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

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 (gamma-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 G(1) 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 G(1) synchronized cells. In contrast, NCS-induced DSBs were equally distributed in all cell cycle phases in both asynchronous and G(1) synchronized cells. Moreover, Cr(VI)-induced gamma-H2AX foci formation was restricted to PCNA-positive cells, whereas NCS-induced gamma-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 gamma-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 interrelated mechanisms of Cr(VI) toxicity and carcinogenesis.

[1]  S. Patierno,et al.  Complexities of chromium carcinogenesis: role of cellular response, repair and recovery mechanisms. , 2003, Mutation research.

[2]  Ji-Hye Park,et al.  DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner. , 2003, Nucleic acids research.

[3]  C. McGowan,et al.  Running into problems: how cells cope with replicating damaged DNA. , 2003, Mutation research.

[4]  Nicholas F. Marko,et al.  Does metabolic radiolabeling stimulate the stress response? Gene expression profiling reveals differential cellular responses to internal beta vs. external gamma radiation , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[5]  R. Bonner,et al.  Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks , 2003, Nature Cell Biology.

[6]  Judith L Campbell,et al.  Dna2 Helicase/Nuclease Causes Replicative Fork Stalling and Double-strand Breaks in the Ribosomal DNA of Saccharomyces cerevisiae* , 2003, Journal of Biological Chemistry.

[7]  A. Sancar,et al.  Identification and Characterization of Human MUS81-MMS4 Structure-specific Endonuclease* , 2003, Journal of Biological Chemistry.

[8]  Y. Pommier,et al.  Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 in Response to Replication-dependent DNA Double-strand Breaks Induced by Mammalian DNA Topoisomerase I Cleavage Complexes* , 2003, Journal of Biological Chemistry.

[9]  S. Patierno,et al.  Chromium (VI) Activates Ataxia Telangiectasia Mutated (ATM) Protein , 2003, The Journal of Biological Chemistry.

[10]  T. Bessho.  Induction of DNA Replication-mediated Double Strand Breaks by Psoralen DNA Interstrand Cross-links* , 2003, The Journal of Biological Chemistry.

[11]  D. E. Pritchard,et al.  Critical role of chromium (Cr)-DNA interactions in the formation of Cr-induced polymerase arresting lesions. , 2002, Biochemistry.

[12]  K. Brown,et al.  ATM Is Activated in Response toN-Methyl-N′-nitro- N-nitrosoguanidine-induced DNA Alkylation* , 2002, The Journal of Biological Chemistry.

[13]  Michel C. Nussenzweig,et al.  Genomic Instability in Mice Lacking Histone H2AX , 2002, Science.

[14]  Weiya Ma,et al.  Requirement of ATM in UVA-induced Signaling and Apoptosis* , 2002, The Journal of Biological Chemistry.

[15]  Junjie Chen,et al.  Histone H2AX Is Phosphorylated in an ATR-dependent Manner in Response to Replicational Stress* , 2001, The Journal of Biological Chemistry.

[16]  Michael M. Murphy,et al.  ATM Phosphorylates Histone H2AX in Response to DNA Double-strand Breaks* , 2001, The Journal of Biological Chemistry.

[17]  A. Kuzminov.  Single-strand interruptions in replicating chromosomes cause double-strand breaks , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Bibikova,et al.  Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication. , 2001, Molecular cell.

[19]  Debasis Bagchi,et al.  Chromium (VI)‐induced oxidative stress, apoptotic cell death and modulation of p53 tumor suppressor gene , 2001, Molecular and Cellular Biochemistry.

[20]  Liying Wang,et al.  Cr(VI) increases tyrosine phosphorylation through reactive oxygen species-mediated reactions , 2001, Molecular and Cellular Biochemistry.

[21]  Liying Wang,et al.  On the mechanism of Cr (VI)‐induced carcinogenesis: Dose dependence of uptake and cellular responses , 2001, Molecular and Cellular Biochemistry.

[22]  Xianglin Shi,et al.  CR (VI) induces cell growth arrest through hydrogen peroxide‐mediated reactions , 2001, Molecular and Cellular Biochemistry.

[23]  M. Jung,et al.  Role of ATM in Oxidative Stress-mediated c-Jun Phosphorylation in Response to Ionizing Radiation and CdCl2 * , 2001, The Journal of Biological Chemistry.

[24]  M. Kastan,et al.  The many substrates and functions of ATM , 2000, Nature Reviews Molecular Cell Biology.

