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DNA Methylation Inhibitor 5-Aza-2'-Deoxycytidine Induces Reversible Genome-Wide DNA Damage That Is Distinctly Influenced by DNA Methyltransferases 1 and 3B

Department of Biochemistry and Molecular Biology and University of Florida Shands Cancer Center Program in Cancer Genetics, Epigenetics and Tumor Virology, University of Florida, Box 100245, Gainesville, Florida 32610

Received 2 October 2007/ Accepted 25 October 2007

Genome-wide DNA methylation patterns are frequently deregulated in cancer. There is considerable interest in targeting the methylation machinery in tumor cells using nucleoside analogs of cytosine, such as 5-aza-2'-deoxycytidine (5-azadC). 5-azadC exerts its antitumor effects by reactivation of aberrantly hypermethylated growth regulatory genes and cytoxicity resulting from DNA damage. We sought to better characterize the DNA damage response of tumor cells to 5-azadC and the role of DNA methyltransferases 1 and 3B (DNMT1 and DNMT3B, respectively) in modulating this process. We demonstrate that 5-azadC treatment results in growth inhibition and G₂ arrest—hallmarks of a DNA damage response. 5-azadC treatment led to formation of DNA double-strand breaks, as monitored by formation of -H2AX foci and comet assay, in an ATM (ataxia-telangiectasia mutated)-dependent manner, and this damage was repaired following drug removal. Further analysis revealed activation of key strand break repair proteins including ATM, ATR (ATM-Rad3-related), checkpoint kinase 1 (CHK1), BRCA1, NBS1, and RAD51 by Western blotting and immunofluorescence. Significantly, DNMT1-deficient cells demonstrated profound defects in these responses, including complete lack of -H2AX induction and blunted p53 and CHK1 activation, while DNMT3B-deficient cells generally showed mild defects. We identified a novel interaction between DNMT1 and checkpoint kinase CHK1 and showed that the defective damage response in DNMT1-deficient cells is at least in part due to altered CHK1 subcellular localization. This study therefore greatly enhances our understanding of the mechanisms underlying 5-azadC cytotoxicity and reveals novel functions for DNMT1 as a component of the cellular response to DNA damage, which may help optimize patient responses to this agent in the future.

Published ahead of print on 8 November 2007.

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