Promotes Chromosome Instability Research Articles

Kendrin Is a Novel Substrate for Separase Involved in the Licensing of Centriole Duplication

The centrosome, consisting of a pair of centrioles surrounded by pericentriolar material, directs the formation of bipolar spindles during mitosis. Aberrant centrosome number can promote chromosome instability, which is implicated in tumorigenesis. Thus, centrosome duplication needs to be tightly regulated to occur only once per cell cycle. Separase, a cysteine protease that triggers sister chromatid separation, is involved in centriole disengagement, which licenses centrosomes for the next round of duplication. However, at least two questions remain unsolved: what is the substrate relevant to the disengagement, and how does separase, activated at anaphase onset, act on the disengagement that occurs during late mitosis. Here, we show that kendrin, also named pericentrin, is cleaved by activated separase at a consensus site in vivo and in vitro, and this leads to the delayed release of kendrin from the centrosome later in mitosis. Furthermore, we demonstrate that expression of a noncleavable kendrin mutant suppresses centriole disengagement and subsequent centriole duplication. Based on these results, we propose that kendrin is a novel and crucial substrate for separase at the centrosome, protecting the engaged centrioles from premature disengagement and thereby blocking reduplication until the cell passes through mitosis.

RB: mitotic implications of a tumour suppressor

RB, a well known tumour suppressor that functions in the control of cell cycle progression and proliferation, has recently been shown to have additional functions in the maintenance of genomic stability, such that inactivation of RB family proteins promotes chromosome instability (CIN) and aneuploidy. Several studies have provided potential explanations for these phenomena that occur following RB loss, and they suggest that this new function of RB may contribute to its role in tumour suppression.

4-Hydroxy-2-Nonenal Mediates Genotoxicity and Bystander Effects Caused by Enterococcus faecalis–Infected Macrophages

Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide and promotes chromosome instability via macrophage-induced bystander effects. We investigated the ability of 4-hydroxy-2-nonenal (4-HNE), a diffusible breakdown product of ω-6 polyunsaturated fatty acids, to mediate these effects. 4-HNE was purified from E faecalis-infected macrophages; its genotoxicity was assessed in human colon cancer (HCT116) and primary murine colon epithelial (YAMC) cell lines. 4-HNE induced G(2)-M cell cycle arrest, led to formation γH2AX foci, and disrupted the mitotic spindle in both cell lines. Binucleate tetraploid cells that formed after incubation with 4-HNE were associated with the activation of stathmin and microtubule catastrophe. Silencing glutathione S-transferase α4, a scavenger of 4-HNE, increased the susceptibility of epithelial cells to 4-HNE-induced genotoxicity. Interleukin-10 knockout mice colonized with superoxide-producing E faecalis developed inflammation and colorectal cancer, whereas colonization with a superoxide-deficient strain resulted in inflammation but not cancer. 4-HNE-protein adducts were found in the lamina propria and macrophages in areas of colorectal inflammation. 4-HNE can act as an autochthonous mitotic spindle poison in normal colonic epithelial and colon cancer cells. This finding links the macrophage-induced bystander effects to colorectal carcinogenesis.

Genistein, isoflavonoids in soybeans, prevents the formation of excess radiation-induced centrosomes via p21 up-regulation

The centrosome is a cytoplasmic organelle which duplicates once during each cell cycle, and the presence of excess centrosomes promote chromosome instability through chromosome missegregation following cytokinesis. Ionizing radiation (IR) can induce extra centrosomes by permitting the continuation of CDK2/Cyclin-A/E-mediated centrosome duplication when cells are arrested in the cell cycle after irradiation. The work described here shows that, in addition to IR, extra centrosomes were induced in human U2OS and mouse NIH3T3 cells after treatment with agents which include DNA adduct-forming chemicals: benzopyrene (BP), 4-nitroquinoline 1-oxide (4NQO), a DNA cross linker: cis-diamminedichloro-platinum (cisplatin), topoisomerase inhibitors: camptothecin, etoposide, genistein, and ultra-violet light (UV). These agents were divided into two categories with respect to the regulation of p21, which is an inhibitor of CDK2/Cyclin-A/E: specifically, p21 was up-regulated by an IR exposure and treatment with topoisomerase inhibitors. However, UV, BP, 4NQO and cisplatin down-regulated p21 below basal levels. When cells were irradiated with IR in combination with all of these agents, except genistein, enhanced induction of extra centrosomes was observed, regardless of the nature of p21 expression. Genistein significantly suppressed the frequency of IR-induced extra centrosomes in a dose-dependent manner, and 20 μg/ml of genistein reduced this frequency to 66%. Consistent with this, genistein substantially up-regulated p21 expression over the induction caused by IR alone, while other agents down-regulated or marginally affected this. This suggests the inhibitory effect of genistein on the induction of extra centrosomes occurs through the inactivation of CDK2/Cyclin-A/E via p21 up-regulation. This hypothesis is supported by the observation that p21 knockdown with siRNA reduced the activity of CDK2/Cyclin-A/E and restored the enhanced effect of a combined treatment with genistein and IR. These results demonstrate the preventive effect of genistein and a crucial role for p21 in IR-induced excess centrosomes.

