Zhu W G, Lakshmanan R R, Beal M D, Otterson G A
Department of Internal Medicine and the Comprehensive Cancer Center, The Ohio State University, Columbus 43210-1240, USA.
Cancer Res. 2001 Feb 15;61(4):1327-33.
Histone acetylation has long been associated with transcriptional activation, whereas conversely, deacetylation of histones is associated with gene silencing and transcriptional repression. Here we report that inhibitors of histone deacetylase (HDAC), depsipeptide and trichostatin A, induce apoptotic cell death in human lung cancer cells as demonstrated by DNA flow cytometry and Western immunoblot to detect cleavage of poly(ADP-ribose) polymerase. This HDAC inhibitorinduced apoptosis is greatly enhanced in the presence of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC). The HDAC inhibitor-induced apoptosis appears to be p53 independent, because no change in apoptotic cell death was observed in H1299 cells that expressed exogenous wild-type p53 (H1299 cells express no endogenous p53 protein). To further investigate the mechanism of DAC-enhanced, HDAC inhibitor-induced apoptosis, we analyzed histone H3 and H4 acetylation by Western immunoblotting. Results showed that depsipeptide induced a dose-dependent acetylation of histones H3 and H4, which was greatly increased in DAC-pretreated cells. By analyzing the acetylation of specific lysine residues at the amino terminus of histone H4 (Ac-5, Ac-8, Ac-12, and Ac-16), we found that the enhancement of HDAC inhibitor-induced acetylation of histones in the DAC-pretreated cells was not lysine site specific. These results demonstrate that DNA methylation status is an important determinant of apoptotic susceptibility to HDAC inhibitors.
长期以来，组蛋白乙酰化一直与转录激活相关，相反，组蛋白去乙酰化则与基因沉默和转录抑制相关。在此我们报告，组蛋白去乙酰化酶（HDAC）抑制剂、缩肽和曲古抑菌素A可诱导人肺癌细胞发生凋亡性细胞死亡，这通过DNA流式细胞术和蛋白质免疫印迹法检测聚（ADP - 核糖）聚合酶的裂解得以证实。在DNA甲基转移酶抑制剂5 - 氮杂 - 2'-脱氧胞苷（DAC）存在的情况下，这种HDAC抑制剂诱导的凋亡会大大增强。HDAC抑制剂诱导的凋亡似乎不依赖p53，因为在表达外源性野生型p53的H1299细胞（H1299细胞不表达内源性p53蛋白）中未观察到凋亡性细胞死亡的变化。为了进一步研究DAC增强的HDAC抑制剂诱导凋亡的机制，我们通过蛋白质免疫印迹法分析了组蛋白H3和H4的乙酰化情况。结果显示，缩肽诱导组蛋白H3和H4发生剂量依赖性乙酰化，在DAC预处理的细胞中这种乙酰化大大增加。通过分析组蛋白H4氨基末端特定赖氨酸残基（Ac - 5、Ac - 8、Ac - 12和Ac - 16）的乙酰化情况，我们发现DAC预处理细胞中HDAC抑制剂诱导的组蛋白乙酰化增强并非赖氨酸位点特异性的。这些结果表明，DNA甲基化状态是对HDAC抑制剂凋亡敏感性一个重要的决定因素。
