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The DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐azadC) induces reversible genome‐wide DNA damage that is distinctly influenced by DNA methyltransferases (DNMTs) 1 and 3B
Author(s) -
Robertson Keith D,
Sankpal Umesh,
Brown Kevin D
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a660-c
Subject(s) - dna damage , dna methylation , methyltransferase , dna repair , dnmt3b , cancer research , chemistry , dna , epigenetics , microbiology and biotechnology , biology , methylation , biochemistry , gene , gene expression
Genome‐wide DNA methylation patterns are frequently deregulated in cancer therefore there is significant interest in targeting the DNA methylation machinery in tumor cells using nucleoside analogs of cytosine, such as 5‐azadC. 5‐azadC exerts its anti‐tumor effects by two mechanisms; reactivation of aberrantly hypermethylated growth regulatory genes and cytoxicity resulting from DNA damage. Objective. To better characterize the DNA damage response of tumor cells to 5‐azadC and the role of DNMT1 and DNMT3B in modulating this process. Results. 5‐azadC treatment results in depletion of soluble DNMTs, growth inhibition, and G2 arrest ‐ hallmarks of a DNA damage response. 5‐azadC treatment led to formation of DNA double strand breaks in an ATM‐dependent manner and this damage was repaired following drug removal. Further analysis revealed activation and relocalization of key strand break repair proteins including ATM, ATR, CHK1, BRCA1, NBS1, and RAD51 by western blotting and immunofluorescence. Significantly, DNMT1 deficient cells demonstrated profound defects in these responses, including complete lack of gammaH2AX induction and blunted p53 activation, while DNMT3B deficient cells generally showed milder defects. Conclusions. This study provides the first detailed analysis of the cytotoxic mechanism of action of 5‐azadC which may help to optimize patient responses to this agent. This work was supported by NIH grants R01CA114229 and K22CA084535 (KDR).