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

目的 研究组蛋白乙酰化对Colo-320和SW1116人结肠癌细胞系p21^WAF1和p16^INKRA基因表达的影响。方法 培养人结肠癌细胞系SW1116和Colo-320,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p21^WAF1和p16^INKRA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-320细胞中均有较弱的p16^INKRA表达;两种结肠癌细胞系p21^WAF1表达缺如。5-aza-dC干预后,p16^INKRA表达增强,相反p21^WAF1仍无明显表达。当该两个细胞系经TSA或NaBu处理后,p21^WAF1转录水平明显上调,并诱导p21^WAF1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p21^WAF1基因相关染色质乙酰化水平,上调p21^WAF1基因的转录,并相应地导致结肠癌细胞生长停滞;而p16^INKRA基因表达主要受甲基化调节。

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

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.