RABL6A, a novel RAB-like protein, controls centrosome amplification and chromosome instability in primary fibroblasts.
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ABSTRACT:
SUBMITTER: Zhang X
PROVIDER: S-EPMC3839920 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundIncorrect segregation of whole chromosomes or parts of chromosome leads to aneuploidy commonly observed in cancer. The correct centrosome duplication, assuring assembly of a bipolar mitotic spindle, is essential for chromosome segregation fidelity and preventing aneuploidy. Alteration of p53 and pRb functions by expression of HPV16-E6 and E7 oncoproteins has been associated with centrosome amplification. However, these last findings could be the result of targeting cellular proteins in addition to pRb by HPV16-E7 oncoprotein. To get a more detailed picture on the role of pRb in chromosomal instability and centrosome amplification, we analyzed the effects of the acute loss of retinoblastoma gene function in primary conditional Rb deficient mouse embryonic fibroblasts (MEFs). Moreover, since pRb is a transcriptional repressor, microarray analysis was done on pRb-competent and pRb-deficient MEFs to evaluate changes in expression of genes for centrosome homeostasis and for correct mitosis.ResultsAcute loss of pRb induces centrosome amplification and aneuploidy in the vast majority of cells analyzed. A time course analysis shows a decrease of cells with amplified centrosomes after 40 days from the adenoviral infection. At this time only 12% of cells still show amplified centrosomes. Interestingly, cells with pRb constitutive loss show a similar percentage of cells with amplified centrosomes. DNA-Chip analyses in MEFs wt (mock infected) and pRb depleted (Ad-Cre infected) cells reveal differential expression of genes controlling both centrosome duplication and mitotic progression.ConclusionOur findings suggest a direct link between pRb status, centrosome amplification and chromosomal instability, and define specific mitotic genes as targets whose gene expression has to be altered to achieve or maintain aneuploidy.
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Project description:Glioblastoma is characterised by invasive growth and a high degree of radioresistance. Survivin, a regulator of chromosome segregation, is highly expressed and known to induce radioresistance in human gliomas. In this study, we examined the effect of survivin suppression on radiosensitivity in malignant glioma cells, while focusing on centrosome aberration and chromosome instability (CIN). We suppressed survivin by small interfering RNA transfection, and examined the radiosensitivity using a clonogenic assay and a trypan blue exclusion assay in U251MG (p53 mutant) and D54MG (p53 wild type) cells. To assess the CIN status, we determined the number of centrosomes using an immunofluorescence analysis, and the centromeric copy number by fluorescence in situ hybridisation. As a result, the radiosensitisation differed regarding the p53 status as U251MG cells quickly developed extreme centrosome amplification (=CIN) and enhanced the radiosensitivity, while centrosome amplification and radiosensitivity increased more gradually in D54MG cells. TUNEL assay showed that survivin inhibition did not lead to apoptosis after irradiation. This cell death was accompanied by an increased degree of aneuploidy, suggesting mitotic cell death. Therefore, survivin inhibition may be an attractive therapeutic target to overcome the radioresistance while, in addition, proper attention to CIN (centrosome number) is considered important for improving radiosensitivity in human glioma.
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Project description:Earlier studies of invasive breast tumors have shown that 60-80% are aneuploid and approximately 80% exhibit amplified centrosomes. In this study, we investigated the relationship of centrosome amplification with aneuploidy, chromosomal instability, p53 mutation, and loss of differentiation in human breast tumors. Twenty invasive breast tumors and seven normal breast tissues were analyzed by fluorescence in situ hybridization with centromeric probes to chromosomes 3, 7, and 17. We analyzed these tumors for both aneuploidy and unstable karyotypes as determined by chromosomal instability. The results were then tested for correlation with three measures of centrosome amplification: centrosome size, centrosome number, and centrosome microtubule nucleation capacity. Centrosome size and centrosome number both showed a positive, significant, linear correlation with aneuploidy and chromosomal instability. Microtubule nucleation capacity showed no such correlation, but did correlate significantly with loss of tissue differentiation. Centrosome amplification was detected in in situ ductal carcinomas, suggesting that centrosome amplification is an early event in these lesions. Centrosome amplification and chromosomal instability occurred independently of p53 mutation, whereas p53 mutation was associated with a significant increase in centrosome microtubule nucleation capacity. Together, these results demonstrate that independent aspects of centrosome amplification correlate with chromosomal instability and loss of tissue differentiation and may be involved in tumor development and progression. These results further suggest that aspects of centrosome amplification may have clinical diagnostic and/or prognostic value and that the centrosome may be a potential target for cancer therapy.
