6-Thioguanine Perturbs Cytosine Methylation at the CpG Dinucleotide Site by DNA Methyltransferases <i>in Vitro</i> and Acts as a DNA Demethylating Agent <i>in Vivo</i>
2009-03-17T00:00:00Z (GMT) by
Thiopurines are among the most successful chemotherapeutic agents for treating a number of human diseases including acute lymphoblastic leukemia. The mechanisms through which the thiopurines elicit their cytotoxic effects remain unclear. We postulate that the incorporation of 6-thioguanine into the CpG site may perturb the methyltransferase-mediated cytosine methylation at this site, thereby interfering with the epigenetic pathways of gene regulation. To gain biochemical evidence for this hypothesis, we assessed, by using a restriction enzyme digestion coupled with LC-MS/MS method, the impact of 6-thioguanine on cytosine methylation mediated by two DNA methyltransferases, human DNMT1 and bacterial <i>Hpa</i>II. Our results revealed that the incorporation of 6-thioguanine into the CpG site could affect the methylation of the cytosine residue by both methyltransferases and the effect on cytosine methylation is dependent on the position of 6-thioguanine with respect to the cytosine to be methylated. The presence of 6-thioguanine at the methylated CpG site enhanced the DNMT1-mediated methylation of the opposing cytosine in the complementary strand, whereas the presence of 6-thioguanine at the unmethylated CpG site abolished almost completely the methylation of its 5′ adjacent cytosine by both DNMT1 and <i>Hpa</i>II. We further demonstrated that the treatment of Jurkat T cells, which were derived from acute lymphoblastic leukemia, with 6-thioguanine could result in an appreciable drop in the level of global cytosine methylation. These results showed that 6-thioguanine, after being incorporated into DNA, may perturb the epigenetic pathway of gene regulation.