke extract alters the cell cycle via the phospholipid transfer protein/transforming growth factor-β1/CyclinD1/CDK4 pathway.

    Science.gov (United States)

    Chai, Xue-Min; Li, You-Lun; Chen, Hong; Guo, Shu-Liang; Shui, Li-Li; Chen, Ya-Juan

    2016-09-05

    This study was aimed to investigate the effect of phospholipid transfer protein (PLTP) on cigarette smoke extract (CSE)-induced alteration of the cell cycle and the possible mechanism. Male Wistar rats and the rat alveolar epithelial cell line (RLE-6TN) were exposed to normal air or different concentrations of CSE. Then PLTP siRNA was transfected into cells and an inhibitor of transforming growth factor-β1 (TGF-β1) was administered prior to CSE exposure. Histological changes and cell cycle stage were recorded, as were the expression levels of PLTP, TGF-β1, CyclinD1 and CDK4. Resulting morphological changes included diffuse interstitial substance incrassation and elevated alveolar rupturing. Flow cytometry analysis revealed an increase in the number of cells in the G1 phase in a time- and dose-related manner. Both PLTP and TGF-β1 were up-regulated at protein and mRNA levels, whereas CyclinD1 and CDK4 expression was down-regulated after CSE exposure. Furthermore, PLTP siRNA significantly suppressed CSE-induced TGF-β1 expression, resulting in up-regulation of CyclinD1 and CDK4, but the TGF-β1 inhibitor was not able to abrogate CSE-induced PLTP over-expression. In conclusion, PLTP may operate upstream of the TGF-β1/CyclinD1/CDK4 pathway and may mediate the CSE-induced G1 arrest in RLE-6TN cells. Our work provides some new insight into the relation between PLTP and cell cycle progression. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. 细胞周期蛋白在喉癌组织中的表达及意义%Expression and Significance of Cyclins in Laryngeal Carcinomas

    Institute of Scientific and Technical Information of China (English)

    刘馨莲; 殷舞; 李淑蓉; 孙静

    2011-01-01

    目的 探讨喉癌组织中细胞周期蛋白(Cyclin D1、Cyclin A、Cyclin B1、Cyclin E)的表达规律及其与喉癌临床病理参数的关系.方法 采用免疫组化技术,检测Cyclin D1、Cyclin A、Cyclin B1、Cyclin E在67例喉癌组织和32例癌旁正常组织中的表达情况.结果 Cyclin D1、Cyclin A、Cyclin B1、Cyclin E在癌旁正常组织中呈低表达.随着喉癌分化程度的降低,Cyclin D1、Cyclin A、Cyclin B1、Cyclin E表达阳性率明显升高,高分化者、中分化者与低分化者比较有显著性差异(P<0.05).结论 通过免疫组织化学法分析细胞周期的数据,有利于肿瘤分级,且细胞周期数据与预后相关.%Objective To investigate the expression and significance of Cyclin Dl ,Cyclin A,Cyclin B1 and Cyclin E in laryngeal carcinomas, and study the correlation between parameters and clinicopathological features. Methods Expression of Cyclin Dl,Cyclin A,Cyclin Bl and Cyclin E were analyzed by immunohistochemistry staining in 67 cases of laryngeal carcinomas and 32 peri-cancer normal larynx tissues. Results Cyclin Dl, Cyclin A, Cyclin Bl and Cyclin E were low Expressed in pericancer normal larynx tissues. With the decreasing of pathological classification, the expression of Cyclin Dl、Cyclin A、Cyclin Bl  and Cyclin E in laryngeal carcinomas increased accordingly. The difference between group highly-differentiated、moderately-differentiated and poorly-differentiated were significant (P < 0. 05 ). Conclusion Cell cycle phase can be analyzed by immunohistochemical stain,and it is beneficial for tumor classification and helpful to predict the prognosis.

