PTEN诱导膀胱癌BIU-87细胞凋亡机制研究

PTEN induces apoptosis through its phosphatase activity in human bladder transitional carcinoma cells BIU-87

目的研究抑癌基因PTEN对人膀胱癌细胞株BIU-87细胞凋亡的影响,并探讨其相关机制。方法将携带野生型PTEN基因及两种突变型基因C124A-PTEN和G129E-PTEN的真核表达载体转染BIU-87细胞,Western blot检测PTEN蛋白表达及蛋白激酶B(PKB/Akt)、焦点黏附激酶(FAK)磷酸化水平的变化;应用流式细胞仪技术和激光共聚焦显微镜技术,分析转染和对照细胞在黏附状态下的凋亡情况。结果与对照组细胞相比,转染野生型PTEN的BIU-87细胞(WT-PT/87)FAK和Akt的磷酸化水平分别降低了59%(P<0.01)和89%(P<0.01),G129E-PT/87细胞内磷酸化FAK和磷酸化Akt的水平分别下降了62%(P<0.01)和46%(P<0.05),而C124APT/87细胞中FAK和Akt的磷酸化水平均无明显变化。WT-PT/87与G129E-PT/87细胞凋亡率分别升至(18.5±2.1)%(t=17.34,P<0.01)和(10.1±0.5)%(t=14.58,P<0.01),明显高于对照细胞,而C124A-PT/87与GFP/87细胞凋亡率无明显升高。结论抑癌基因PTEN诱导膀胱癌细胞的凋亡与其脂质磷酸酶抑制Akt的磷酸化有关。其蛋白磷酸酶活性参与调节Akt磷酸化,其机制可能与调节FAK的磷酸化有关。

Objective To investigate the effect and mechanism of tumor suppressor gene PTEN on apoptosis of human bladder transitional carcinoma cells BIU-87.Methods BIU-87 cells were transfected with GFP plasmids containing wild-type PTEN or mutant PTEN gene （G129E-PTEN, C124A-PTEN） in vitro. The PTEN expression and the phosphorylation level of focal adhesion kinase （FAK） and protein kinase B （Akt） were detected by Western blot. Flow cytometry assay and laser scanning confocal microscopy were used to analyze apoptosis on transfected cells.Results Compared with the control, the phosphorylation level of FAK and Akt in the cells transfected with wild-type PTEN decreased 59% （ P 〈0.01） and 89% （ P 〈0.01）, G129E-PT/87 （62%, P 〈0.01; 46%, P 〈0.05）, respectively; whereas the apoptosis rates increased to （18.5±2.1）% （ t =17.34, P 〈0.01） and （10.1±0.5）% （ t =14.58, P 〈 0.01）. However, in C124A-PT/87, neither the phosphorylation of FAK and Akt nor the anoikis rates had obviously changed, although the PTEN expression enhanced remarkably in comparison with the control cells.Conclusions Activity tumor suppressor gene PTEN can dephosphrylate FAK through its protein phosphatase and inhibit phosphorylation of Akt with a consequence of apoptosis of human bladder transitional carcinoma cells BIU-87 through its lipid phosphatase activity.