Chromium induces chromosomal instability,which is partly due to deregulation of BubR1 and Emi1, two APC/C inhibitors

Disruption of cell cycle checkpoints and interference with the normal cell cycle progression frequently result in cell death or malignant transformation. Hexavalent chromium [Cr(VI)] is a well-known carcinogen that has been implicated in the occurrence of many types of human malignancies, including lung cancer. However, the exact mechanism by which Cr(VI) causes malignant transformation in the lung remains unknown. We have demonstrated that chronic exposure to a noncytotoxic concentration of Cr(VI) induced a variety of chromosomal abnormalities, including premature sister chromatid separation, chromosomal breakage and the presence of lagging/misaligned chromosomes. After treatment with nocodazole, both HeLa and normal lung bronchial epithelial cells were arrested at mitosis. However, Cr(VI) significantly compromised M-phase arrest induced by nocodazole. Cr(VI) suppressed BubR1 activation and reduced expression of Emi1, leading to an unscheduled activation of APC/C. Consistent with this observation, Cr(VI) treatment caused enhanced polyubiquitination of geminin during mitotic release, while it deregulated the activity of Cdt1, a DNA replication licensing factor. Combined, these results suggest that Cr(VI)-induced chromosomal instability is partly due to a perturbation of APC/C activities, leading to chromosomal instability.Key words: chromium, checkpoint, chromosome instability, APC/C, BubR1, Emi1     

Protein tyrosine phosphatase (PTP) inhibition enhances chromosomal stability after genotoxic stress: Decreased chromosomal instability (CIN) at the expense of enhanced genomic instability (GIN)?

Inappropriate survival signaling after DNA damage may facilitate clonal expansion of genetically compromised cells, and it is known that protein tyrosine phosphatase (PTP) inhibitors activate key survival pathways. In this study we employed the genotoxicant, hexavalent chromium [Cr(VI)], which is a well-documented carcinogen of occupational and environmental concern. Cr(VI) induces a complex array of DNA damage, including DNA double strand breaks (DSBs). We recently reported that PTP inhibition bypassed cell cycle arrest and abrogated Cr(VI)-induced clonogenic lethality. Notably, PTP inhibition resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of DNA damage may lead to genomic instability (GIN), via cell cycle checkpoint bypass. The aim of the present study was to determine the effect of PTP inhibition on DNA DSB formation and chromosomal integrity after Cr(VI) exposure. Diploid human lung fibroblasts were treated with Cr(VI) in the presence or absence of the PTP inhibitor, sodium orthovanadate, for up to 24h, and cells were analyzed for DNA DSBs and chromosomal damage. Cr(VI) treatment induced a rapid increase in DNA DSBs, and a significant increase in total chromosomal damage (chromatid breaks and gaps) after 24h. In sharp contrast, PTP inhibition abrogated both DNA DSBs and chromosomal damage after Cr(VI) treatment. In summary, PTP inhibition in the face of Cr(VI) genotoxic stress decreases chromosomal instability (CIN) but increases mutagenesis, which we postulate to be a result of error-prone DNA repair.     

Characterisation of the human APC1, the largest subunit of the anaphase-promoting complex

Accurate segregation of sister chromatids during mitosis is necessary to avoid the aneuploidy found in many cancers. The spindle checkpoint, which monitors the metaphase to anaphase transition, has been shown to be defective in cancers with chromosomal instability. This checkpoint regulates the anaphase-promoting complex or cyclosome (APC/C), a cell cycle ubiquitin ligase regulating among other things sister chromatid separation. We have previously investigated the mouse Apc1 protein (previously also called Tsg24), the largest subunit of the APC/C. We have now sequenced a full-length human APC1 cDNA, mapped its chromosomal location, and analysed its intron-exon boundaries. We have also investigated the RNA and protein expression of the Apc1 and other APC/C components in normal and cancer cells and the relative occurrence of expressed sequence tags (ESTs) representing APC subunits from different tissues. The different APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they perform their functions as part of a complex. A difference from this pattern is however seen for the APC6, which in some cases is more strongly expressed, suggesting a special function for this protein in certain tissues and cell types.     