  17. A minicircuitry of microRNA-9-1 and RUNX1-RUNX1T1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia.

    Science.gov (United States)

    Fu, Lin; Shi, Jinlong; Liu, Anqi; Zhou, Lei; Jiang, Mengmeng; Fu, Huaping; Xu, Keman; Li, Dandan; Deng, Ailing; Zhang, Qingyi; Pang, Yifan; Guo, Yujie; Hu, Kai; Zhou, Jiansuo; Wang, Yapeng; Huang, Wenrong; Jing, Yu; Dou, Liping; Wang, Lili; Xu, Kailin; Ke, Xiaoyan; Nervi, Clara; Li, Yonghui; Yu, Li

    2017-02-01

    MicroRNA-9-1(miR-9-1) plays an important role in the mechanism that regulates the lineage fate of differentiating hematopoietic cells. Recent studies have shown that miR-9-1 is downregulated in t (8; 21) AML. However, the pathogenic mechanisms underlying miR-9-1 downregulation and the RUNX1-RUNX1T1 fusion protein, generated from the translocation of t (8; 21) in AML, remain unclear. RUNX1-RUNX1T1 can induce leukemogenesis through resides in and functions as a stable RUNX1-RUNX1T1-containing transcription factor complex. In this study, we demonstrate that miR-9-1 expression increases significantly after the treatment of RUNX1-RUNX1T1 (+) AML cell lines with decitabine (a DNMT inhibitor) and trichostatin A (an HDAC inhibitor). In addition, we show that RUNX1-RUNX1T1 triggers the heterochromatic silencing of miR-9-1 by binding to RUNX1-binding sites in the promoter region of miR-9-1 and recruiting chromatin-remodeling enzymes, DNMTs, and HDACs, contributing to hypermethylation of miR-9-1 in t (8; 21) AML. Furthermore, because RUNX1, RUNX1T1, and RUNX1-RUNX1T1 are all regulated by miR-9-1, the silencing of miR-9-1 enhances the oncogenic activity of these genes. Besides, overexpression of miR-9-1 induces differentiation and inhibits proliferation in t (8; 21) AML cell lines. In conclusion, our results indicate a feedback circuitry involving miR-9-1 and RUNX1-RUNX1T1, contributing to leukemogenesis in RUNX1-RUNX1T1 (+) AML cell lines. © 2016 UICC.

  18. Magnetic Resonance Water Proton Relaxation in Protein Solutions and Tissue: T1ρ Dispersion Characterization

    Science.gov (United States)

    Chen, Enn-Ling; Kim, Raymond J.

    2010-01-01

    Background Image contrast in clinical MRI is often determined by differences in tissue water proton relaxation behavior. However, many aspects of water proton relaxation in complex biological media, such as protein solutions and tissue are not well understood, perhaps due to the limited empirical data. Principal Findings Water proton T1, T2, and T1ρ of protein solutions and tissue were measured systematically under multiple conditions. Crosslinking or aggregation of protein decreased T2 and T1ρ, but did not change high-field T1. T1ρ dispersion profiles were similar for crosslinked protein solutions, myocardial tissue, and cartilage, and exhibited power law behavior with T1ρ(0) values that closely approximated T2. The T1ρ dispersion of mobile protein solutions was flat above 5 kHz, but showed a steep curve below 5 kHz that was sensitive to changes in pH. The T1ρ dispersion of crosslinked BSA and cartilage in DMSO solvent closely resembled that of water solvent above 5 kHz but showed decreased dispersion below 5 kHz. Conclusions Proton exchange is a minor pathway for tissue T1 and T1ρ relaxation above 5 kHz. Potential models for relaxation are discussed, however the same molecular mechanism appears to be responsible across 5 decades of frequencies from T1ρ to T1. PMID:20052404

  19. SirT1 is required in the male germ cell for differentiation and fecundity in mice

    Science.gov (United States)

    Bell, Eric L.; Nagamori, Ippei; Williams, Eric O.; Del Rosario, Amanda M.; Bryson, Bryan D.; Watson, Nicki; White, Forest M.; Sassone-Corsi, Paolo; Guarente, Leonard

    2014-01-01

    Sirtuins are NAD+-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis. PMID:25142464

  20. Deletion of glycine transporter 1 (GlyT1) in forebrain neurons facilitates reversal learning: enhanced cognitive adaptability?

