Gadd45表达对肿瘤细胞HCT116生长的抑制作用及其机理

目的利用Gadd45可诱导细胞系探讨Gadd45对肿瘤细胞生长抑制作用的分子机理。方法用四环素撤除表达系统以及克隆形成实验、流式细胞检测、TUNEL法检测、Western印迹检测等方法研究Gadd45表达对肿瘤细胞HCT116生长的抑制作用及其机理。结果用四环素撤除表达系统,建立了稳定传代的Gadd45可诱导细胞系HCT116。撤除四环素,HCT116细胞的Gadd45能够被诱导高表达。实验结果证实,在我们建立的细胞系中,Gadd45诱导高表达对细胞生长的抑制率高于85％。流式细胞检测表明,Gadd45高表达有细胞G2-M期阻滞作用。同时,TUNEL法检测到Gadd45诱导的细胞凋亡,Western印迹检测观察到PARP和半胱氨蛋白水解酶3蛋白质剪切激活。结论Gadd45高表达可以抑制HCT116细胞生长,其机理主要是通过Gadd45诱导细胞G2-M期阻滞和激活细胞凋亡途径起作用。

Suppression of HCT116 growth of cells by Gadd45

OBJECTIVE: To study the mechanism of suppression of growth of HCT116 human colon carcinoma cells by Gadd45 gene. METHODS: HCT116 human colon carcinoma cells were transfected with pTRE-Gadd45 vector so as to establish Gadd45-inducible cell line that was cultured in medium with tetracycline. Then tetracycline was withdrawn. The number of cell clones was counted. Flow cytometry was used to detect the percentages of cells at the G1, S, and G2-M phases. TUNEL technique was used to detect the apoptosis of cells. Western blotting was used to analyze the expression of Gadd45 protein, and apoptosis-related proteins: poly-ADP-ribose polymerase (PARP), and caspase 3 protein. RESULTS: Gadd45 protein was not expressed in the HCT116 cells cultured in the medium with tetracycline, however, it was expressed with a gradually increased level in the cells cultured in the medium from which tetracycline was withdrawn, The clone formation rate of HCT116 cells was 100% in the medium with tetracycline, however, was only 14.2% in the medium with tetracycline withdrawal, with a suppression rate of more than 85%. The percentage of cells in G(2)-M phase was significantly increased in the cells cultured in the medium with tetracycline withdrawal. 24-36 hours after the withdrawal of tetracycline, PARP and caspase 3 protein were activated remarkably. CONCLUSION: High expression of Gadd45 inhibits the growth of HCT116 cells, through inducing G2-M arrest and activating apoptotic pathway.