Cdk1-phosphorylated CUEDC2 promotes spindle checkpoint inactivation and chromosomal instability

Aneuploidy and chromosomal instability are major characteristics of human cancer. These abnormalities can result from defects in the spindle assembly checkpoint (SAC), which is a surveillance mechanism for accurate chromosome segregation through restraint of the activity of the anaphase-promoting complex/cyclosome (APC/C). Here, we show that a CUE-domain-containing protein, CUEDC2, is a cell-cycle regulator that promotes spindle checkpoint inactivation and releases APC/C from checkpoint inhibition. CUEDC2 is phosphorylated by Cdk1 during mitosis. Depletion of CUEDC2 causes a checkpoint-dependent delay of the metaphase-anaphase transition. Phosphorylated CUEDC2 binds to Cdc20, an activator of APC/C, and promotes the release of Mad2 from APC/C-Cdc20 and subsequent APC/C activation. CUEDC2 overexpression causes earlier activation of APC/C, leading to chromosome missegregation and aneuploidy. Interestingly, CUEDC2 is highly expressed in many types of tumours. These results suggest that CUEDC2 is a key regulator of mitosis progression, and that CUEDC2 dysregulation might contribute to tumour development by causing chromosomal instability.     

Constitutive Activation of Epidermal Growth Factor Receptor Promotes Tumorigenesis of Cr(VI)-transformed Cells through Decreased Reactive Oxygen Species and Apoptosis Resistance Development

Hexavalent chromium (Cr(VI)) compounds are well-established lung carcinogens. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates cell survival, tumor invasion, and angiogenesis. Our results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) is able to cause malignant cell transformation. These transformed cells exhibit apoptosis resistance with reduced poly ADP-ribose polymerase cleavage (C-PARP) and Bax expression and enhanced expressions of Bcl-2 and Bcl-xL. These transformed cells also exhibit reduced capacity of reactive oxygen species (ROS) generation along with elevated expression of antioxidant manganese superoxide dismutase 2 (SOD2). The expression of this antioxidant was also elevated in lung tumor tissue from a worker exposed to Cr(VI) for 19 years. EGFR was activated in Cr(VI)-transformed BEAS-2B cells, lung tissue from animals exposed to Cr(VI) particles, and human lung tumor tissue. Further study indicates that constitutive activation of EGFR in Cr(VI)-transformed cells was due to increased binding to its ligand amphiregulin (AREG). Inhibition of EGFR or AREG increased Bax expression and reduced Bcl-2 expression, resulting in reduced apoptosis resistance. Furthermore, inhibition of AREG or EGFR restored capacity of ROS generation and decreased SOD2 expression. PI3K/AKT was activated, which depended on EGFR in Cr(VI)-transformed BEAS-2B cells. Inhibition of PI3K/AKT increased ROS generation and reduced SOD2 expression, resulting in reduced apoptosis resistance with commitment increase in Bax expression and reduction of Bcl-2 expression. Xenograft mouse tumor study further demonstrates the essential role of EGFR in tumorigenesis of Cr(VI)-transformed cells. In summary, the present study suggests that ligand-dependent constitutive activation of EGFR causes reduced ROS generation and increased antioxidant expression, leading to development of apoptosis resistance, contributing to Cr(VI)-induced tumorigenesis.     

Multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of Cr(VI)-transformed lung cells