    Science.gov (United States)

    Singer, Philipp; Boison, Detlev; Möhler, Hanns; Feldon, Joram; Yee, Benjamin K

    2009-10-01

    Local availability of glycine near N-methyl-D-aspartate receptors (NMDARs) is partly regulated by neuronal glycine transporter 1 (GlyT1), which can therefore modulate NMDAR function because binding to the glycine site of the NMDAR is necessary for channel activation. Disrupting GlyT1 in forebrain neurons has been shown to enhance Pavlovian conditioning and object recognition memory. Here, the authors report that the same genetic manipulation facilitated reversal learning in the water maze test of reference memory, but did not lead to any clear improvement in a working memory version of the water maze test. Facilitation in a nonspatial discrimination reversal task conducted on a T maze was also observed, supporting the conclusion that forebrain neuronal GlyT1 may modulate the flexibility in (new) learning and relevant mnemonic functions. One possibility is that these phenotypes may reflect reduced susceptibility to certain forms of proactive interference. This may be relevant for the suggested clinical application of GlyT1 inhibitors in the treatment of cognitive deficits, including schizophrenia, which is characterized by cognitive inflexibility in addition to the positive symptoms of the disease.

  1. Influence of BMSCs-derived exosome on proliferation and invasion of mouse breast cancer cells 4 T1 and the mechanism%BMSCs 来源的外泌体对小鼠乳腺癌细胞4 T1增殖、侵袭的影响及机制探讨

    Institute of Scientific and Technical Information of China (English)

    王丹丹; 陈建中; 亢春彦

    2015-01-01

    inhibitor of PI3K/Akt signaling pathway) group.4T1+vehicle group was added with 400μL se-rum-free medium, 4T1+exosome group was added with 400 μL serum-free medium containing 400 μg/mL exosome and 4T1+exosome+Y294002 group was added with 400 μL serum-free medium containing 400 μg/mL exosome and 5 μg/mL Y294002.We used the MTT, cell scratch test and Western blotting to examine the effect of BMSCs-derived exosome on the proliferation, migration and invasion abilities as well as the PI3K/Akt signaling pathway of 4T1 cells.Results The in-hibition rates of cell proliferation of 4T1+vehicle group, 4T1+exosome group and 4T1+exosome+Y294002 group were 0.713%±0.05%, 0.401%±0.03%and 0.459%±0.80%, respectively.Significant difference was found between 4T1+exosome group and 4T1+vehicle group, 4T1+exosome+Y294002 group (all P<0.05).The cell migration distance of 4T1+vehicle group, 4T1+exosome group and 4T1+exosome+Y294002 group was (388.0 ±36.1), (295.0 ± 34.2) and (275.0 ±63.5) μm, respectively.Significant difference was found between 4T1+exosome group and 4T1+vehicle group, 4T1+exosome+Y294002 group (all P<0.05).The OD values of p-AKT and β-catenin of 4T1+exo-some group were 0.3 ±0.11 and 0.3 ±0.08, they were 1.1 ±0.41 and 0.7 ±0.08 in 4T1+vehicle group, 0.4 ±0.13 and 0.3 ±0.07 in the 4T1+exosome+Y294002 group.Significant difference was found between 4T1+exosome group and 4T1+vehicle group, 4T1+exosome+Y294002 group (all P<0.05).Conclusion BMSCs-derived exosome can promote the proliferation, migration and invasion abilities of mouse breast cancer cells 4T1, which may be related to the up-regulation of PI3K/Akt signaling pathway.

  2. The expression of cyclin-dependent kinase inhibitors p15, p16, p21, and p27 during ovarian follicle growth initiation in the mouse

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    Bayrak Aykut

    2003-05-01

    Full Text Available Abstract Background Cyclins regulate the cell cycle in association with cyclin dependent kinases (CDKs. CDKs are under inhibitory control of cyclin dependent kinase inhibitors (CDKIs. Method In this study we tested the expression of CDKIs p15, p16, p21 and p27 by immunohistochemistry to determine the role of CDKIs in the initiation of primordial follicle growth. Ovaries were collected from 60-day-old cycling B6D2F1/J mice (n = 16. Results Expression of p15, p16, p21 and p27 did not vary in granulosa and theca cells by the follicle stage. However, p16 staining was stronger (++ in the oocytes of all primordial, and 57.4 ± 3.1% of primary follicles compared to the remaining primary and more advanced follicles (+. Interestingly, primary follicles with weaker (+ oocyte staining for p16 had significantly larger mean follicle diameter compared to the primary and primordial follicles with stronger (++ oocyte staining (55.6 ± 2.1 vs. 32.0 ± 1.0 and 26.5 ± 0.7 μm, respectively, p Conclusions These preliminary findings suggest that the initiation of oocyte growth, which seems to lead follicle growth, is associated with diminished p16 expression in the mouse ovary. Further studies are needed to investigate the factors that regulate the expression of p16 in the oocyte, which might also govern the initiation of primordial follicle growth.