Escherichia coli damages host DNA

A genotoxin produced by strains of Escherichia coli induces double-strand breaks in host DNA and promotes chromosome instability and mutations.

Loss of p53 and MCT-1 Overexpression Synergistically Promote Chromosome Instability and Tumorigenicity

MCT-1 oncoprotein accelerates p53 degradation by means of the ubiquitin-dependent proteolysis. Our present data show that induction of MCT-1 increases chromosomal translocations and deregulated G(2)-M checkpoint in response to chemotherapeutic genotoxin. Remarkably, increases in chromosome copy number, multinucleation, and cytokinesis failure are also promoted while MCT-1 is induced in p53-deficient cells. In such a circumstance, the Ras-mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-mitogen-activated protein kinase signaling activity and the expression of metastatic molecules are amplified. Given a p53-silencing background, MCT-1 malignantly transforms normal breast epithelial cells that are satisfactory for stimulating cell migration/adhesion and tumorigenesis. Detailed analyses of MCT-1 oncogenicity in H1299 p53-null lung cancer cells have shown that ectopically expressed MCT-1 advances xenograft tumorigenicity and angiogenesis, which cannot be completely suppressed by induction of p53. MCT-1 counteracts mutually with p53 at transcriptional levels. Clinical validations confirm that MCT-1 mRNA levels are differentially enriched in comparison between human lung cancer and nontumorigenic tissues. The levels of p53 mRNA are comparatively reduced in a subset of cancer specimens, which highly present MCT-1 mRNA. Our results indicate that synergistic promotions of chromosomal imbalances and oncogenic potency as a result of MCT-1 expression and p53 loss play important roles in tumor development.

GSK-3 inhibitors induce chromosome instability

BackgroundSeveral mechanisms operate during mitosis to ensure accurate chromosome segregation. However, during tumour evolution these mechanisms go awry resulting in chromosome instability. While several lines of evidence suggest that mutations in adenomatous polyposis coli (APC) may promote chromosome instability, at least in colon cancer, the underlying mechanisms remain unclear. Here, we turn our attention to GSK-3 – a protein kinase, which in concert with APC, targets β-catenin for proteolysis – and ask whether GSK-3 is required for accurate chromosome segregation.ResultsTo probe the role of GSK-3 in mitosis, we inhibited GSK-3 kinase activity in cells using a panel of small molecule inhibitors, including SB-415286, AR-A014418, 1-Azakenpaullone and CHIR99021. Analysis of synchronised HeLa cells shows that GSK-3 inhibitors do not prevent G1/S progression or cell division. They do, however, significantly delay mitotic exit, largely because inhibitor-treated cells have difficulty aligning all their chromosomes. Although bipolar spindles form and the majority of chromosomes biorient, one or more chromosomes often remain mono-oriented near the spindle poles. Despite a prolonged mitotic delay, anaphase frequently initiates without the last chromosome aligning, resulting in chromosome non-disjunction. To rule out the possibility of "off-target" effects, we also used RNA interference to selectively repress GSK-3β. Cells deficient for GSK-3β exhibit a similar chromosome alignment defect, with chromosomes clustered near the spindle poles. GSK-3β repression also results in cells accumulating micronuclei, a hallmark of chromosome missegregation.ConclusionThus, not only do our observations indicate a role for GSK-3 in accurate chromosome segregation, but they also raise the possibility that, if used as therapeutic agents, GSK-3 inhibitors may induce unwanted side effects by inducing chromosome instability.

Overexpression of Hepatitis C Virus NS5A Protein Induces Chromosome Instability via Mitotic Cell Cycle Dysregulation

Hepatocellular carcinoma (HCC) is a common primary cancer associated with high incidences of genetic variations including chromosome instability. Moreover, it has been demonstrated that hepatitis C virus (HCV) is one of the major causes of HCC. However, no previous work has assessed whether HCV proteins are associated with the induction of chromosome instability. Here, we found that liver cell lines constitutively expressing full-length or truncated versions of the HCV genome show a high incidence of chromosome instability. In particular, the overexpression of HCV NS5A protein in cultured liver cells was found to promote chromosome instability and aneuploidy. Further experiments showed that NS5A-induced chromosome instability is associated with aberrant mitotic regulations, such as, an unscheduled delay in mitotic exit and other mitotic impairments (e.g. multi-polar spindles). Thus, our results indicate that HCV NS5A protein may be directly involved in the induction of chromosome instability via mitotic cell cycle dysregulation, and provide novel insights into the molecular mechanisms of HCV-associated hepatocarcinogenesis.