B-cell lymphoma-2 (Bcl-2) is an antiapoptotic protein known to be important in the regulation of apoptosis in various cell types. However, its role in malignant transformation and tumorigenesis of human lung cells is not well understood. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation; however, the role of Bcl-2 in the transformation is unclear. Using a gene silencing approach, we showed that Bcl-2 plays an important role in the malignant properties of Cr(VI)-transformed cells. Downregulation of Bcl-2 inhibited the invasive and proliferative properties of the cells as well as their colony forming and angiogenic activities, which are upregulated in the transformed cells as compared to control cells. Furthermore, animal studies showed the inhibitory effect of Bcl-2 knockdown on the tumorigenesis of Cr(VI)-transformed cells. The role of Bcl-2 in malignant transformation and tumorigenesis was confirmed by gene silencing experiments using human lung carcinoma NCI-H460 cells. These cells exhibited aggressive malignant phenotypes similar to those of Cr(VI)-transformed cells. Knockdown of Bcl-2 in the H460 cells inhibited malignant and tumorigenic properties of the cells, indicating the general role of Bcl-2 in human lung tumorigenesis. Ingenuity Pathways Analysis (IPA) revealed potential effectors of Bcl-2 in tumorigenesis regulation. Additionally, using IPA together with ectopic expression of p53, we show p53 as an upstream regulator of Bcl-2 in Cr(VI)-transformed cells. Together, our results indicate the novel and multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of human lung epithelial cells chronically exposed to Cr(VI).     

ATR regulates hexavalent chromium-induced S-phase checkpoint through phosphorylation of SMC1

Hexavalent chromium (Cr[VI]) is an industrial waste product known to cause nasal and lung cancer in exposed workers. Intracellularly, Cr[VI] undergoes a series of enzymatic reductions resulting in the formation of reactive chromate intermediates and oxygen free radicals. These metabolites react with DNA to cause numerous types of genomic lesions, but the cellular response to these genotoxic insults is poorly understood. Recently, we demonstrated that in response to DNA damage induced by Cr[VI], an ataxia-telangiectasia mutated (ATM) and structural maintenance of chromosomal protein 1 (SMC1)-dependent S-phase checkpoint is activated. Interestingly, this checkpoint response was only ATM-dependent in cells exposed to low doses of Cr[VI], we demonstrate that the ATM and Rad3 related kinase, ATR, is required to activate the S-phase checkpoint. In response to all doses of Cr[VI], ATR is activated and phosphorylates SMC1 to facilitate the checkpoint. Further, chromatin binding ability of Rad17 is required for this process. Taken together, these results indicate that the Rad17-ATR-SMC1 pathway is essential for Cr[VI]-induced S-phase checkpoint activation.     

AKT1 mediates bypass of the G1/S checkpoint after genotoxic stress in normal human cells

Certain forms of hexavalent chromium [Cr(VI)] are human carcinogens. Our recent work has shown that a broad range protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SOV), abrogated both Cr(VI)-induced growth arrest and clonogenic lethality. Notably, SOV enhanced Cr(VI) mutation frequency, ostensibly through forced survival of genetically damaged cells. In the present study, co-treatment with this PTP inhibitor bypassed the Cr(VI)-induced G1/S checkpoint arrest in diploid human lung fibroblasts (HLF). Moreover, the PTP inhibitor abrogated the Cr(VI)-induced decrease in the expression of key effectors of the G1/S checkpoint [Cyclin D1, phospho Ser 807/811 Rb (pRB), p27]. Cr(VI)-induced G1 arrest was associated with the cytoplasmic appearance of pRb and the nuclear localization of p27, both of which were reversed by the PTP inhibitor. The PTP inhibitor’s reversal of G1/S checkpoint effector localization after Cr exposure was found to be Akt1-dependent, as this was abrogated by transfection with either akt1 siRNA or an Akt1-kinase dead plasmid. Furthermore, Akt1 activation alone was sufficient to induce G1/S checkpoint bypass and to prevent Cr(VI)-induced changes in pRb and p27 localization. In conclusion, this work establishes Akt1 activation to be both sufficient to bypass the Cr(VI)-induced G1/S checkpoint, as well as necessary for the observed PTP inhibitor effects on key mediators of the G1/S transition. The potential for Akt to bypass G1/S checkpoint arrest in the face of genotoxic damage could increase genomic instability, which is a hallmark of neoplastic progression.     