  3. DYRK1A-mediated Cyclin D1 Degradation in Neural Stem Cells Contributes to the Neurogenic Cortical Defects in Down Syndrome

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    Sònia Najas

    2015-02-01

    Full Text Available Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome, this is associated with a deficit in cortical neurons that arises during prenatal development. However, the pathogenic mechanisms that cause this deficit have not yet been defined. Here we show that the human DYRK1A kinase on chromosome 21 tightly regulates the nuclear levels of Cyclin D1 in embryonic cortical stem (radial glia cells, and that a modest increase in DYRK1A protein in transgenic embryos lengthens the G1 phase in these progenitors. These alterations promote asymmetric proliferative divisions at the expense of neurogenic divisions, producing a deficit in cortical projection neurons that persists in postnatal stages. Moreover, radial glial progenitors in the Ts65Dn mouse model of Down syndrome have less Cyclin D1, and Dyrk1a is the triplicated gene that causes both early cortical neurogenic defects and decreased nuclear Cyclin D1 levels in this model. These data provide insights into the mechanisms that couple cell cycle regulation and neuron production in cortical neural stem cells, emphasizing that the deleterious effect of DYRK1A triplication in the formation of the cerebral cortex begins at the onset of neurogenesis, which is relevant to the search for early therapeutic interventions in Down syndrome.

  4. Transcriptome analysis of tetraploid cells identifies cyclin D2 as a facilitator of adaptation to genome doubling in the presence of p53.

    Science.gov (United States)

    Potapova, Tamara A; Seidel, Christopher W; Box, Andrew C; Rancati, Giulia; Li, Rong

    2016-10-15

    Tetraploidization, or genome doubling, is a prominent event in tumorigenesis, primarily because cell division in polyploid cells is error-prone and produces aneuploid cells. This study investigates changes in gene expression evoked in acute and adapted tetraploid cells and their effect on cell-cycle progression. Acute polyploidy was generated by knockdown of the essential regulator of cytokinesis anillin, which resulted in cytokinesis failure and formation of binucleate cells, or by chemical inhibition of Aurora kinases, causing abnormal mitotic exit with formation of single cells with aberrant nuclear morphology. Transcriptome analysis of these acute tetraploid cells revealed common signatures of activation of the tumor-suppressor protein p53. Suppression of proliferation in these cells was dependent on p53 and its transcriptional target, CDK inhibitor p21. Rare proliferating tetraploid cells can emerge from acute polyploid populations. Gene expression analysis of single cell-derived, adapted tetraploid clones showed up-regulation of several p53 target genes and cyclin D2, the activator of CDK4/6/2. Overexpression of cyclin D2 in diploid cells strongly potentiated the ability to proliferate with increased DNA content despite the presence of functional p53. These results indicate that p53-mediated suppression of proliferation of polyploid cells can be averted by increased levels of oncogenes such as cyclin D2, elucidating a possible route for tetraploidy-mediated genomic instability in carcinogenesis.

  5. Cyclin-dependent kinase inhibitors inspired by roscovitine: purine bioisosteres.

    Science.gov (United States)

    Jorda, Radek; Paruch, Kamil; Krystof, Vladimír

    2012-01-01

    Roscovitine is a synthetic inhibitor of cyclin-dependent kinases that is currently undergoing clinical trials as a candidate drug for some oncological indications. Its discovery prompted many research teams to further optimize its structure or to initiate their own related but independent studies. This article reviews known roscovitine bioisosteres that have been prepared as CDK inhibitors using different core heterocycles. The individual bioisostere types have been described and explored to a different extent, which complicates direct comparisons of their biochemical activity - only six direct analogs containing different purine bioisosteres have been prepared and evaluated side by side with roscovitine. Only four types of bioisosteres have demonstrated improved biological properties, namely pyrazolo[ 1,5-a]-1,3,5-triazines, pyrazolo[1,5-a]pyrimidines, pyrazolo[1,5-a]pyridines and pyrazolo[4,3-d]pyrimidines.

