Abstract
The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.
Highlights
IntroductionGlobal hypomethylation is accompanied by hypermethylated and transcriptionally silenced tumor suppressor genes
Aberrant DNA methylation is a major hallmark of cancer [1,2,3]
We used genome-scale Infinium analysis to systematically characterize the demethylation responses after AZA and DAC treatment in two human cancer cell lines. To this end we investigated methylation levels of more than 27,000 CG dinucleotides representing more than 14,000 genes [19] in HCT116 colon cancer cells and in HL-60 myeloid leukemia cells
Summary
Global hypomethylation is accompanied by hypermethylated and transcriptionally silenced tumor suppressor genes. These so-called epimutations contribute to the loss of proliferation control in cancer cells [4,5,6]. The archetypal DNMT inhibitors 5azacytidine (azacytidine, AZA) and 29-deoxy-5-azacytidine (decitabine, DAC) have been approved for the treatment of myelodysplastic syndrome, a preleukemic bone marrow disorder. Despite their use in the clinic and in numerous preclinical studies, the knowledge of the mode of action of these drugs is still incomplete [11]
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