Involvement of the p38 MAP kinase in Cr(VI)-induced growth arrest and apoptosis

Hexavalent chromium [Cr(VI)] is a carcinogenic genotoxin commonly found in industry and the environment. DNA damage resulting from Cr(VI) exposure triggers numerous stress responses, including activation of cell cycle checkpoints and initiation of apoptosis. Mechanisms controlling these responses, while extensively studied, have yet to be fully elucidated. Here, we demonstrate that the p38 mitogen-activated protein kinase (MAPK) is activated by Cr(VI) exposure and that inhibition of p38 function using the selective inhibitor SB203580 results in abrogation of S-phase and G2 cell cycle checkpoints in response to Cr(VI). Also, we observe that inhibition of p38 results in decreased cell survival and increased percentage of apoptotic cells following Cr(VI) treatment. Taken together, these results indicate that p38 function is critical for optimal stress response induced by Cr(VI) exposure.     

Overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase/Akt

Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the hMre11 complex, a central player associated with double strand break repair. We previously demonstrated that c-Myc directly activates NBS1 expression. Here we have shown that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation. Repression of endogenous NBS1 levels using short interference RNA reduces the transformation activity of two tumor cell lines. Increased NBS1 expression is observed in 40-52% of non-small cell lung carcinoma, hepatoma, and esophageal cancer samples. NBS1 overexpression stimulates phosphatidylinositol (PI) 3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as glycogen synthase kinase 3beta and mammalian target of rapamycin in different cell lines and tumor samples. Transformation induced by NBS1 overexpression can be inhibited by a PI3-kinase inhibitor (LY294002). Repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Rat1a cells overexpressing NBS1. These results indicate that overexpression of NBS1 is an oncogenic event that contributes to transformation through the activation of PI3-kinase/Akt.     

Deficient repair of particulate hexavalent chromium-induced DNA double strand breaks leads to neoplastic transformation

Hexavalent chromium (Cr(VI)) is a potent respiratory toxicant and carcinogen. The most carcinogenic forms of Cr(VI) are the particulate salts such as lead chromate, which deposit and persist in the respiratory tract after inhalation. We demonstrate here that particulate chromate induces DNA double strand breaks in human lung cells with 0.1, 0.5, and 1 microg/cm(2) lead chromate inducing 1.5, 2, and 5 relative increases in the percent of DNA in the comet tail, respectively. These lesions are repaired within 24 h and require Mre11 expression for their repair. Particulate chromate also caused Mre11 to co-localize with gamma-H2A.X and ATM. Failure to repair these breaks with Mre11-induced neoplastic transformation including loss of cell contact inhibition and anchorage-independent growth. A 5-day exposure to lead chromate induced loss of cell contact inhibition in a concentration-dependent manner with 0, 0.1, 0.5, and 1 microg/cm(2) lead chromate inducing 1, 78, and 103 foci in 20 dishes, respectively. These data indicate that Mre11 is critical to repairing particulate Cr(VI)-induced double strand breaks and preventing Cr(VI)-induced neoplastic transformation.     

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

This study shows that the cell cycle E3 ubiquitin ligase APC/C is a regulator of several chromatin regulatory proteins, including the multivalent epigenetic reader and writer UHRF1. Perturbing UHRF1 ubiquitylation and degradation alters cell cycle and DNA methylation patterning, pointing to a key role for cell cycle degradation in shaping chromatin environments.     

A process independent of the anaphase-promoting complex contributes to instability of the yeast S phase cyclin Clb5

Proteolytic destruction of many cyclins is induced by a multi-subunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C). In the budding yeast Saccharomyces cerevisiae, the S phase cyclin Clb5 and the mitotic cyclins Clb1-4 are known as substrates of this complex. The relevance of APC/C in proteolysis of Clb5 is still under debate. Importantly, a deletion of the Clb5 destruction box has little influence on cell cycle progression. To understand Clb5 degradation in more detail, we applied in vivo pulse labeling to determine the half-life of Clb5 at different cell cycle stages and in the presence or absence of APC/C activity. Clb5 is significantly unstable, with a half-life of approximately 8-10 min, at cell cycle periods when APC/C is inactive and in mutants impaired in APC/C function. A Clb5 version lacking its cyclin destruction box is similarly unstable. The half-life of Clb5 is further decreased in a destruction box-dependent manner to 3-5 min in mitotic or G(1) cells with active APC/C. Clb5 instability is highly dependent on the function of the proteasome. We conclude that Clb5 proteolysis involves two different modes for targeting of Clb5 to the proteasome, an APC/C-dependent and an APC/C-independent mechanism. These different modes apparently have overlapping functions in restricting Clb5 levels in a normal cell cycle, but APC/C function is essential in the presence of abnormally high Clb5 levels.     

