I specialize in epigenetic oncology and if I understand your question, then I would say that what needs to be appreciated here is that the potential for global demethylation does not necessarily entail global reactivation; it is a necessary but not sufficient condition. Let me explain.

Adam Karpf and colleagues at the Huntsman Cancer Institute of the University of Utah were the first to eloquently demonstrate the limited gene activation in epithelial cells - both tumor and normal - treated with the DNMT inhibitor 5-AZA (5-aza-2'-deoxycytidine; commercial Decitabine), using genome-wide expression analysis with microarrays to analyze the response of three tumor and one normal epithelial cell line.

[Karpf AR, Lasek AW, Ririe TO, et al. Limited gene activation in tumor and normal epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. Mol Pharmacol 2004 Jan; 65(1):18-27. At: http://molpharm.aspetjournals.org/content/65/1/18.full]

The key - and surprising - finding was that treatment with 5-AZA induced / activated only a limited number of genes (mean, 0.67%; range, 0.17-1.8%) of the 25,940 genes screened in each of the epithelial cell lines tested. This is clearly a far more limited scope of gene expression changes than would be otherwise expected from a DNMT inhibitor, an agent which by definition participates in and perturbs a fundamental transcriptional regulatory mechanism, essentially "the way of all DNMT inhibitors". It would have been expected that DNMT induction of demethylation and reactivation of silenced genes would exhibit a broader spectrum of applicability to gene expression modifications than at a level of less than 1% mean.

These unexpected results led the investigators to try to discover a mechanism that would account for the highly limited extent of gene activation in 5-AZA treated cells. It was seen that 5-AZA-mediated gene activation requires both (1) not only the customary inhibition of DNA methylation but also (2) the presence of trans-factors mediating the activation of hypomethylated target genes.

This is a breakthrough conclusion: that gene expression changes require both the DNMT inhibition activity of 5-AZA as well as transcriptional activators, shown to be essential beyond just promoter demethylation. Thus , any model of 5-AZA-induced gene reactivation requires:

1) the reversal of promoter DNA hypermethylation, AND

2) the presence of (sequence-specific) transcriptional activators competent for activation of the target promoter.

And this in turns posits three distinct states of gene activation: silenced, activated, and a state of permissive. The key mechanism here is that 5-AZA must be seen on this model to be sufficient on its own only to induce a transcriptionally permissive state, but for specific gene activation to occur, sequence-specific transcription activators, aka trans-factors, must be present that are competent for the activation of target promoter. The key ingredient becomes these trans-factors or activators for the conversion of a permissive promoter into an activated one. To sum:

Gene Activation = DNMT inhibition + transcriptional activation.

Constantine Kaniklidis

Director of Medical Research, No Surrender Breast cancer Foundation (NSBCF)

European Association for Cancer research (EACR)

Thanks for the interesting answer.

Hello Constantine Kaniklidis,

Could you please tell me the ideal treatment condition for using HDAC inhibitor/ methyl transferase inhibitor on cells? I have 5 azacytidine which is from sigma. The data sheet says that it is highly unstable in aqueous solutions. But most of the research paper states that they treat cells for more than 48hrs. why is it so?  If it is unstable, what is the point in treating cells beyond 24 hrs? Does these inhibitors require longer time to carry out their function?