Inhibition of Histone Deacetylation Does Not Block Resilencing of p16 after 5-Aza-2′-Deoxycytidine Treatment

Abstract

Epigenetic drugs are in use in clinical trials of various human cancers and are potent at reactivating genes silenced by DNA methylation and chromatin modifications. We report here the analysis of a set of normal fibroblast and cancer cell lines after combination treatment with the DNA methyltransferase inhibitor 5-aza-2 ¶-deoxycytidine (5-aza-CdR) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA). Low doses of the drug combination caused cell cycle arrest, whereas high doses induced apoptosis in T24 bladder carcinoma cells. Both p16 (CDKN2A/INK4) and p21 (CIP1/SDI1/WAF1) expression were induced to similar levels in normal and cancer cells in a dose-dependent fashion after combination treatments. We detected a distinct increase of histone H3 acetylation at lysine 9/14 near the transcription start sites, in both LD419 normal fibroblasts and T24 bladder carcinoma cells, whereas the acetylation changes in the p21 locus were less apparent. Interestingly, the levels of trimethylation of histone H3 on lysine 9, which usually marks inactive chromatin regions and was associated with the p16 promoter in silenced T24 cells, did not change after drug treatments. Furthermore, we provide evidence that the remethylation of the p16 promoter CpG island in T24 cells after 5-aza-CdR treatment cannot be halted by subsequent continuous PBA treatment. The p16 gene is resilenced with kinetics similar to 5-aza-CdR onlytreated cells, which is also marked by a localized loss of histone acetylation at the transcription start site. Altogether, our data provide new insights into the mechanism of epigenetic drugs and have important implications for epigenetic therapy. [Cancer Res 2007;67(1):346-53]

Combined inhibition of DNA methylation and histone acetylation enhances gene re-expression and drug sensitivity in vivo

“…Although belinostat increases the level of re-expression of both MLH1 and MAGE-A1 it does not appear to alter the kinetics of re-expression. This is consistent with the observation that the HDAC inhibitor 4-phenylbutyric acid does not inhibit re-silencing of p16 after decitabine treatment (Egger et al, 2007). Re-expression of MAGE-A1 is transient.…”

“…A study of the combination of decitabine and trichostatin A on MLH1 expression also concluded that the effect of the HDAC inhibitor was not due to a further reduction in DNA methylation (Cameron et al, 1999). It is possible that the HDAC inhibitor allows increased access of the transcription factors to the demethylated gene as a result of increased levels of histone acetylation and the resulting chromatin remodelling (Egger et al, 2007).…”

Dynamics of bivalent chromatin domains upon drug induced reactivation and resilencing in cancer cells

“…Our results suggest that DNA methylation also contributes to maintain the repressive histone code in genes poised for silencing and confirm the role of DNA methylation as the foremost player in preserving the inactivation of silenced genes. 38 Besides that, retention of bivalent signatures probably acts as a memento and is likely to participate in the restoration of the silencing. 6 Another interesting finding arises from the analysis of DNA methylation in different chromatin fractions and in various experimental conditions.…”

Differentiation and adaptation epigenetic networks: Translational research in gastric carcinogenesis

“…For example, p16 inactivation by methylation in cancer cells is very stable. Methylation of p16 CpG islands can be gradually restored within about two-weeks after demethylation induced by DNMT1 inhibitor treatment, with or without combined treatment of HDAC inhibitor [26]. After the p16-methylated cell line AGS is fused with the p16-unmethylated cell line MGC803, we find that the methylation status of p16 CpG islands is very consistent in fusion cells.…”