CAJ | 학술논문

目的阐明鼻咽癌细胞中EB病毒编码的潜伏膜蛋白1(LMP1)活化核转录因子NF-κB的机制.方法利用强力霉素Dox诱导表达LMP1的鼻咽癌细胞株Tet-on-LMP1-HNE2为实验模型,首先应用免疫印迹方法测定Dox诱导后不同时相LMP1的表达动力学以及IκBs蛋白量及功能的改变.进而用间接免疫荧光法检测NF-κB的亚细胞定位.最后采用瞬间共转染及报道基因活性分析分析NF-κB的活性.结果在鼻咽癌细胞Tet-on-LMP1-HNE2中,Dox处理15分钟后LMP1的表达迅速升高并维持与较高水平直至120分钟.LMP1的诱导性表达导致IκBα的磷酸化并降解,但IκBα蛋白总量无改变.继IκBα的磷酸化并降解,NF-κB(P65)自胞浆易位至胞核且活性升高.IκBα的显性负性突变子抑制NF-κB(P65)的核易位及报道活性.LMP1的诱导性表达并未引起IκBβ蛋白水平变化.结论在鼻咽癌细胞Tet-on-LMP1-HNE2中,EB病毒LMP1通过IκBα的磷酸化并降解激活核转录因子NF-κB的活性,并且,LMP1诱导的NF-κB 活性能被IκBα的显性负性突变子完全抑制.IκBβ在此信号传导途径中无改变.LMP1表达前后IκBα蛋白总量维持恒定可能是由于NF-κB的活化迅速启动了IκBα的重头合成这一自身调节环路所致.
목적천명비인암세포중EB병독편마적잠복막단백1(LMP1)활화핵전록인자NF-κB적궤제.방법이용강력매소Dox유도표체LMP1적비인암세포주Tet-on-LMP1-HNE2위실험모형,수선응용면역인적방법측정Dox유도후불동시상LMP1적표체동역학이급IκBs단백량급공능적개변.진이용간접면역형광법검측NF-κB적아세포정위.최후채용순간공전염급보도기인활성분석분석NF-κB적활성.결과재비인암세포Tet-on-LMP1-HNE2중,Dox처리15분종후LMP1적표체신속승고병유지여교고수평직지120분종.LMP1적유도성표체도치IκBα적린산화병강해,단IκBα단백총량무개변.계IκBα적린산화병강해,NF-κB(P65)자포장역위지포핵차활성승고.IκBα적현성부성돌변자억제NF-κB(P65)적핵역위급보도활성.LMP1적유도성표체병미인기IκBβ단백수평변화.결론재비인암세포Tet-on-LMP1-HNE2중,EB병독LMP1통과IκBα적린산화병강해격활핵전록인자NF-κB적활성,병차,LMP1유도적NF-κB 활성능피IκBα적현성부성돌변자완전억제.IκBβ재차신호전도도경중무개변.LMP1표체전후IκBα단백총량유지항정가능시유우NF-κB적활화신속계동료IκBα적중두합성저일자신조절배로소치.
Abstract:Objective To elucidate the mechanisms by which Epstein-Barr virus-encoded latent membrane protein 1 activates NF-κB in nasopharyngeal carcinoma cells.Methods A tetracycline-regulated LMP1-expressing nasopharyngeal carcinoma cell line, Tet-on-LMP1-HNE2, was used as the cell model. The kinetics of the expression of proteins, including LMP1, IκBα and IκBβ, was analyzed by Western blotting. The subcellular localization of NF-κB (p65) was detected by indirect immunofluorescence assay. The NF-κB transactivity was studied by transient transfection and reporter gene assay. Results IκBα was phosphorylated and degraded after the inducible expression of LMP1, although the total protein levels remained stable. The steady-state level of total IκBβ protein may have resulted from the initiation of an autoregulation loop after the activation of NF-κB. No change in the IκBβ level was detected. NF-κB (p65) was translocated from the cytoplasm to the nucleus following degradation of IκBα. After the introduction of the dominant-negative mutant of IκBα (Del 71) into Tet-on-LMP1-HNE2 cells, both nuclear translocation and transactivation of NF-κB induced by LMP1 was significantly inhibited. Conclusions The results indicated that in nasopharyngeal carcinoma cells, LMP1 activated NF-κB via phosphorylation and degradation of IκBα, but not IκBβ. The dominant-negative mutant of IκBα (Del 71) could completely inhibit both the nuclear translocation and transactivation of NF-κB induced by LMP1.