Anaphase-promoting complex/cyclosome controls HEC1 stability

Objective: Chromosome segregation during mitosis requires a physically large proteinaceous structure called the kinetochore to generate attachments between chromosomal DNA and spindle microtubules. It is essential for kinetochore components to be carefully regulated to guarantee successful cell division. Depletion, mutation or dysregulation of kinetochore proteins results in mitotic arrest and/or cell death. HEC1 (high expression in cancer) has been reported to be a kinetochore protein, depletion of which, by RNA interference, results in catastrophic mitotic exit. Materials and methods and results: To investigate how HEC1 protein is controlled post‐translation, we analysed the role of anaphase‐promoting complex/cyclosome (APC/C)‐Cdh1 in degradation of HEC1 protein. In this study, we show that HEC1 is an unstable protein and can be targeted by endogenous ubiquitin‐proteasome system in HEK293T cells. Results of RNA interference and in vivo ubiquitination assay indicated that HEC1 could be ubiquitinated and degraded by APC/C‐hCdh1 E3 ligase. The evolutionally conserved D‐box at the C‐terminus functioned as the degron of HEC1, destruction of which resulted in resistance to degradation mediated by APC/C‐Cdh1. Overexpression of non‐degradable HEC1 (D‐box destroyed) induced accumulation of cyclin B protein in vivo and triggered mitotic arrest. Conclusion: APC/C‐Cdh1 controls stability of HEC1, ensuring normal cell cycle progression.     

Role of the Fancg gene in protecting cells from particulate chromate-induced chromosome instability

Particulate hexavalent chromium (Cr(VI)) is a known human lung carcinogen. Cr(VI)-induced tumors exhibit chromosome instability (CIN), but the mechanisms underlying these effects are unknown. We investigated a possible role for the Fanconi anemia (FA) pathway in particulate Cr(VI)-induced chromosomal damage by focusing on the Fancg gene, which plays an important role in cellular resistance to DNA interstrand crosslinks. We used the isogenic Chinese hamster ovary (CHO) KO40 fancg mutant compared with parental and gene-complemented cells. We found that fancg cells treated with lead chromate had lower intracellular Cr ion levels than control cell lines. Accounting for differences of Cr ion levels between cell lines, we discovered that fancg cells treated with lead chromate had increased cytotoxicity and chromosomal aberrations, which was not observed after restoring the Fancg gene. Chromosomal damage was manifest as increased total chromosome damage and percent metaphases with damage, specifically an increase in chromatid and isochromatid breaks. We conclude that Fancg protects cells from particulate Cr(VI)-induced cytotoxicity and chromosome damage, which is consistent with the known sensitivity of fancg cells to crosslinking damage and the ability of Cr(VI) to produce crosslinks.     

Bub1: Escapades in a Cellular World

The spindle assembly checkpoint is an important surveillance mechanism that ensures high fidelity mitotic chromosome segregation. This is accomplished by monitoring whether sister chromatids lack tension or attachment to spindle microtubules. It is mediated by checkpoint complexes or individual proteins that inhibit the ubiquitin ligase activity of the anaphase-promoting complex/ cyclosome (APC/C) via targeting of the Cdc20 regulatory subunit. The Bub1 kinase is a key spindle checkpoint regulatory protein. Bub1 also plays more pleiotropic roles. Thus, Bub1 is required for assembly of a functional inner centromere, sister chromatid cohesion via targeting of the Shugoshin protein, and metaphase congression. Evidence based on Bub1 mutations in colorectal cancers suggests it might be a driving force in tumorigenesis via generation of chromosomal instability (CIN) and aneuploidy. Recently we reported a surveillance mechanism linking loss of Bub1 to activation of the p53 pathway, specifically premature cell senescence in normal human fibroblasts. Interestingly, SV40 large T antigen (LT) targets Bub1 and this is correlated with oncogenic transformation and compromise of the spindle checkpoint. Future studies on Bub1 combining genetic approaches with analysis of LT perturbations are likely to yield further insight.     