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Project description:Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic disease predominantly caused by alteration or dysregulation of the PKD1 gene, which encodes polycystin-1 (PC1). The disease is characterized by the progressive expansion of bilateral fluid-filled renal cysts that ultimately lead to renal failure. Individual cysts, even within patients with germline mutations, are genetically heterogeneous, displaying diverse chromosomal abnormalities. To date, the molecular mechanisms responsible for this genetic heterogeneity remain unknown. Using a lentiviral-mediated siRNA expression model of Pkd1 hypomorphism, we show that loss of PC1 function is sufficient to produce centrosome amplification and multipolar spindle formation. These events lead to genomic instability characterized by gross polyploidism and mitotic catastrophe. Following these dramatic early changes, the cell population rapidly converges toward a stable ploidy in which centrosome amplification is significantly decreased, though cytological abnormalities such as micronucleation, chromatin bridges and aneuploidy remain common. In agreement with our in vitro findings, we provide the first in vivo evidence that significant centrosome amplification occurs in kidneys from conditional Pkd1 knockout mice at early and late time during the disease progression as well as in human ADPKD patients. These findings establish a novel function of PC1 in ADPKD pathogenesis and a genetic mechanism that may underlie the intrafamilial variability of ADPKD progression.
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Project description:Infections with Epstein-Barr virus (EBV) are associated with cancer development, and EBV lytic replication (the process that generates virus progeny) is a strong risk factor for some cancer types. Here we report that EBV infection of B-lymphocytes (in vitro and in a mouse model) leads to an increased rate of centrosome amplification, associated with chromosomal instability. This effect can be reproduced with virus-like particles devoid of EBV DNA, but not with defective virus-like particles that cannot infect host cells. Viral protein BNRF1 induces centrosome amplification, and BNRF1-deficient viruses largely lose this property. These findings identify a new mechanism by which EBV particles can induce chromosomal instability without establishing a chronic infection, thereby conferring a risk for development of tumours that do not necessarily carry the viral genome.
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Project description:The role of the alternate G protein-coupled estrogen receptor 1 (GPER1) in colorectal cancer (CRC) development and progression is unclear, not least because of conflicting clinical and experimental evidence for pro- and anti-tumorigenic activities. Here, we show that low concentrations of the estrogenic GPER1 ligands, 17β-estradiol, bisphenol A, and diethylstilbestrol cause the generation of lagging chromosomes in normal colon and CRC cell lines, which manifest in whole chromosomal instability and aneuploidy. Mechanistically, (xeno)estrogens triggered centrosome amplification by inducing centriole overduplication that leads to transient multipolar mitotic spindles, chromosome alignment defects, and mitotic laggards. Remarkably, we could demonstrate a significant role of estrogen-activated GPER1 in centrosome amplification and increased karyotype variability. Indeed, both gene-specific knockdown and inhibition of GPER1 effectively restored normal centrosome numbers and karyotype stability in cells exposed to 17β-estradiol, bisphenol A, or diethylstilbestrol. Thus, our results reveal a novel link between estrogen-activated GPER1 and the induction of key CRC-prone lesions, supporting a pivotal role of the alternate estrogen receptor in colon neoplastic transformation and tumor progression.
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Project description:Aneuploidy, frequently observed in premalignant lesions, disrupts gene dosage and contributes to neoplastic progression. Theodor Boveri hypothesized nearly 100 years ago that aneuploidy was due to an increase in centrosome number (multipolar mitoses) and the resultant abnormal segregation of chromosomes. We performed immunocytochemistry, quantitative immunofluorescence, karyotypic analysis, and time-lapse microscopy on primary human diploid epithelial cells and fibroblasts to better understand the mechanism involved in the production of supernumerary centrosomes (more than two microtubule nucleating bodies) to directly demonstrate that the presence of supernumerary centrosomes in genomically intact cells generates aneuploid daughter cells. We show that loss of p16(INK4a) generates supernumerary centrosomes through centriole pair splitting. Generation of supernumerary centrosomes in human diploid epithelial cells was shown to nucleate multipolar spindles and directly drive production of aneuploid daughter cells as a result of unequal segregation of the genomic material during mitosis. Finally, we demonstrate that p16(INK4a) cooperates with p21 through regulation of cyclin-dependent kinase activity to prevent centriole pair splitting. Cells with loss of p16(INK4a) activity have been found in vivo in histologically normal mammary tissue from a substantial fraction of healthy, disease-free women. Demonstration of centrosome dysfunction in cells due to loss of p16(INK4a) suggests that, under the appropriate conditions, these cells can become aneuploid. Gain or loss of genomic material (aneuploidy) may provide the necessary proproliferation and antiapoptotic mechanisms needed for the earliest stages of tumorigenesis.
