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By growing cells in the presence of 3 mM thymidine and 5-azadeoxycytidine up to 20% of DNA cytosines have been substituted with azacytosine. No substitution was obtained on incubating with 5-methyldeoxycytidine. Azacytosine-substituted DNA has a very low level of 5-methylcytosine and cells, which survive azadeoxycytidine treatments maintain this low level of methylation in the absence of the drug. The DNA of such cells is undermethylated fairly evenly in all classes of DNA e.g., satellite and unique DNA. Incubation of cells in azadeoxycytidine leads to high cell mortality which is not related to the lack of DNA methylation but may be linked to the altered interactions of proteins with the substituted DNA. This effect, rather than reduced DNA methylation, may be the cause of differentiative changes observed on treatment of cells with 5-azacytidine.
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