  6. Cdk11-CyclinL Controls the Assembly of the RNA Polymerase II Mediator Complex

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    Julie Drogat

    2012-11-01

    Full Text Available The large Mediator (L-Mediator is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1 complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex.

  7. Cdk11-cyclinL controls the assembly of the RNA polymerase II mediator complex.

    Science.gov (United States)

    Drogat, Julie; Migeot, Valérie; Mommaerts, Elise; Mullier, Caroline; Dieu, Marc; van Bakel, Harm; Hermand, Damien

    2012-11-29

    The large Mediator (L-Mediator) is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator) and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1) complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex.

  8. Cyclin-dependent kinase 5, a node protein in diminished tauopathy: a systems biology approach

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    John Fredy Castro-Alvarez

    2014-09-01

    Full Text Available Alzheimer's disease (AD is the most common cause of dementia worldwide. One of the main pathological changes that occurs in AD is the intracellular accumulation of hyperphosphorylated Tau protein in neurons. Cyclin-dependent kinase 5 (CDK5 is one of the major kinases involved in Tau phosphorylation, directly phosphorylating various residues and simultaneously regulating various substrates such as kinases and phosphatases that influence Tau phosphorylation in a synergistic and antagonistic way. It remains unknown how the interaction between CDK5 and its substrates promotes Tau phosphorylation, and systemic approaches are needed that allow an analysis of all the proteins involved. In this review, the role of the CDK5 signaling pathway in Tau hyperphosphorylation is described, an in silico model of the CDK5 signaling pathway is presented. The relationship among these theoretical and computational models shows that the regulation of Tau phosphorylation by PP2A and GSK3β is essential under basal conditions and also describes the leading role of CDK5 under excitotoxic conditions, where silencing of CDK5 can generate changes in these enzymes to reverse a pathological condition that simulates AD.

  9. Different Patterns of Cyclin D1/CDK4-E2F-1/4 Pathways in Human Embryo Lung Fibroblasts Treated by Benzo[a]pyrene at Different Doses1

    Institute of Scientific and Technical Information of China (English)

    MENG YE; BING-CI LIU; XIANG-LIN SHI; BAO-RONG YOU; HONG-JU DU; XIAO-WEI JIA; FU-HAI SHEN

    2008-01-01

    Objective To investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]P. Methods Human embryo lung fibroblasts(HELFs)were treated with 2 μmol/L or 100 μmol/L B[a]P which were provided with some characteristics of transformed cells (T-HELFs).Cyclin D1,CDK4 and E2F-1/4 expressions were determined by Westem blotting.Flow cytometry was used to detect the distribution of cell cycle.Results After B[a]P treatment,the proportion of the first gap(G1)phase cells decreased.CDK4 and E2F-4 expression did not change significantly.In 2 μmol/L treated cells,a marked overexpression of cyclin D1 and E2F-1 was observed.However,in T-HELFs overexpression was limited to cyclin D1 only,and no overexpression of E2F-1 was observed.The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs (A-D1 and A-K4)and T-HELFs(T-A-D1 and T-A-K4).Atier 2 μmol/L B[a]P treatment,overexpression of E2F-1 was attenuated in A-D1,and E2F-4 expression was decreased significantly in A-K4.In T-A-D1 and T-A-K4,E2F-4 expression was increased significantly,compared with T-HELFs.The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4.Conclusions Cyclin D1/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment.In HELFs treated with 2 μmol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression;however,in HELFs treated with 100 μmol/L B[a]P,both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.

