Ⅰ型磷酸酶抑制亚基-1对人宫颈癌HeLa细胞增殖的抑制作用

PPI1(Inhibitor-1 ofprotein phosphatase 1)是I型磷酸酶的抑制亚基之一,其活化依赖于35位苏氨酸蛋白激酶(PKA)的磷酸化而发挥抑制作用.本研究目的在于探讨PPIl持续活化型突变体的表达对人宫颈癌细胞株增殖的影响及其可能的作用机制.利用PPI1野生型和活化型突变体表达质粒分别转染HeLa细胞,首先通过H~3TdR掺入实验、迁移实验观察PPI1基因对HeLa细胞增殖能力的影响,研究结果表明:在活化型突变体表达的HeLa细胞株中,细胞~3H掺入量和迁移能力明显受抑.其次通过流式细胞术、Giemsa染色法分析PPIl对HeLa细胞的细胞周期的影响;FACS分析表明HeLa细胞G2/M期比例明显升高;经脱氧胸苷同步化后,该组细胞进入有丝分裂期明显滞后.最后利用Western blot分析PPI1对MAPK信号转导通路的影响,Western blot分析显示该组细胞的ERK磷酸化水平明显下降.研究表明PPI1活化型突变体的表达可抑制人宫颈癌细胞株的增殖,这与其诱导HeLa细胞G2/M期停滞、有丝分裂的进入延缓有关,其中涉及到MAPK信号转导通路的活化受抑制.

Effect of PPI1 on Proliferation of Human Cervix Carcinoma Cell Line HeLa

Inhibitor-1 of protein phosphatase 1 (PPI1) is one of the regulatory subunits of protein phosphatase I.When phosphorylated by protein kinase A (PKA) at Thr~(-35),PPIl inhibited PPl activity.In this research,we attempted to reveal the effects of expression of activated mutant of PPIl (PPIl/sIl) on cell proliferation and to explore its possible mechanism.Here,we employed the expression plasmids harbouring with wild type and activated mutant of PPI1 gene to transfecte into human cervix carcinoma cell line HeLa,respectively.Firstly,cell proliferation was evaluated by ~3H]-TdR incorporation assay and migration assay.The result showed that the ~3H]-thymidine incorporation and migration were impaired in HeLa-PPI1/sI1 cells.The analysis of cell cycle of HeLa cells was analyzed by flow cytometry and Giemsa staining presented that the proportion of cells in G2/M phase was higher than the references in transient transfected HeLa cells with activated mutant of PPIl.After released from thymidine arrest,in HeLa-PPI1/sIl cells,the entry of mitosis was delayed,which was demonstrated by Giemsa staining,The further research on detecting the effects of PPI1 on MAPK signal transduction by immunoblotting.The result indicated that the level of ERK phosphorylation was declined sharply.To sum up,the expression of PPI1/sI1 suppresses proliferation of human cervix carcinoma cell line HeLa by inducing G2/M arrest and the delay in mitotic entry,which might involve in the inhibition of ERK activation.