p33^(ING1b)增强骨肉瘤细胞U2OS对足叶乙甙的敏感性  

p33^(ING1b) Enhances Chemosensitivity of Osteosarcoma Cell U2OS to Etoposide

作　　者：朱晋军[1] 廖威明[1] 李佛保[1] 朱孝峰[2] 周军民[2] 刘宗潮[2] 

机构地区：[1]中山大学附属第一医院骨科骨肿瘤研究中心,广东广州510080 [2]中山大学肿瘤防治中心肿瘤研究所,广东广州510060

出　　处：《癌症》2004年第6期640-644,共5页Chinese Journal of Cancer

基　　金：国家自然科学基金(No.30070766)~~

摘　　要：背景与目的:作为一个新的抑癌基因,ING1与p53有许多相似的生物学功能,如细胞生长抑制、DNA修复、凋亡和化疗敏感性等。本实验目的在于研究p33ING1b对骨肉瘤细胞药物敏感性的影响并探讨其作用机制。方法:瞬时转染p33ING1b进入骨肉瘤细胞株U2OS后,用足叶乙甙(etoposide,VP-16)处理24h,然后采用台盼蓝拒染法计数活细胞数并计算细胞生长抑制率,流式细胞仪、DAPI染色等方法检测细胞凋亡率,Westernblot技术检测p53、p21WAF1、MDM2和Bax蛋白的表达。结果:台盼蓝染色结果表明,瞬时转染p33ING1b进入骨肉瘤细胞株U2OS后,再用VP-16处理24h,细胞生长抑制率明显增加(63.1±5.1)%。流式细胞计数和DAPI染色检测结果表明,细胞凋亡率明显增加(62.7%)。Westernblot检测结果显示,外源性p33ING1b高表达明显提高了p53及其下游基因p21WAF1和Bax蛋白的表达水平,转染p33ING1b进入骨肉瘤细胞株U2OS后,再用VP-16处理24h,p53、p21和Bax蛋白表达增加。在各实验组中,MDM2的蛋白表达没有明显变化。结论:p33ING1b能够上调p53蛋白,并上调p53下游因子——p21WAF1和Bax的表达水平,通过p53依赖性凋亡信号通路,提高骨肉瘤细胞U2OS对VP-16的敏感性。BACKGROUND &OBJECTIVE: As a new tumor suppressor gene, ING1 shared many biological functions with p53, such as cell cycle arrest, DNA repair, apop tosis, and chemosensitivity. The aim of this study was to investigate the effect of p33ING1b on chemosensitivity of osteosarcoma cells and its mechanism. METHOD S: p33ING1b was overexpressed in osteosarcoma cell line U2OS through transient t ransfection. After transfection, U2OS cells were treated with etoposide for 24 h ours, then cell growth inhibitory rates were detected by trypan blue exclusion a ssay, and apoptosis was assessed using flow cytometry analysis and fluorescent m icroscopy. Furthermore, the protein expression of p53, p21WAF1, MDM2 and Bax wer e determined by Western blot analysis. RESULTS: After transient transfection wit h p33ING1b vector for 24 hours, U2OS cells were treated with 20 靏/ml VP-16 fo r 24 hours. The results showed that the cell growth inhibitory rates strongly in creased [(63.1±5.1)%], and etoposide-induced apoptosis was increased(62.7 %). Ectopic overexpression of p33ING1b increased the protein expression of p53 and strongly enhanced the expression of endogenous p21WAF1 and Bax. Moreover, a fter transfection and treatment with 20 靏/ml VP-16, the protein expression of p53, p21WAF1, and Bax strongly increased compared with other groups. The protei n expression of MDM2 showed no significant difference. CONCLUSION: These observa tions suggest that p33ING1b up-regulates p53 protein, and cooperate with p53 in stimulating expression of p21WAF1 and Bax gene, thus to enhance etoposide-indu ced apoptosis via p53-dependent pathways.

关 键 词：骨肉瘤 p33^ISG1b 足叶乙甙 P53 化疗敏感性 

分 类 号：R738.1[医药卫生—肿瘤]