We are examining the role of methylation and its effects on chromatin structure, on DNA-protein binding and on gene expression, especially in relation to mechanisms of mutagenesis and carcinogenesis. 5-Azacytidine (5AC) alters eukaryotic gene expression by modulating the methylation patterns of CpG sequences in DNA. We used mutation as a measure of gene expression and examined the mutagenic activity of a series of cytidine analogs, including 5AC. We found that their ability to induce resistance in mammalian cells correlated with their reported ability to induce differentiation in C3H10T1/2 cells. Of 148 6TG clones analyzed, 68 demonstrated classical lesions (point mutations and deletions) in the structural gene. Eighty-one showed no mutation in the structural gene suggesting that either there must be a mutation in the promoter region or other (epigenetic) events are responsible for 6-thioguanine resistance. In order to further examine the effect of changes in chromatin methylation patterns, we examined the effects of 5-azacytidine on mitosis and on micronucleus formation in mammalian cells. 5-Azacytidine induced micronuclei at mutagenic concentrations. The induced micronuclei contained mostly chromosomal fragments. The micronuclei were induced only when the compound was added while the cells were in S phase. Supravital UV microscopy revealed that chromatid bridges were observed in anaphase and, in some cases, were sustained into interphase. These observations indicate that 5-azacytidine is predominantly a clastogen through its incorporation into DNA.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Intramural Research (Z01)
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