PUMA在结肠癌细胞中的表达及诱导细胞凋亡中的作用

目的:通过5-Fu诱导携带有野生型p53基因的HCT116和携带有突变性p53基因的HT-29两种结肠癌细胞系,比较两株细胞在各时间点凋亡水平和PUMA mRNA表达情况的差异,探讨PUMA对细胞凋亡的作用及诱导其表达的基本途径。方法:用逆转录聚合酶链反应(RT—PCR)检测不同结肠癌细胞株HT-29、HCT116在抗肿瘤药物5-Fu作用下不同时间点结肠癌细胞株内PUMA mRNA表达水平的差异,用吖啶橙/溴化乙啶(AO/EB)荧光染色检测各时间点细胞的凋亡水平,分析其与PUMAmRNA表达水平之间的关系。结果:携带有野生型P53基因的结肠癌细胞株HCT116在5-Fu作用下6h即可出现PUMA mRNA的表达,随着药物作用时间的延长其表达强度增加,并且与细胞凋亡水平呈正相关;含有突变型P53基因的结肠癌细胞株HT-29在5-Fu作用下无PUMA的表达。结论:通过5-Fu诱导细胞凋亡出现的PUMA表达需要野生型P53基因,突变型P53基因无法诱导PUMA的表达。PUMA与结肠癌细胞凋亡水平呈正相关,是一种促凋亡蛋白。

The Expression of PUMA in Colon Carcinoma Cells and its Effect in Cell Apoptosis

Objective:To investigate the cell apoptosis effect of PUMA(=p53 up-regulated modulator of apoptosis)and its basic channel to be generated by inducing two colon carcinoma cell lines to apoptosis by 5-Fu(5-fluorouracil),and then com- pare the apoptosis levels and the expression of PUMA mRNA in different time spots after treated by 5-Fu.Methods:PUMA mRNA expression was detected by RT-polymerase chain reaction in HCT116 contained wt p53 and HT-29 cells contained mut p53 genes by using the DNA-damaging agent 5-Fu in different time spots,and apoptosis levels were detected by acridine orange/ ethidium bromide double staining(AO/EB)method at 1the same time.Results:6 hours after using the 5-Fu,PUMA mRNA expression was found to be induced in HCT116 cell following treatment with 5-Fu and enhanced apparently during the drug ac- tion time.Furthermore,the PUMA mRNA expression directly correlated with the cell apoptosis levels.No expression of the mRNA of PUMA was found in HT-29 within the 48 hours after treated by 5-Fu.Conclusions:The expression of PUMA is mainly de- pendent on intact p53;PUMA,induced by 5-Fu,may be a direct mediator of p53-associated apoptosis.PUMA would promote apoptosis in the early time and is likely to play a role in mediating cell death.