表型遗传修饰对人结肠癌细胞周期和抑癌基因表达的调控

Regulation of cell cycle progression and expression of tumor suppressor genes in human colonic cancer cells by epigenetics

目的 分析和探讨同一结肠癌细胞系DNA甲基化和组蛋白乙酰化对抑癌基因表达和细胞周期的影响。方法 培养结肠癌细胞HT 2 9、SW 1116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trichostatinA(TSA)及丁酸盐干预细胞。提取基因组DNA和RNA ,部分行甲基化特异性PCR(MSP)检测 p16 INK4A基因启动子区甲基化情况 ;以RT PCR研究p16 INK4A和p2 1WAF1mRNA表达水平 ;同时以流式细胞仪分析SW 1116和Colo 32 0细胞周期。结果 干预前 ,HT 2 9、SW 1116和Colo 32 0三种结肠癌细胞系中均有较弱的 p16 INK4A表达 ;SW 1116和Colo 32 0细胞的p2 1WAF1表达缺如。对于HT 2 9细胞 ,1μmol/L的 5 aza dC干预 2 4h后 ,p16 INK4A基因启动子区甲基化水平明显降低而mRNA水平显著增高 ,10 μmol/L干预时则无显著改变。在SW 1116和Colo 32 0细胞中 ,5 aza dC干预后 p16 INK4A表达增强 ,且以 10 μmol/L或 5 μmol/L浓度干预 2 4h者为最明显 ,相反p2 1WAF1仍无明显表达。该两个细胞系经TSA或丁酸盐处理后 ,p2 1WAF1转录水平显著上调。另外 ,该两个细胞系中 ,5 aza dC并不能改变细胞周期 ,而TSA或丁酸盐使细胞阻滞于G1期。结论 三种人结肠癌细胞中 ,p16 INK4A基因的表达均?

Objective To investigate the effects of DNA methylation and histone acetylation on cell cycle progression and expression of tumor suppressor genes in human colonic cancer cells. Methods Three colonic cancer cell lines(HT 29,SW1116 and Colo 320) were treated with the DNA methylation inhibitor, 5 aza 2' deoxycytidine(5 aza dC) and/or the histone deacetylase(HDAC) inhibitor, trichostatin A(TSA)and sodium butyrate. The methylation status of the promoter of the p16 INK4A gene was assayed by methylation specific PCR(MSP). The expression of p16 INK4A and p21 WAF1 were analyzed by RT PCR. Cell cycle distribution was studied by flow cytometry. Results p16 INK4A expression was weakly detected in three colon cancer cells (HT 29,SW1116 and Colo 320) and p21 WAF1 expression was not detected in SW1116 and Colo 320 cells before treatment. The methylation status of the promoter of the p16 INK4A gene was significantly decreased and mRNA expression was markedly increased in HT 29 cells after treatment with 1 μmol/L but not 10 μmol/L of 5 aza dC for 24 hours. In SW1116 and Colo 320 cells, the expression of p16 INK4A was obviously enhanced at 10 μmol/L or 5 μmol/L of 5 aza dC for 24 hours. However, p21 WAF1 gene expression has not been detected. Interestingly, after treatment of TSA or sodium butyrate, the transcription of p21 WAF1 was significantly up regulated in these two cell lines. In addition, 5 aza dC did not affect cell cycle distribution, but TSA or sodium butyrate blocked cells mainly in the G 1 phases. Conclusions The expression of p16 INK4A gene was regulated by DNA methylation in three human colonic cancer cells. The expression of p21 WAF1 gene was regulated by histone acetylation in SW1116 and Colo 320. In these two cell lines, histone hyperacetylation causes a G 1 cell cycle arrest.