Chromosome instability in T-cells cultured in the presence of pancuronium or fentanyl.

Genomic instability is recognized as a cause of cellular apoptosis and certain drugs that exhibit a proapoptotic effect are also able to induce chromosome damage. Since we found in recent experiments that drugs such as pancuronium and fentanyl exerted an apoptogenic effect on T cells, we studied the capacity of those agents to promote chromosome instability, i.e. chromosome aberrations (CA) and telomeric associations (tas) in peripheral blood lymphocytes. Lymphocytes from healthy donors were cultured with pancuronium or fentanyl, using two different concentrations for each drug: 20 and 200 ng/ml for pancuronium and 10 and 30 ng/ml for fentanyl, respectively. Cells were exposed to each concentration of these drugs either for 24 or 48 h. The higher concentration chosen was the same at which we detected the proapoptotic effect in our previous works. Cytogenetic analysis was performed by means of a standard technique and chromosome aberrations or telomeric associations were blindly evaluated by two independent observers. The chromosome aberrations we observed in treated cells were not significantly different from control lymphocytes. However, an unusual rate of telomeric associations (P < 0.001) was detected in cells exposed to both pancuronium and fentanyl, at each concentration tested and at each exposure time of the study. Fentanyl and pancuronium do not have a direct clastogenic effect on T cultures, but at the same concentrations at which we demonstrated their apoptogenic power, these drugs are able to increase genomic instability through inducing an elevated rate of telomeric associations. Such a capacity could exploit in peripheral T cells the same mitochondrion-mediated signal pathway of apoptosis death.

Hyperploidy induced by drugs that inhibit formation of microtubule promotes chromosome instability.

Antimicrotubule drugs (AMDs), such as taxol and vincristine, are the most important addition to the chemotherapeutic armamentarium against human cancers. It has been shown that prolonged AMD treatment induces hyperploidy in G1-checkpoint-defective cancer cells and that these hyperploid cells subsequently undergo apoptosis. However, a fraction of these hyperploid cells are able to survive the prolonged mitotic stress and resume cell-cycle progression. We established hyperploid clones that escaped from cell death after AMD treatment from two glioma cell lines, U251MG and U87MG. Subtractive comparative genomic hybridization (CGH) analysis revealed that clones derived from U87MG mainly had chromosome number changes, but that those from U251MG showed both numerical and structural chromosomal changes. Furthermore, numerous aberrations identified in U251MG clones were remarkably chromosome-specific, which may have been due to clonal selection for cells that have an advantage in growth and/or survival. All clones derived from both cell lines had abnormalities in chromosome segregation, and karyotypes of clones were more heterogeneous than those of parental cells, suggesting that cells having a higher chromosome number are subject to asymmetric chromosome segregation, resulting in a heterogeneous karyotype. All clones derived from U87MG and U251MG increased both centric and acentromeric micronuclei, suggesting the presence of chromosome structural abnormality. AMD treatment induces hyperploid formation and chromosome instability in checkpoint-deficient cancer cells.

The influence of interstitial telomeric sequences on chromosome instability in human cells

Although most telomere repeat sequences are found at the ends of chromosomes, some telomeric repeat sequences are also found at intrachromosomal locations in mammalian cells. Several studies have found that these interstitial telomeric repeat sequences can promote chromosome instability in rodent cells, either spontaneously or following ionizing radiation. In the present study we describe the extensive cytogenetic analysis of three different human cell lines with plasmids containing telomeric repeat sequences integrated at interstitial sites. In two of these cell lines, Q18 and P8SX, instability has been detected in the chromosome containing the integrated plasmid, involving breakage/fusion/bridge cycles or amplification of the plasmid DNA, respectively. However, the data suggest that the instability observed is characteristic of the general instability in these cell lines and that the telomeric repeat sequences themselves are not responsible. Consistent with this interpretation, the chromosome containing an integrated plasmid with 500 bp of telomeric repeat sequences is highly stable in the third cell line, SNG28, which has a relatively stable genome. The stability of the chromosome containing the integrated plasmid sequences in SNG28 makes this an excellent cell line to study the effect of ionizing radiation on the stability of interstitial telomeric sequences in human cells.