  10. Systematic review on the surgical treatment for T1 gallbladder cancer

    Institute of Scientific and Technical Information of China (English)

    Seung Eun Lee; Jin-Young Jang; Chang-Sup Lim; Mee Joo Kang; Sun-Whe Kim

    2011-01-01

    AIM: To evaluate the efficacy of simple and extended cholecystectomy for mucosa (T1a) or muscularis (T1b)gallbladder (GB) cancer.METHODS: Original studies on simple and extended cholecystectomy for T1a or T1b GB cancer were searched from MEDLINE (PubMed), Cochrane Library, EMBase,and CancerLit using the search terms of GB, cancer/carcinoma/tumor/neoplasm.RESULTS: Twenty-nine out of the 2312 potentially relevant publications met the eligibility criteria. Of the 1266patients with GB cancer included in the publications,706 (55.8%) and 560 (44.2%) had T1a and T1b GB cancer, respectively. Simple cholecystectomy for T1a and T1b GB cancer was performed in 590 (83.6%) and 375 (67.0%) patients, respectively (P < 0.01). In most series, the treatment of choice was simple cholecystectomy for T1a GB cancer patients with a 5-year survival rate of 100%. Lymph node metastasis was detected in 10.9% of the T1b GB cancer patients and in 1.8% of the T1a GB cancer patients, respectively (P < 0.01).Eight patients (1.1%) with T1a GB cancer and 52 patients (9.3%) with T1b GB cancer died of recurrent GB cancer (P < 0.01).CONCLUSION: Simple cholecystectomy represents the adequate treatment of T1a GB cancer. There is no definite evidence that extended cholecystectomy is advantageous over simple cholecystectomy for T1b GB cancer.

  11. High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

    DEFF Research Database (Denmark)

    Kongsfelt, Iben Boutrup; Byskov, Kristina; Pedersen, Lasse Ebdrup

    2014-01-01

    The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells......, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing...... the lowest PiT1 mRNA levels. The mechanism behind the role of PiT1 in increased cell proliferation is not known. We, however, found that compared to control cells, cultures of NIH3T3 cells overexpressing PiT1 upon seeding showed increased cell number after 24 h and had shifted more cells from G0/G1 to S+G2/M...

  12. Ultra-low field T1 vs. T1rho at 3T and 7T: study of rotationally immobilized protein gels and animal brain tissues

    Science.gov (United States)

    Dong, Hui; Inglis, Ben; Barr, Ian; Clarke, John

    2015-03-01

    Clinical magnetic resonance imaging (MRI) machines operating in static fields of typically 1.5 T or 3 T can capture information on slow molecular dynamics utilizing the so-called T1rho technique. This technique, in which a radiofrequency (RF) spin-lock field is applied with microtesla amplitude, has been used, for example, to determine the onset time of stroke in studies on rats. The long RF pulse, however, may exceed the specific absorption rate (SAR) limit, putting subjects at risk. Ultra-low-field (ULF) MRI, based on Superconducting Quantum Interference Devices (SQUIDs), directly detects proton signals at a static magnetic field of typically 50-250 μT. Using our ULF MRI system with adjustable static field of typically 55 to 240 μT, we systematically measured the T1 and T2 dispersion profiles of rotationally immobilized protein gels (bovine serum albumin), ex vivo pig brains, and ex vivo rat brains with induced stroke. Comparing the ULF results with T1rho dispersion obtained at 3 T and 7 T, we find that the degree of protein immobilization determines the frequency-dependence of both T1 and T1rho. Furthermore, T1rho and ULF T1 show similar results for stroke, suggesting that ULF MRI may be used to image traumatic brain injury with negligible SAR. This research was supported by the Henry H. Wheeler, Jr. Brain Imaging Center and the Donaldson Trust.

  13. T1rho mapping of entire femoral cartilage using depth- and angle-dependent analysis

    Energy Technology Data Exchange (ETDEWEB)

    Nozaki, Taiki; Kaneko, Yasuhito; Yu, Hon J.; Yoshioka, Hiroshi [University of California Irvine, Department of Radiological Sciences, Orange, CA (United States); Kaneshiro, Kayleigh [University of California Irvine, School of Medicine, Irvine, CA (United States); Schwarzkopf, Ran [University of California Irvine, Department of Orthopedic Surgery, Irvine, CA (United States); Hara, Takeshi [Gifu University Graduate School of Medicine, Department of Intelligent Image Information, Division of Regeneration and Advanced Medical Sciences, Gifu (Japan)

    2016-06-15

    To create and evaluate normalized T1rho profiles of the e