Inhibition of histone deacetylation does not block resilencing of p16 after 5-aza-2′-deoxycytidine treatment
Inhibition of histone deacetylation does not block resilencing of p16 after 5-aza-2′-deoxycytidine treatment
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DOI：
10.1158/0008-5472.can-06-2845
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发表时间：
2007-01-01
期刊：
影响因子：
11.2
通讯作者：
Jones, Peter A.
中科院分区：
文献类型：
--
作者：
Egger, Gerda;Aparicio, Ana M.;Jones, Peter A.
表观遗传药物被用于各种人类癌症的临床试验，在重新激活因DNA甲基化和染色质修饰而沉默的基因方面效果显著。我们在此报告了一组正常成纤维细胞和癌细胞系在DNA甲基转移酶抑制剂5-aza-2'-脱氧胞苷（5-aza-CdR）和历史悠久的去乙酰化酶抑制剂4-苯基丁酸（PBA）联合治疗后的分析。低剂量联合用药引起T24膀胱癌细胞周期阻滞，高剂量联合用药引起T24膀胱癌细胞凋亡。在联合治疗后，p16 （CDKN2A/INK4）和p21 （CIP1/SDII/WAF1）的表达在正常细胞和癌细胞中以剂量依赖的方式被诱导至相似水平。我们发现，在LD419正常成纤维细胞和T24膀胱癌细胞中，转录起始位点附近赖氨酸9/14的组蛋白H3乙酰化明显增加，而p21位点的乙酰化变化不太明显。有趣的是，赖氨酸9上的组蛋白H3三甲基化水平在药物治疗后没有改变，赖氨酸9通常标记非活性染色质区域，并与沉默的T24细胞中的p16启动子相关。此外，我们提供的证据表明，在5-aza-CdR处理后，T24细胞中p16启动子CpG岛的再甲基化不能通过随后的连续PBA处理来阻止。p16基因的恢复动力学与仅处理5-aza-CdR的细胞相似，这也以转录起始位点组蛋白乙酰化的局部缺失为标志。总之，我们的数据为表观遗传药物的机制提供了新的见解，并对表观遗传治疗具有重要意义。
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 historic 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/SDII/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 only-treated 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.