Cell cycle-specific reactivation of an inactive X-chromosome locus by 5-azadeoxycytidine.

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Abstract
Three cytidine analogs contain modifications in the 5-position of the cytosine ring (5-azacytidine, 5-aza-2''-deoxycytidine and pseudoisocytidine); the analogs induced the expression of human hypoxanthine/guanine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) gene (HPRT) from a structurally normal inactive human X-chromosome retained in a mouse-human somatic cell hybrid. From 0.1-8% of the cells surviving treatment with these analogs were able to form colonies in selective medium (hypoxanthine/aminopterin/thymidine/glycine medium), but 2 other analogs, 5-fluoro-2''-deoxycytidine and 5,6-dihydro-5-azacytidine, did not induce HPRT expression. The inactive X-chromosome present in the hybrid was late replicating, and experiments with synchronized cells showed that the induction of HPRT expression by 5-aza-2''-deoxycytidine occurred maximally in cells treated in the latter half of the S phase. Two division cycles were required after analog treatment for the highest frequency of expression of the induced gene. Because the analogs are powerful inhibitors of the methylation of cytosine residues in DNA, the results imply that demethylation of specific DNA sequences may be required for the reexpression of human HPRT.