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1.
The ataxia telangiectasia mutated (ATM) protein plays a central role in early stages of DNA double strand break (DSB) detection and controls cellular responses to this damage. Although hypersensitive to ionizing radiation-induced clonogenic lethality, ataxia telangiectasia cells are paradoxically deficient in their ability to undergo ionizing radiation-induced apoptosis. This contradiction illustrates the complexity of the central role of ATM in DNA damage response and the need for further understanding. Certain hexavalent chromium (Cr(VI)) compounds are implicated as occupational respiratory carcinogens at doses that are both genotoxic and cytotoxic. Cr(VI) induces a broad spectrum of DNA damage, but Cr(VI)-induced DSBs have not been reported. Here, we examined the role of ATM in the cellular response to Cr(VI) and found that Cr(VI) activates ATM. We also show that physiological targets of ATM, p53 Ser-15 and Chk2 Thr-68, were phosphorylated by Cr(VI) exposure in an ATM-dependent fashion. We found that ATM-/- cells were markedly resistant to Cr(VI)-induced apoptosis but considerably more sensitive to Cr(VI)-induced clonogenic lethality than wild type cells, indicating that resistance to Cr(VI)-induced apoptosis did not confer a selective survival advantage. However, analysis of long term growth arrest revealed a striking difference: ATM-/- cells were markedly less able to recover from Cr(VI)-induced growth arrest. This indicates that terminal growth arrest is the fate of these apoptosis-resistant cells. In summary, ATM is involved in cellular response to a complex genotoxin that may not directly induce DSBs. Our data suggest that ATM is a major signal initiator for genotoxin-induced apoptosis but, paradoxically, also contributes to maintenance of cell survival by facilitating recovery/escape from terminal growth arrest. The results also strongly suggest that terminal growth arrest is not merely an extended or even irreversible form of checkpoint arrest, but instead an independent and unique cell fate pathway.  相似文献   

2.
Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr-DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA-Cr-protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr-DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr-DNA adducts processed by NER, the incision of CrCl(3) [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl(3)) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 microM we observed approximately 2 Cr(III)-DNA adducts per plasmid. At this same concentration of Cr(III) we found that approximately 17% of the plasmid DNA contained ICLs ( approximately 0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 microM) was incubated with Bca UvrABC we observed approximately 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)-DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.  相似文献   

3.
The cellular responses to carcinogen exposure influence cellular fate, which in turn modulates the neoplastic response. Certain hexavalent chromium [Cr(VI)] compounds are implicated as occupational respiratory carcinogens at doses that are both genotoxic and cytotoxic. We examined the mechanism of Cr(VI)-induced apoptosis in normal human fibroblasts (BJ) immortalized by human telomerase gene transfection (BJ-hTERT), and we assessed the spectrum of cumulative cellular fates [(a) regaining of replicative potential; (b) terminal growth arrest; or (c) apoptosis] for a narrow range of increasingly genotoxic doses of Cr(VI). Exposure of BJ-hTERT cells to Cr(VI) resulted in a dose-dependent increase in apoptosis that involved mitochondrial disruption as evidenced by mitochondrial membrane depolarization and cytochrome c release. The initial response to Cr(VI) exposure was inhibition of cell cycle progression. At the lowest dose tested (1 microM; 32% clonogenic survival), the cell cycle inhibition led to terminal growth arrest but no apoptosis. The fraction of terminally growth arrested cells increased as the dose was increased to 3 microM but then decreased at 4, 5, and 6 microM as apoptosis became the predominant cell fate. Our results suggest that cell populations exposed to Cr(VI) have a different spectrum of responses, depending on the extent of DNA damage, and that the regaining of replicative potential after relatively higher genotoxic exposures may be attributable to either escape from, or resistance to, terminal growth arrest or apoptosis.  相似文献   

4.
The genotoxicity associated with the metabolic reduction of hexavalent chromium [Cr(VI)] is complex and can impede DNA polymerase-mediated replication in vitro. The exact biochemical nature of Cr-induced polymerase arresting lesions (PALs) is not understood, but is believed to involve the formation of Cr-DNA interstrand cross-links (ICLs). The aim of this investigation was to determine the dependence of direct Cr-DNA interactions on the development of PALs in DNA treated with trivalent Cr [Cr(III)] or with Cr(VI) in the presence of ascorbic acid (Asc), a major intracellular reductant, using an in vitro, acellular system. The formation of Cr-DNA adducts, ICLs, and PALs was maximal at Asc:Cr(VI) molar ratios of 0.5-2, but gradually decreased at higher ratios. EDTA, a Cr(III) chelator, significantly decreased Cr-DNA binding and ICL and PAL formation. Co-treatment of DNA with Cr(VI)/Asc and mannitol, a Cr(V) chelator, selectively inhibited the formation of mono/bifunctional DNA adducts and PALs produced by Cr(VI) reduction, but had no effect on Cr(III)-DNA binding or Cr(III)-induced polymerase arrest. Blocking Cr-DNA phosphate interaction by preincubation of DNA with MgCl(2) abrogated DNA binding and ICL and PAL production. DNA strand breaks and abasic sites may lead to the in vitro arrest of DNA polymerases; however, we failed to detect significant increases in the frequency of these lesions following Cr(VI)/Asc treatment. These data indicate that the bifunctional adduction of Cr to DNA phosphates (ICLs) constitutes a major PAL. Furthermore, the generation of DNA strand breaks and abasic sites by Cr(VI) reduction is insufficient to explain PALs observed in vitro.  相似文献   

