人结肠癌细胞中组蛋白乙酰化对细胞周期调节基因表达的影响  

Regulation of histone acetylation on the expression of cell cycle-associated genes in human colon cancer cell lines

作　　者：陈萦晅[1] 房静远[1] 陆娟[1] 邱德凯[1] 

机构地区：[1]上海第二医科大学附属仁济医院,上海市消化疾病研究所200001

出　　处：《中华医学杂志》2004年第4期312-317,共6页National Medical Journal of China

基　　金：国家自然科学基金资助项目 (3 0 170 413 ) ;高等学校全国优秀博士学位论文作者专项基金资助项目 (199946) ;上海市教委"曙光计划"课题经费资助项目 (0 2SG45 )

摘　　要：目的　研究组蛋白乙酰化对Colo 32 0和SW1116人结肠癌细胞系p2 1WAF1和p16 INK4A基因表达的影响。方法　培养人结肠癌细胞系SW1116和Colo 32 0 ,分别以去甲基化制剂 5 氮脱氧胞苷 (5 aza dC)和 /或组蛋白脱乙酰化酶 (HDAC)抑制剂曲古抑菌素 (TSA)及丁酸钠 (NaBu)干预细胞。运用流式细胞术检测细胞周期变化 ;以定量逆转录 聚合酶链反应 (RT PCR)法研究控制细胞周期的基因p2 1WAF1和p16 INK4A的表达 ;染色质免疫沉淀技术分析基因相关染色质乙酰化组蛋白的水平。结果　TSA或NaBu使人结肠癌细胞阻滞于G1期 ,而 5 aza dC并不能改变细胞周期。正常情况下 ,SW1116和Colo 32 0细胞中均有较弱的p16 INK4A表达 ;两种结肠癌细胞系p2 1WAF1表达缺如。 5 aza dC干预后 ,p16 INK4A表达增强 ,相反p2 1WAF1仍无明显表达。当该两个细胞系经TSA或NaBu处理后 ,p2 1WAF1转录水平明显上调 ,并诱导p2 1WAF1基因相关染色质乙酰化组蛋白H3和H4的积聚。结论　两种人结肠癌细胞系中 ,HDAC抑制剂通过选择性增加p2 1WAF1基因相关染色质乙酰化水平 ,上调p2 1WAF1基因的转录 ,并相应地导致结肠癌细胞生长停滞 ;而p16 INK4A基因表达主要受甲基化调节。Objective To investigate the effects of histone acetylation on the expression of p21 WAF1 and p16 INK4A genes in two human colon cancer cell lines. Methods Two colon cancer cell lines (SW1116 and Colo -320) were treated with the DNA methyltransferase (DNMT) inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC) and/or the histone deacetylase (HDAC) inhibitor, trichostatin A(TSA)or sodium butyrate(NaBu). The cell cycle distribution was studied by flow cytometry (FCM). The expression of p21 WAF1 and p16 INK4A genes mRNA was detected by real-time RT-PCR. The level of acetylated histones in chromatin associated with the p21 WAF1 and p16 INK4A genes was examined by chromatin immunoprecipitation (ChIP) assay. Results TSA or NaBu blocked cells mainly in the G 1 phase, whereas 5-aza-dC treatment failed to affect cell cycle distribution. Expression of p16 INK4A was detected slightly and p21 WAF1 was not expression in SW1116 and Colo-320 cells before treatment. In SW1116 and Colo-320 cells, the expression of p16 INK4A gene was markedly increased after treatment of 5-aza-dC, although 5-aza-dC treatment did not activate the expression of p21 WAF1 gene. Treatment of TSA and NaBu resulted in the significant over-expression of p21 WAF1 in these two cell lines and induced an accumulation of acetylate histones H3 and H4 in chromatin associated with p21 WAF1 gene. Conclusion In these two human colon cancer cell lines, HDAC inhibitors stimulate the p21 WAF1 gene expression by selectively increasing the degree of acetylation of the gene-associated histones, and induce a G 1 cell cycle arrest. The expression of the p16 INK4A gene is regulated by DNA methylation.

关 键 词：结肠癌 组蛋白乙酰化酶 细胞周期 基因表达调控 表观遗传修饰 

分 类 号：R735.3[医药卫生—肿瘤]