Ⅰ型磷酸酶抑制亚基-1对人宫颈癌HeLa细胞凋亡的影响  

Effect of Inhibitor-1 of Protein PhosphataseⅠ on Apoptosis of Human Cervical Carcinoma Cell Line HeLa

作　　者：曾麒燕[1] 秦雪[2] 张红[1] 

机构地区：[1]广西医科大学生物化学与分子生物学教研室,广西南宁530021 [2]广西医科大学第一附属医院临床医学实验中心,广西南宁530027

出　　处：《癌症》2007年第11期1177-1182,共6页Chinese Journal of Cancer

基　　金：广西自然科学基金(No.0542077)~~

摘　　要：背景与目的:Ⅰ型磷酸酶抑制亚基-1(inhibitor-1 of protein phosphataseⅠ,PPI1)依赖于35位苏氨酸的磷酸化而发挥抑制作用。本研究目的在于观察PPI1野生型和持续活化型突变体的表达对宫颈癌细胞株的凋亡及凋亡刺激敏感性的影响,并初步探讨其可能的作用机制。方法:通过Lipofectamine介导,用PPI1野生型和持续活化型突变体表达质粒分别转染HeLa细胞,以未转染的及转染空载体的HeLa细胞为对照组,用RT-PCR和Western blot鉴定目的蛋白的表达;通过克隆形成实验观察PPI1对细胞克隆形成能力的影响,用Hoechst33258荧光染色、亚二倍体分析法分析PPI1对HeLa细胞凋亡的影响,用流式细胞术分析转染PPI1后的HeLa细胞对肿瘤坏死因子(tumor-necrosis factor,TNF)凋亡刺激的敏感性,用Western blot分析PPI1对凋亡相关蛋白表达及MAPK信号转导通路的影响。结果:PPI1野生型和持续活化型突变体在HeLa细胞中能够有效表达。荧光染色和亚二倍体分析均表明PPI1持续活化型突变体的表达使细胞凋亡增多,并能明显抑制HeLa细胞的克隆形成能力。Western blot分析表明,PPI1持续活化型突变体可使促凋亡分子P53、P27、BAK表达上调,而抗凋亡分子Bcl-2和Bcl-xL的表达下调。流式细胞术分析表明PPI1持续活化型突变体表达的HeLa细胞在TNF-α刺激后细胞凋亡率为19.06%,而对照组为2.67%和1.89%。Westernblot分析表明,TNF刺激后,在JNK信号转导通路中,该组细胞JNK的磷酸化程度比对照组明显增强,而且持续的时间也较长;对于p38信号转导通路,则在TNF刺激30min后,p38出现磷酸化,而其他组均无。结论:PPI1持续活化型突变体的表达可诱导宫颈癌细胞株的凋亡,并能明显增强HeLa细胞对TNF凋亡刺激的敏感性,其中涉及到JNK、p38信号转导通路的活化增强。BACKGROUND ＆ OBJECTIVE, Inhibitor-1 of protein phosphatase Ⅰ （PPI1） is one of the regulatory subunits of protein phosphatase 1 （PP1）. When phosphorylated by protein kinase A （PKA） at Thr-35, PPI1 inhibits the activity of PP1. This study was to observe the effects of wild type and activated mutant of PPI1 （PPI1/wt and PPI1/sI1） on the apoptosis of human cervical carcinoma cell line HeLa, and to explore its possible mechanism. METHODS： Plasmids containing PPI1/wt or PPI1/sI1 were transfected into HeLa cells. Stable cell clones expressing PPI1 protein （HeLa-PPI1/wt and HeLa-PPI1/sI1） were selected by G418. Untransfected and mock-transfected HeLa cells were used as control. The expression of PPI1/wt and PPI1/sI1 were confirmed by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. Cell clonogenic ability was detected by colony formation assay. The morphologic alterations of the cells were observed with Hoechst 33258 staining. The apoptosis before and after stimulation of tumor-necrosis factor （TNF） was detected by flow cytometry. The expression of apoptosis-related proteins in the cells was detected by Western blot. RESULTS： PPI1/wt and PPI1/sI1 were efficiently expressed in HeLa cells. The clonogenic ability of HeLa-PPI1/sI1 cells was inhibited. The apoptosis of HeLa-PPI1/sI1 cells were enhanced. The expression of P53, P27 and BAK were up-regulated, while the expression of Bcl-2 and Bcl-xL were down-regulated. After TNF stimulation, the apoptosis rate was 19.06% for HeLa-PPI1/sI1 cells, 2.67% for HeLa-mock cells, and 1.89% for untransfected HeLa cells. PPI1/sI1 enhanced the phosphorylation of JNK, and induced the activation of P38 at 30 min after TNF stimulation. CONCLUSION： PPI1/sI1 could induce apoptosis of HeLa cells, and enhance the sensitivity of HeLa cells to TNF stimulation, which might enhance the activation of JNK and p38 signal transduction.

关 键 词：Ⅰ型磷酸酶 抑制亚基-1 HELA细胞 细胞凋亡 信号转导 肿瘤坏死因子 

分 类 号：R737.33[医药卫生—肿瘤]