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Project description:Cell division cycle 6 (Cdc6) plays key roles in regulating DNA replication, and activation and maintenance of cell cycle check points. In addition, Cdc6 exerts oncogenic properties via genomic instability associated with incomplete DNA replication. This study aimed to examine the effects of Cdc6 on pancreatic cancer (PC) cells. Our results showed that Cdc6 expression was higher in clinical PC specimens (based on analysis of the GEPIA database) and cell lines, and the high Cdc6 expression was associated with poorer survival in The Cancer Genome Atlas-PC cohort. In addition, Cdc6-depleted PC cells significantly inhibited cell proliferation and colony formation, delayed G2/M cell cycle progression, and increased expression of p-histone H3 and cyclin A2 levels. These observations could be explained by Cdc6 depletion leading to multipolar and split spindles via centrosome amplification and microtubule disorganization which eventually increases chromosome missegregation. Furthermore, Cdc6-depleted PC cells showed significantly increased apoptosis, which was consistent with increased caspase-9 and caspase-3 activation. Collectively, our results demonstrated that Cdc6-depleted PC cells are arrested in mitosis and eventually undergo cell death by induced multipolar spindles, centrosome aberrations, microtubule disorganization, and chromosome instability. In conclusion, Cdc6 may be a potential biomarker and therapeutic target for PC.
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Project description:Overexpression of the low molecular weight isoforms (LMW-E) of cyclin E induces chromosome instability; however, the degree to which these tumor-specific forms cause genomic instability differs from that of full-length cyclin E (EL), and the underlying mechanism(s) has yet to be elucidated. Here, we show that EL and LMW-E overexpression impairs the G(2)-M transition differently and leads to different degrees of chromosome instability in a breast cancer model system. First, the most significant difference is that EL overexpression prolongs cell cycle arrest in prometaphase, whereas LMW-E overexpression reduces the length of mitosis and accelerates mitotic exit. Second, LMW-E-overexpressing cells are binucleated or multinucleated with amplified centrosomes, whereas EL-overexpressing cells have the normal complement of centrosomes. Third, LMW-E overexpression causes mitotic defects, chromosome missegregation during metaphase, and anaphase bridges during anaphase, most of which are not detected on EL induction. LMW-E induces additional mitotic defects in cooperation with p53 loss in both normal and tumor cells. Fourth, LMW-E-overexpressing cells fail to arrest in the presence of nocodazole. Collectively, the mitotic defects mediated by LMW-E induction led to failed cytokinesis and polyploidy, suggesting that LMW-E expression primes cells to accrue chromosomal instability by shortening the length of mitosis. Lastly, LMW-E expression in human breast cancer tissues correlates with centrosome amplification and higher nuclear grade. These results suggest that LMW-E overexpression leads to higher centrosome numbers in breast cancer, which is a prerequisite for genomic instability.
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Project description:The centrosome is involved in cytoplasmic microtubule organization during interphase and in mitotic spindle assembly during cell division. Centrosome amplification (abnormal proliferation of centrosome number) has been observed in several types of cancer and in precancerous conditions. Therefore, it is important to elucidate the mechanism of centrosome amplification in order to understand the early stage of carcinogenesis. Primary cells could be used to better understand the early stage of carcinogenesis rather than immortalized cells, which tend to have various genetic and epigenetic changes. Previously, we demonstrated that a poly(ADP-ribose) polymerase (PARP) inhibitor, 3-aminobenzamide (3AB), which is known to be nontoxic and nonmutagenic, could induce centrosome amplification and chromosomal aneuploidy in CHO-K1 cells. In this study, we compared primary mouse embryonic fibroblasts (MEF) and immortalized MEF using 3AB. Although centrosome amplification was induced with 3AB treatment in immortalized MEF, a more potent PARP inhibitor, AG14361, was required for primary MEF. However, after centrosome amplification, neither 3AB in immortalized MEF nor AG14361 in primary MEF caused chromosomal aneuploidy, suggesting that further genetic and/or epigenetic change(s) are required to exhibit aneuploidy. The DNA-damaging agents doxorubicin and γ-irradiation can cause cancer and centrosome amplification in experimental animals. Although doxorubicin and γ-irradiation induced centrosome amplification and led to decreased p27Kip protein levels in immortalized MEF and primary MEF, the phosphorylation ratio of nucleophosmin (Thr199) increased in immortalized MEF, whereas it decreased in primary MEF. These results suggest that there exists a yet unidentified pathway, different from the nucleophosmin phosphorylation pathway, which can cause centrosome amplification in primary MEF.
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