title: 6-Thioguanine perturbs cytosine methylation at the CpG dinucleotide site by DNA methyltransferases in vitro and acts as a DNA demethylating agent in vivo.
abstract: 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 HpaII. 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 HpaII. 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.
authors.affiliation: Department of Chemistry, University of California, Riverside, California 92521-0403, USA.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Wang
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D003596:Cytosine / Q000378:metabolism*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D000077209:Decitabine; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013866:Thioguanine / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D001374:Azacitidine; D003596:Cytosine; D004247:DNA; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D015257:DNA-Cytosine Methylases; D000077209:Decitabine; D015226:Dinucleoside Phosphates; D009838:Oligodeoxyribonucleotides; D013866:Thioguanine; C015772:CpG dinucleotide; C029641:DNA modification methylase HpaII; C000619380:DNMT1 protein, human