NF-κB介导的EBV-LMP1在Rat-1细胞转化和成瘤中的作用

背景与目的:EB病毒潜伏性膜蛋白1(latent membrane protein 1,LMP1)是病毒基因组编码的致瘤蛋白.本研究拟探讨LMP1在细胞转化和致瘤过程中的作用机制.方法:采用基因重组方法构建LMP1和显性负突变IкBα逆转录病毒质粒pLNSX-LMP1和pBabe-IкBα,分别与含有转录因子NF-кB启动子的荧光素酶表达质粒共转染293细胞,单光子检测仪测定LMP1对NF-кB的活化作用及IкBα对NF-кB的抑制作用;同时将2种重组病毒载体分别导入PA317细胞包装,获取2种逆转录病毒(retrovirus,RV),单独(RV-LMP1)或先后(先RV-LMP1后RV-IкBα)感染体外培养的Rat1细胞,检测转导细胞的集落形成能力及裸鼠成瘤能力.结果:当pBabe-IкBα与pLNSX-LMP1以1∶1共转染,IкBα能使LMP1活化NF-кB的作用降低75%;当以3∶1共转染,LMP1的活化作用几乎全部被抑制.IкBα能明显抑制RV-LMP1感染细胞的恶性表型:平皿克隆形成数从368±7和287±17分别降至59±6和8±2(n=3,P＜0.001);软琼脂集落形成数从477±13和347±10分别降至61±15和95±7(n=3,P＜0.001);裸鼠成瘤能力显著降低,瘤体积自(1.61 0.23)cm3降至(0.20±0.08)cm3(n=5,P＜0.001).结论:EB病毒LMP1促Rat1细胞恶性转化作用主要依赖NF-кB的活化.

Effects of Epstein-Barr virus latent membrane protein 1 mediated by nuclear factor-kappaB on transformation and tumorigenesis of rat-1 cells

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is an oncoprotein coded by EBV genome. This study was to investigate the effects of EBV LMP1 on transformation and tumorigenesis of Rat-1 cells. METHODS: Retrovirus plasmids pLNSX-LMP1 and pBabe-IkappaBalpha, constructed by gene recombination technique, were cotransfected respectively with nuclear factor-kappaB luciferase reporter (pNF-kappaB-luc) into 293 cells. The actions of LMP1 in activating NF-kappaB and IkappaBalpha in inhibiting NF-kappaB were measured by luciferase activity assay. Moreover, pLNSX-LMP1 and pBabe-IkappaBalpha were transfected respectively into the ecotropic retrovirus packaging cell line PA317 to generate LMP1 retrovirus (RV-LMP1) and IkappaBalpha retrovirus (RV-IkappaBalpha). After Rat-1 cells were infected by RV-LMP1 alone or RV-LMP1 combined RV-IkappaBalpha, their malignant transformation phenotype was detected by colony forming assay and nude mice tumorigenicity assay. RESULTS: When pLNSX-LMP1 and pBabe-IkappaB were cotransfected at a ratio of 1:1, IkappaBalpha inhibited LMP1-mediated NF-kappaB activation by 75%? and at a ratio of 3:1, it almost completely inhibited LMP1-mediated NF-kappaB activation. IkappaBalpha obviously inhibited LMP1-mediated malignant phenotype of Rat-1 cells:colony formation number on plates were significantly decreased from (368+/-7)/well and (287+/-17)/well to (59+/-6)/well and (8+/-2)/well (P<0.001). Foci in soft agarose were decreased from (477+/-13)/well and (347+/-10)/well to (61+/-15)/well and (95+/-7)/well (P<0.001). The ability of tumorigenicity in nude mice was markedly decreased: tumor volume was decreased from (1.61+/-0.23) cm3 to (0.20+/-0.08) cm3 (P<0.001). CONCLUSION: EBV-LMP1 could lead to transformation and tumorigenesis of Rat-1 cells by activating NF-kappaB.