[25]  B. Michel,et al.  RuvABC‐dependent double‐strand breaks in dnaBts mutants require RecA , 2000, Molecular microbiology.

[26]  P. McHugh,et al.  Defining the Roles of Nucleotide Excision Repair and Recombination in the Repair of DNA Interstrand Cross-Links in Mammalian Cells , 2000, Molecular and Cellular Biology.

[27]  V. Yamazaki,et al.  A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage , 2000, Current Biology.

[28]  Myron F. Goodman,et al.  The importance of repairing stalled replication forks , 2000, Nature.

[29]  B. Merrill,et al.  A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants. , 1999, Genetics.

[30]  E. Rogakou,et al.  Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo , 1999, The Journal of cell biology.

[31]  D. E. Pritchard,et al.  Sensitive quantitation of chromium-DNA adducts by inductively coupled plasma mass spectrometry with a direct injection high-efficiency nebulizer. , 1998, Toxicological sciences : an official journal of the Society of Toxicology.

[32]  S. Patierno,et al.  Arrest of replication by mammalian DNA polymerases α and β caused by chromium‐DNA lesions , 1998 .

[33]  S. Ehrlich,et al.  RuvAB Acts at Arrested Replication Forks , 1998, Cell.

[34]  Y Taya,et al.  Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. , 1998, Science.

[35]  J. Singh,et al.  Differential sensitivity of chromium-mediated DNA interstrand crosslinks and DNA-protein crosslinks to disruption by alkali and EDTA. , 1998, Toxicological sciences : an official journal of the Society of Toxicology.

[36]  E. Rogakou,et al.  DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139* , 1998, The Journal of Biological Chemistry.

[37]  W Popp,et al.  Elevated DNA single-strand breakage frequencies in lymphocytes of welders exposed to chromium and nickel. , 1998, Carcinogenesis.

[38]  B. Michel,et al.  DNA double‐strand breaks caused by replication arrest , 1997, The EMBO journal.

[39]  Y. Shiloh,et al.  Rapamycin resistance in ataxia-telangiectasia. , 1996, Oncogene.

[40]  G. Bubley,et al.  CARCINOGENESIS: Chromium(VI) treatment of normal human lung cells results in guanine-specific DNA polymerase arrest, DNA-DNA cross-links and S-phase blockade of cell cycle , 1996 .

[41]  C. White,et al.  Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells. , 1995, The American journal of physiology.

[42]  S. Patierno,et al.  Base-specific arrest of in vitro DNA replication by carcinogenic chromium: relationship to DNA interstrand crosslinking. , 1994, Carcinogenesis.

[43]  S. Patierno,et al.  Preferential formation and repair of chromium-induced DNA adducts and DNA--protein crosslinks in nuclear matrix DNA. , 1994, Carcinogenesis.

[44]  S. Patierno,et al.  Dna polymerase arrest by adducted trivalent chromium , 1994, Molecular carcinogenesis.

[45]  T. Thatcher,et al.  Phylogenetic analysis of the core histones H2A, H2B, H3, and H4. , 1994, Nucleic acids research.

[46]  R. Bravo,et al.  Cyclin/PCNA is the auxiliary protein of DNA polymerase-δ , 1987, Nature.

[47]  W. Bonner,et al.  Histone 2A, a heteromorphous family of eight protein species. , 1980, Biochemistry.

[48]  T. Thatcher,et al.  Phylogenetic analysis of the core histones H 2 A , H 2 B , H 3 , and H 4 , 2005 .

[49]  D. Petering,et al.  Model reactions of Cr (VI) with DNA mediated by thiol species , 2004, Molecular and Cellular Biochemistry.

[50]  T. Kashimoto,et al.  Detection of dichromate (VI)-induced DNA strand breaks and formation of paramagnetic chromium in multiple mouse organs. , 2001, Toxicology and applied pharmacology.

[51]  S. Patierno,et al.  Arrest of replication by mammalian DNA polymerases alpha and beta caused by chromium-DNA lesions. , 1998, Molecular carcinogenesis.

[52]  J. Celis,et al.  Nuclear patterns of cyclin (PCNA) antigen distribution subdivide S-phase in cultured cells--some applications of PCNA antibodies. , 1986, Leukemia research.

[53]  S. Okada,et al.  Alterations in ribonucleic acid synthesis by chromium (III). , 1981, Journal of inorganic biochemistry.