Generation of S phase-dependent DNA double-strand breaks by Cr(VI) exposure: Involvement of ATM in Cr(VI) induction of γ-H2AX

Document Type

Journal Article

Publication Date

11-1-2004

Journal

Carcinogenesis

Volume

25

Issue

11

DOI

10.1093/carcin/bgh242

Abstract

Certain hexavalent chromium [Cr(VI)] compounds are implicated as occupational respiratory carcinogens. Cr(VI) induces a broad spectrum of DNA damage, but Cr(VI)-induced DNA double-strand breaks (DSBs) have not been reported. Previously we found that Cr(VI) activates the ataxia telangiectasia mutated (ATM) kinase. ATM is activated specifically in response to DSBs. Therefore, the objective of this study was to investigate DSB induction by Cr(VI) exposure with the overarching hypothesis that S phase-dependent DSBs are produced by Cr(VI) exposure. To test this hypothesis, normal human fibroblasts were treated with either Cr(VI) or neocarzinostatin (NCS). DSBs were analyzed by both comet assay under neutral conditions, which detects primarily DNA DSBs, and phosphorylation of histone H2AX (γ-H2AX) and the resultant formation of nuclear foci, which are considered to be indicative of DSBs. Induction of DSBs was observed after Cr(VI) exposure, however, the Cr(VI)-induced DSBs were abrogated by G1 synchronization. Furthermore, our data showed that Cr(VI)-induced DSBs were only observed in the S phase population, whereas no significant DSBs were observed in Cr(VI)-treated G1 synchronized cells. In contrast, NCS-induced DSBs were equally distributed in all cell cycle phases in both asynchronous and G1 synchronized cells. Moreover, Cr(VI -induced γ-H2AX foci formation was restricted to PCNA-positive cells, whereas NCS-induced γ-H2AX foci formed in both PCNA-positive and PCNA-negative cells. These results indicate that Cr(VI -induced DSBs are S phase-dependent. Finally, our data showed that Cr(VI -induced γ-H2AX production was significantly decreased in ATM-/- cells compared with ATM+/+ cells. Taken together, these results suggest that Cr(VI)-induced activation of ATM involves the formation of S phase-dependent DSBs. Examining the mechanism of Cr(VI)-induced DSBs will aid in understanding the inter-related mechanisms of Cr(VI) toxicity and carcinogenesis. © Oxford University Press 2004; all rights reserved.

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