5-Azacytidine inhibits the induction of transient TK-deficient cells by 5-bromodeoxyuridine. A novel hypothesis for the facilitation of hypermethylation by 5-bromodeoxyuridine.

This paper examines the mechanism by which 5-bromodeoxyuridine (BrdUrd) induces a high frequency of transient trifluorothymidine (F3TdR)-resistant variants in the TK6 human lymphoblast cell line (a TK +/- heterozygote). This phenomenon has previously been termed 'pseudomutation' (Liber et al., 1985). We now report that 5-azacytidine (5-AzaC), an inhibitor of DNA methylation, reverses BrdUrd-induced pseudomutation in a dose-dependent manner. The inhibition by 5-AzaC is highly specific and does not appear to involve nucleotide pool perturbations. 5-AzaC inhibits the pseudomutagenic effect (transient trifluorothymidine resistance in a thymidine kinase heterozygote), but not the stable mutagenic effect (stable 6-thioguanine resistance or trifluorothymidine resistance in a hypoxanthine-guanine phosphoribosyltransferase-proficient cell) induced by BrdUrd. 5-AzaC did not affect the induction nor expression of mutation induced by several other chemical mutagens at either the tk or hgprt loci. Inhibition of pseudomutation by 5-AzaC did not appear to be caused by a number of potential confounding factors. Although significant changes in the levels of DNA methylation were detected by HPLC analysis in BrdUrd-treated cells, the dose response for inhibition of pseudomutation by 5-AzaC was correlated with a significant decrease in 5-methylcytidine levels. These results and additional data in the literature have led us to postulate a novel mechanism in which the substitution of BrdUrd in a TpG dinucleotide(s) may serve as a substrate for non-heritable methylation and hence transiently inactivate tk gene expression.