FCCC LOGO Faculty Publications
Cairns P
5'-azacytidine expression arrays
Methods Mol Biol. 2009 ;507 :165-74
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Epigenetic silencing of a gene can be reversed, resulting in reactivation of expression, by drugs such as the DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5Aza-dC, azacytidine). This drug is added to cell culture media and is incorporated into the new strand during DNA replication in the cell. 5Aza-dC forms a covalent complex with the active sites of the DNA methyltransferase, depleting methyltransferase activity, which results in generalized demethylation. Until recently, global analyses of gene methylation in cancer cells were largely restricted to array or gel-based comparisons of the methylation status of CpG islands between normal and tumor cell DNA. An expression microarray-based screen has the advantage of a more genome-wide analysis with a better gene annotation and, coupled with a reactivation strategy, has the further advantage that it should preferentially identify reexpression of epigenetically silenced genes over methylated CpG islands that do not influence transcription. However, the direct reactivation of methylated genes, as well as secondary effects of azacytidine treatment, can lead to a cascade of deregulation in downstream unmethylated gene expression. A validation strategy is therefore the key for efficient identification of genes methylated in the wild-type cultured tumor cells. An azacytidine-based reactivation approach can only be used on cell lines so validation should include analysis of primary tumors. The potential of this approach for the identification of new hypermethylated genes and pathways has been demonstrated in bladder, colorectal, esophageal, and most other cancer types.
Cairns, Paul United States Methods in molecular biology (Clifton, N.J.) Methods Mol Biol. 2009;507:165-74.