DNA损伤后细胞凋亡与周期停滞中Δ40P53的作用  

The role of Δ40P53 in apoptosis and cell cycle arrest after DNA damage

作　　者：刘道洁[1] 徐树莹[1] 丁渭[1] 宋凤丽[1] 汪志鹏[2] 张超[1] 乔录新[1] 

机构地区：[1]首都医科大学附属北京佑安医院北京市肝病研究所,100069 [2]首都医科大学基础医学院

出　　处：《北京医学》2013年第6期427-430,共4页Beijing Medical Journal

基　　金：国家自然科学基金面上项目(81272266)

摘　　要：目的探讨HCT116p53-/-细胞Δ40P53在脱氧核糖核酸(DNA)损伤时细胞凋亡和细胞周期停滞中的作用,明确Δ40P53与野生型P53的不同作用。方法荧光素酶报告基因和免疫印迹法检测Δ40P53在DNA损伤后转录活性和表达情况,流式细胞技术检测DNA损伤后细胞周期停滞情况,乳酸脱氢酶(LDH)检测分析DNA损伤后细胞凋亡情况,分析Δ40P53的生物学功能。结果 HCT116p53+/+细胞中bax和p21基因的启动子活性在甲烷磺酸甲酯(MMS)刺激时比无MMS刺激时高3～5倍,HCT116p53-/-细胞中bax和p21基因的启动子活性在有无MMS刺激时无显著不同;免疫印迹表明有和无MMS刺激的细胞Δ40P53保持在较高水平,野生型P53经MMS刺激过度表达;LDH法检测50μg/ml的MMS刺激后52.2%的HCT116p53+/+细胞凋亡,而只有32.5%的HCT116p53-/-细胞凋亡,差异有统计学意义(t=84.2,P<0.05);流式细胞仪检测MMS处理后88%的HCT116p53-/-细胞进入G2/S期细胞,66%的HCT116p53+/+细胞进入G2/S期细胞。结论经MMS刺激的HCT116p53-/-细胞的Δ40P53不能诱导细胞凋亡和细胞周期阻滞。Objective To investigate the role of △40P53 in apoptosis and cell cycle arrest after DNA damage in HCTll6 p53-/- cell, to identify the difference between △40P53 and wild-type P53. Methods Luciferase reporter gene and immunoblotting were used to detect transcriptional activity and expression of △40P53 after DNA damage; flow cytometry was used to confirm cell cycle arrest after DNA damage, lactate dehydrogenase detection was used to analyze cell apop- tosis after DNA damage, then biological functions of △40P53 was analyzed. Results The bax and p21 promoters activity had 3-5 folds higher in MMS stress than no stress at HCTll6 p53＋/＋. However, bax and p21 promoters activity had no significant difference between MMS and no-MMS stress at HCT116 p53-/- cells; immuno-blotting showed that △40P53 was keep in high level in both no-MMS and MMS stress and wtP53 was induced over expression by MMS; HCT116 p53-/- and HCTll6 p53＋/＋ cells were treated with 50 μg/ml MMS for 24 h and then cell death was detected by LDH method, 52.2% of HCT116 p53＋/＋ cells died, while only 32.5% of HCT116 p53-/- cells died, with significant difference （t = 84.2, P 〈 0.05）; flow cytometry showed that 88% of HCT116 p53-/- cell went into G2/S phase after cells were treated by DNA damage agent-MMS, only 66% of HCTll6 p53＋/＋ cell went into G2/S phase after cells were treated by DNA damage agent-MMS. Conclusion △40P53 in HCTll6 p53-/- cells failed to induce cell apoptosis and cell cycle arrest following MMS stress.

关 键 词：P53蛋白异构体(Δ40P53) DNA损伤 细胞凋亡 细胞周期停滞 

分 类 号：Q255[生物学—细胞生物学]