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.
Call
K M
KM
Division of Toxicology, Whitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge 02139.
Thilly
W G
WG
D001374:Azacitidine / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000494:pharmacology*; D002453:Cell Cycle; D002851:Chromatography, High Pressure Liquid; D003562:Cytidine / Q000494:pharmacology; D004247:DNA / Q000187:drug effects*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008745:Methylation / Q000187:drug effects; D009154:Mutation / Q000187:drug effects; D013937:Thymidine Kinase / Q000096:biosynthesis
D003841:Deoxycytidine; D003562:Cytidine; D004247:DNA; C016569:5-methyldeoxycytidine; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; D001374:Azacitidine