鼻咽癌细胞中EB病毒编码的潜伏膜蛋白1活化cyclinD1的表达  

The Epstein-Barr Virus Encoded Latent Membrane Protein 1(EBV-LMP1) Activates Expression of CyclinD1

作　　者：赵晓荣[1] 王承兴[1] 罗非君[1] 顾焕华[1] 唐敏[1] 夏林庆[1] 邓琳[1] 易薇[1] 邓锡云[1] 曹亚[1] 

机构地区：[1]中南大学湘雅医学院肿瘤研究所,长沙410078

出　　处：《生物化学与生物物理进展》2001年第5期704-710,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金杰出青年基金 (3 95 2 5 0 2 2 );国家重点基础研究发展规划 (973 )项目 (G19980 5 12 0 1)~~

摘　　要：为了探讨EB病毒编码的潜伏膜蛋白 1(EBV LMP1)促进细胞增殖 ,参与EBV相关疾病致瘤的分子机制 ,研究了LMP1在鼻咽癌细胞中调节cyclinD1表达 ,进而影响细胞周期行进及细胞恶性表型改变 ,并初步确定了LMP1发挥该功能的结构域 .利用已建株的Tet on LMP1 HNE2鼻咽癌细胞系 ,蛋白质印迹实验分析LMP1诱导cyclinD1蛋白质表达的表达动力学 ,包括时间效应及剂量效应 ;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照 ,确定LMP1活化cyclinD1表达的结构域 .同时结合基因诱导表达及反义寡聚核酸技术阻断基因表达的实验方法 ,进一步确定LMP1上调的cyclinD1功能 ,即对细胞周期行进及细胞恶性表型的影响 .结果表明LMP1确实可以诱导cyclinD1的表达 (2～ 4倍 ) ,且诱导具有时间依赖性及剂量依赖性 ;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照 ,结合报道基因分析法 ,确定与空白载体细胞系比较 ,野生型LMP1从转录水平可反式激活cyclinD1报道基因活性约 11 2倍 ,其中CTAR1及CTAR2均可活化cyclinD1表达 ,但以CTAR2为主 ,与野生型LMP1诱导cyclinD1反式激活活性比较 ,CTAR1缺失导致cyclinD1报道基因活性下降 2 3 6 % ,CTAR2缺失导致cyclinD1活性下降约 80 7% ,C端均缺失时cyclinD1活性只?Although LMP1 is expressed in the majority of Nasopharyngeal carcinoma (NPC), the effect of LMP1 on cellular gene expression and its contribution to the cell growth and the development of malignancy is largely unknown. CyclinD1 expression activated by LMP1 was studied. A dual-stable LMP1 integrated NPC cell line with Tet-on regulating system, designated as Tet-on-LMP1 HNE2 was used to gain insight into the cell kinetics of the induction of cyclinD1 with Western blotting. The expression of LMP1 in Tet-on-LMP1-HNE2 was tightly regulated by tetracycline or its derivation, doxycycline. LMP1 has two essential signaling domains with the carboxy terminus, termed C-terminal activation regions1 (CTAR1) and CTAR2. With cell lines stably expressed wild type LMP1, the vector or various deletion mutants and reporter gene assay, the activation essential domains of LMP1 activation cyclinD1 was also identified. The progression of cell cycle was determined by flow cytometry and soft agar assay was done to indicate that the cyclinD1 induced by LMP1 is functional. The results indicates that LMP1 induced cyclin D1 protein expression in both dose-dependent and time-dependent manner with Western blotting analysis in Tet-on-LMP1-HEN2 cell line. Reporter gene assay revealed that wild type LMP1 also can induce cyclinD1 expression at the transcriptional level via trans-activation compared to the control (11.2 fold). LMP1 deletion mutants lacking either CTAR1 or CTAR2 or both the CTAR1 and CTAR2 deletion mutants had a decreased ability to induce cyclin D1 expression (76.4%, 19.3%, 17.7%). The results of flow cytometry analysis pointed to a cell cycle arrest at the G0/G1 phase compared to doxycycline negative Tet-on-LMP1-HNE2 (66.42% to 56.55%). Compared with cultured with sense PS-ODN-LMP1 (30.48%), cultured with antisense PS-ODN-LMP1 and cyclinD1 showed profound decrease in colony formation ( 15.21%, 21.76%). This is the first report showing that cyclin D1 expression could be activated by a viral protein, LMP-1. This novel finding may thu

关 键 词：EB病毒 潜伏膜蛋白1 周期蛋白D1 鼻咽癌细胞 

分 类 号：R739.63[医药卫生—肿瘤] R373[医药卫生—临床医学]