摘要目的 探讨p53凋亡刺激蛋白2(ASPP2)的磷酸化状态对ASPP2/p53凋亡通路活性的影响.方法 结肠癌HCT116细胞分别转染野生型ASPP2表达质粒(Aw)和非磷酸化突变型ASPP2质粒(Am),使细胞过表达野生型和非磷酸化突变型ASPP2蛋白.以奥沙利铂诱导HCT116细胞凋亡.annexin V/PI双染细胞后,以流式细胞术分析HCT116细胞的凋亡水平.Western blot法检测HCT116细胞中ASPP2蛋白的表达量和磷酸化状态.免疫共沉淀法检测ASPP2蛋白与p53的结合情况.结果 奥沙利铂能诱导HCT116结肠癌细胞凋亡以及ASPP2 ser92和ser361两个位点发生磷酸化.Aw和Am质粒转染的p53+/+HCT116细胞的凋亡率分别为(3.8±1.0)％和(3.9±1.2)％,与绿色荧光蛋白(GFP)质粒转染的p53+/+HCT116细胞的凋亡率[(4.0±0.8)％]差异无统计学意义(P＞0.05).但经过奥沙利铂处理后,Aw质粒转染的p53+/+HCT116细胞的凋亡率为(46.7±3.9)％,明显高于Am和GFP质粒转染的p53+/+HCT116细胞[分别为(40.1±10.2)％和(37.1±6.9)％,P＜0.05],而Am和GFP质粒转染组p53+/+HCT116细胞的凋亡率差异无统计学意义(P＞0.05).磷酸化的ASPP2通过p53依赖途径促进奥沙利铂诱导的HCT116细胞凋亡,而ASPP2的磷酸化状态影响其与p53的结合能力.结论 在奥沙利铂诱导的结肠癌HCT116细胞凋亡过程中,ASPP2的磷酸化状态调节p53的促凋亡功能.

abstractsObjective To investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2) phosphorylation status in the regulation of ASPP2-p53 apoptotic pathway activity.Methods Cells were individually transfected with green fluorescent protein (GFP)-encoding vector,constitutively nonphosphorylatable ASPP2 mutant-ASPP2 (Am)-encoding vector,and wild type ASPP2 (Aw)-encoding vector) plasmids,respectively,to make them overexpressing phosphorylated and non-phosphorylated ASPP2 proteins,respectively.Cell apoptosis was induced by oxaliplatin.The apoptosis rate of cells was determined by flow cytometry after staining with FITC-conjugated annexin V and PI.ASPP2 protein level and its phosphorylation status were observed by Western blot.The interaction between ASPP2 and p53 was observed by immunoprecipitation assay.Results Oxaliplatin induced cell apoptosis and caused phosphorylation of ASPP2 at ser92/ser361 in the HCT116 cells.The apoptosis rate of Aw and Am plasmids-transfected cells were (3.8 ± 1.0) ％ and (3.9 ± 1.2) ％ respectively,statistically with a non-significant difference (P ＞ 0.05) in comparison with that of the GFP plasmid-transfected cells [(4.0 ± 0.8) ％].After oxaliplatin treatment,the apoptosis rate of Aw plasmid-transfected cells was (46.7 ± 3.9) ％,significantly higher than that of the Am and GFP plasmid-transfected cells [(40.1 ± 10.2)％ and (37.1 ±6.9)％,respectively,P ＜0.05],however,there was no statistically significant difference (P ＞ 0.05) between Am and GFP plasmid-transfected cells.These results indicate that phosphorylated ASPP2 promoted the oxaliplatin-induced apoptosis of HCT116 cells through a p53-dependent pathway.Phosphorylation status of ASPP2 influenced its binding activity to p53.Conclusion Phosphorylation status of ASPP2 modulates p53 apoptotic function in oxaliplatin-induced apoptosis of colorectal cancer HCT116 cells.