5.
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.  相似文献   

6.
Hexavalent chromium (Cr(VI)) is a widespread environmental contaminant and a known human carcinogen, generally causing bronchial cancer. Recent studies have shown that the particulate forms of Cr(VI) are the potent carcinogens. Particulate Cr(VI) is known to induce a spectrum of DNA damage such as DNA single strand breaks, Cr-DNA adducts, DNA-protein crosslinks and chromosomal aberrations. However, particulate Cr(VI)-induced DNA double strand breaks (DSBs) have not been reported. Thus, the aim of this study was to determine if particulate Cr(VI)-induces DSBs in human bronchial cells. Using the single cell gel electrophoresis assay (comet assay), showed that lead chromate-induced concentration dependent increases in DSBs with 0.1, 0.5, 1 and 5 microg/cm2 lead chromate inducing a 20, 50, 67 and 109% relative increase in the tail integrated intensity ratio, respectively. Sodium chromate at concentrations of 1, 2.5 and 5 microM induced 38, 78 and 107% relative increase in the tail integrated intensity ratio, respectively. We also show that genotoxic concentrations of lead chromate activate the ataxia telangiectasia mutated (ATM) protein, which is thought to play a central role in the early stages of DSB detection and controls cellular responses to this damage. The H2A.X protein becomes rapidly phosphorylated on residue serine 139 in cells when DSBs are introduced into the DNA by ionizing radiation. By using immunofluorescence, we found that lead chromate-induced concentration-dependent increases in phosphorylated H2A.X (r-H2A.X) foci formation with 0.1, 0.5, 1, 5 and 10 microg/cm2 lead chromate inducing a relative increase in the number of cells with r-H2A.X foci formation of 43, 51, 115 and 129%, respectively.  相似文献   

7.
Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr–DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA–Cr–protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr–DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr–DNA adducts processed by NER, the incision of CrCl3 [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl3) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 μM we observed 2 Cr(III)–DNA adducts per plasmid. At this same concentration of Cr(III) we found that 17% of the plasmid DNA contained ICLs (0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 μM) was incubated with Bca UvrABC we observed 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)–DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.  相似文献   

8.
Some hexavalent chromium (Cr(VI))-containing compounds are human lung carcinogens. While ample information is available on the genetic lesions produced by Cr, surprisingly little is known regarding the cellular mechanisms involved in the removal of Cr-DNA adducts. Nucleotide excision repair (NER) is a highly versatile pathway that is responsive to a variety of DNA helix-distorting lesions. Binary Cr-DNA monoadducts do not produce a significant degree of helical distortion. However, these lesions are unstable due to the propensity of Cr(III) to form DNA adducts (DNA interstrand crosslinks, DNA-protein/amino acid ternary adducts) which may serve as substrates for NER. Therefore, the focus of this study was to determine the role of NER in the processing of Cr-DNA damage using normal (CHO-AA8) and NER-deficient [UV-5 (XP-D); UV-41 (ERCC4/XP-F)] hamster cells. We found that both UV-5 and UV-41 cells exhibited an increased sensitivity towards Cr(VI)-induced clonogenic lethality relative to AA8 cells and were completely deficient in the removal of Cr-DNA adducts. In contrast, repair-complemented UV-5 (expressing hamster XPD) and UV-41 (expressing human ERCC4) cells exhibited similar clonogenic survival and removed Cr-DNA adducts to a similar extent as AA8 cells. In order to extend these findings to the molecular level, we examined the ability of Cr(III)-damaged DNA to induce DNA repair synthesis in cell extracts. Repair synthesis was observed in reactions using extracts derived from AA8, or repair-complemented, but not NER-deficient cells. Cr(III)-induced repair resynthesis was sensitive to inhibition by the DNA polymerase δ/ε inhibitor, aphidicolin, but not 2′,3′-dideoxythymidine triphosphate (ddTTP), a polymerase β inhibitor. These results collectively suggest that NER functions in the protection of cells from Cr(VI) lethality and is essential for the removal of Cr(III)-DNA adducts. Consequently, NER may represent an important mechanism for preventing Cr(VI)-induced mutagenesis and neoplastic transformation.  相似文献   

9.
10.
Wakeman TP  Kim WJ  Callens S  Chiu A  Brown KD  Xu B 《Mutation research》2004,554(1-2):241-251
Hexavalent chromium (Cr[VI]) is a common industrial waste product, an environmental pollutant, and a recognized human carcinogen. Following cellular uptake, Cr[VI] can cause DNA damage, however, the mechanisms by which mammalian cells respond to Cr-induced DNA damage remain to be elucidated. Using single cell gel electrophoresis (e.g., Comet Assay) and immunofluoresence microscopy to detect the presence of gamma-H2AX foci, we find that Cr[VI] induces DNA double-strand breaks similar to ionizing radiation (IR). We also demonstrated that ataxia telangiectasia mutated (ATM) is activated in response to Cr[VI] and exposure to Cr[VI] triggers a dose and ATM-dependent S-phase arrest. Further, we document that ATM is required for phosphorylation of the structural maintenance of chromosome protein 1 (SMC1). Finally, we find that ATM-dependent phosphorylation of SMC1 is required to facilitate S-phase cell-cycle arrest in response to Cr[VI] exposure. Collectively, these results indicate that the ATM-SMC1 pathway plays a critical role in cellular response to Cr[VI].  相似文献   

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