Mediator of DNA Damage Checkpoint 1 (MDC1) Regulates Mitotic Progression

Human mediator of DNA damage checkpoint 1 (hMDC1) is an essential component of the cellular response to DNA double strand breaks. Recently, hMDC1 has been shown to associate with a subunit of the anaphase-promoting complex/cyclosome (APC/C) (Coster, G., Hayouka, Z., Argaman, L., Strauss, C., Friedler, A., Brandeis, M., and Goldberg, M. (2007) J. Biol. Chem. 282, 32053–32064), a key regulator of mitosis, suggesting a possible role for hMDC1 in controlling normal cell cycle progression. Here, we extend this work to show that hMDC1 regulates normal metaphase-to-anaphase transition through its ability to bind directly to the APC/C and modulate its E3 ubiquitin ligase activity. In support of a role for hMDC1 in controlling mitotic progression, depletion of hMDC1 by small interfering RNA results in a metaphase arrest that appears to be independent of both BubR1-dependent signaling pathways and ATM/ATR activation. Mitotic cells lacking hMDC1 exhibit markedly reduced levels of APC/C activity characterized by reduced levels of Cdc20, and a failure of Cdc20 to bind the APC/C and CREB-binding protein. We suggest therefore that hMDC1 functionally regulates the normal metaphase-to-anaphase transition by modulating the Cdc20-dependent activation of the APC/C.     

XRCC1 protects cells from chromate-induced chromosome damage, but does not affect cytotoxicity

Hexavalent chromium Cr(VI) is a well known human carcinogen. This genotoxic metal induces DNA strand breaks and chromosome damage. However, the relationship between these lesions is uncertain. Our study focused on examining the role of XRCC1 in sodium chromate-induced cytotoxicity and chromosomal aberrations in Chinese Hamster Ovary (CHO) cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant) and H9T3 (EM9 complemented with human XRCC1 gene). Results show that concentration-dependent decreases in relative survival are similar in all three cell lines, indicating that XRCC1 is not crucial for protecting cells from sodium chromate-induced cytotoxicity. Similarly the frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total number of Cr(VI)-induced chromosome aberrations was exacerbated by XRCC1 deficiency and the spectrum of chromosome damage changed dramatically. Specifically, chromatid and isochromatid lesions were the most prominent aberrations induced in the cell lines and XRCC1 was essential to reduce the formation of chromatid lesions. In addition, XRCC1 deficiency caused a dramatic increase in the number of chromatid exchanges indicating that it is involved in protection from Cr(VI)-induced chromosome instability.     

XRCC1 protects cells from chromate-induced chromosome damage, but does not affect cytotoxicity

Hexavalent chromium Cr(VI) is a well known human carcinogen. This genotoxic metal induces DNA strand breaks and chromosome damage. However, the relationship between these lesions is uncertain. Our study focused on examining the role of XRCC1 in sodium chromate-induced cytotoxicity and chromosomal aberrations in Chinese Hamster Ovary (CHO) cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant) and H9T3 (EM9 complemented with human XRCC1 gene). Results show that concentration-dependent decreases in relative survival are similar in all three cell lines, indicating that XRCC1 is not crucial for protecting cells from sodium chromate-induced cytotoxicity. Similarly the frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total number of Cr(VI)-induced chromosome aberrations was exacerbated by XRCC1 deficiency and the spectrum of chromosome damage changed dramatically. Specifically, chromatid and isochromatid lesions were the most prominent aberrations induced in the cell lines and XRCC1 was essential to reduce the formation of chromatid lesions. In addition, XRCC1 deficiency caused a dramatic increase in the number of chromatid exchanges indicating that it is involved in protection from Cr(VI)-induced chromosome instability.