141 resultados para cell cycle checkpoint
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Background/Aims: The chromosome locus 3p21.3 is a
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Cell division depends on the fine control of both microtubule dynamics and microtubule organisation. The microtubule bundling protein MAP65 is a 'midzone MAP' essential for the integrity of the anaphase spindle and cell division. Arabidopsis thaliana MAP65-1 (AtMAP65-1) binds and bundles microtubules by forming 25 nm cross-bridges. Moreover, as AtMAP65-1 bundles microtubules in interphase, anaphase and telophase but does not bind microtubules in prophase or metaphase, its activity through the cell cycle must be under tight control. Here we show that AtMAP65-1 is hyperphosphorylated during prometaphase and metaphase and that CDK and MAPK are involved in this phosphorylation. This phosphorylation inhibits AtMAP65-1 activity. Expression of nonphosphorylatable AtMAP65-1 has a negative effect on mitotic progression resulting in excessive accumulation of microtubules in the metaphase spindle midzone causing a delay in mitosis. We conclude that normal metaphase spindle organisation and the transition to anaphase is dependent on inactivation of AtMAP65-1.
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An understanding of the mechanisms underlying the development of resistance to chemotherapy treatment is a gateway to the introduction of novel therapies and improved outcomes for women presenting with ovarian cancer (OC). The desired apoptotic death post-chemotherapy depends on an intact and fully functioning cell cycle machinery.
In this study we demonstrate that stable expression of miR-433 renders OC cells more resistant to paclitaxel treatment. Interestingly, only cells with the highest miR-433 survived paclitaxel suggesting the possible role of miR-433 in cancer recurrence. Importantly, for the first time we demonstrate that miR 433 induces cellular senescence, exemplified by a flattened morphology, the downregulation of phosphorylated Retinoblastoma (p Rb) and increased β galactosidase activity. Surprisingly, miR 433 induced senescence was independent of two well recognised senescent drivers: p21 and p16. Further in silico analysis followed by in vitro experiments identified CKD6 as a novel miR-433 target gene possibly explaining the observed p21 and p16-independent induction of cellular senescence. Another in silico identified miR-433 target gene was CDC27, a protein involved in the regulation of the cell cycle during mitosis. We demonstrate that the overexpression of pre-miR-433 leads to the downregulation of CDC27 in vitro revealing a novel interaction between miR-433 and CDC27, an integral cell cycle regulating protein.
Interestingly, miR-433 expressing cells also demonstrated an ability to impact their tumour microenvironment. We show that miR-433 is present in exosomes released from miR-433 overexpressing and high miR-433 naïve cells. Moreover, growth condition media (GCM) harvested from cells with high miR-433 have higher levels of IL-6 and IL-8, two key cytokines involved in the senescence associated secretory phenotype (SASP). Importantly, GCM from miR-433-enriched cells repressed the growth of co-cultured cells with initial studies showing a GCM-dependent induction of chemoresistance.
In conclusion, data in this study highlights how the aberrant expression miR-433 contributes to chemoresistance in OC cells. We postulate that standard chemotherapy, particularly paclitaxel, used to treat women with OC may have an attenuated ability to kill cells harbouring increased levels of miR-433, allowing for a subsequent chemoresistant phenotype post-therapy.
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Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies.
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BackgroundThe recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear.Design and MethodsThe expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays.ResultsWe found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples.ConclusionsIn conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival.
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The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle ('Cell Cycle Common'), versus those that were specifically enriched in a subset of cell cycle phases ('Phase Restricted'). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide desaturase 1 was identified as an AR-regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention.
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Background Ten to twenty per cent of breast tumours exhibit a basallike genetic profile and these tumours carry a poor prognosis. Breast tumours which contain germline mutations for BRCA1 commonly exhibit a molecular profile similar to basal breast tumours. BRCA1 is a tumour suppressor gene which is mutated in up to 5–10% of breast cancer cases and is involved in multiple cellular processes including DNA damage control, cell cycle checkpoint control, apoptosis, ubiquitination and transcriptional regulation.
Methods Microarray-based profiling was carried out using the HCC1937EV and HCC1937BR breast cancer cell lines. Basal gene and protein expression levels were analysed by qRT-PCR and western blotting. ChIP analyses were performed and demonstrated that BRCA1 regulates basal gene expression through a transcriptional mechanism involving c-myc.
Results We have previously carried out microarray-based expression profiling to examine differences in gene expression when BRCA1 is reconstituted in BRCA1 mutated HCC1937 breast cancer cells. We observed that p-cadherin and the cytokeratin 5 and cytokeratin 17 genes, which are strongly correlated with the basal phenotype, are differentially expressed when BRCA1 is reconstituted. In addition, qRT-PCR and ChIP analysis of BRCA1 reconstituted cells show that BRCA1 represses the expression of these basal genes by a transcriptional mechanism. Furthermore, abrogation of endogenous BRCA1 protein in the T47D cell line using siRNA results in reexpression of these basal genes, suggesting that BRCA1 expression levels may be important in basal gene expression. We have also demonstrated that BRCA1 is physically associated with the promoter regions of basal genes through an association with c-myc. Consequently, we have confirmed that siRNA inhibition of c-myc in T47D cells results in re-expression of these genes.
Conclusions Our results suggest that BRCA1 is involved in the transcriptional regulation of genes associated with the basal phenotype and that BRCA1 controls basal gene expression through a transcriptional mechanism involving c-myc. Further work is now concentrating on defining the relationship between BRCA1 and basal gene expression and how this may affect clinical responses to breast cancer chemotherapy.
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Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using GO-G, confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser(15) (p53(Ser15)), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologicafly distinct subpool of p53(Ser15) is ATM dependent and resistant to 26S-proteasomal degradation. p53(Ser15) colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear micro-beam irradiation studies confirm p53 S,,15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser(15) or Ser(18) phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21(WAF) decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis. (Cancer Res 2005; 65(23): 10810-21).
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The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-ß chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-ß, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-ß from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-ß exchange from heterochromatin, promoting DNA repair.
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Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107–Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities
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BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mRNA splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1-related protein complexes and their roles in maintaining genomic stability and tumour suppression.