Cao Wen-jing, Zhang Yao-zhen, Zhang Dong-hua, Li Deng-ju, Tang Jin-zhi
Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China.
Chin Med J (Engl). 2004 Jul;117(7):972-7.
The aim of this study was to explore whether the inhibition of nuclear factor-kappaB (NF-kappaB) activation by mutant IkappaBalpha (S32, 36-->A) can enhance TNF-alpha-induced apoptosis of leukemia cells and to investigate the possible mechanism.
The mutant IkappaBalpha gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IkappaBalpha were obtained by the limiting dilution method. TNF-alpha-induced NF-kappaB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-alpha. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM).
Mutant IkappaBalpha protein was confirmed to exist by Western blot. The results of EMSA showed that NF-kappaB activation by TNF-alpha in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IkappaBalpha repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-alpha, but changed very little in transfected HL-60 cells. The inhibition of NF-kappaB activation by mutant IkappaBalpha enhanced TNF-alpha-induced apoptosis. The cytotoxic effects of TNF-alpha were amplified in a time- and dose-dependent manner.
NF-kappaB activation plays an important role in the resistance to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB by mutant IkappaBalpha could provide a new approach that may enhance the anti-leukemia effects of TNF-alpha or even of other cytotoxic agents.
本研究旨在探讨突变型IκBα（S32, 36→A）对核因子-κB（NF-κB）激活的抑制作用是否能增强肿瘤坏死因子-α（TNF-α）诱导的白血病细胞凋亡，并研究其可能的机制。
采用脂质体介导技术将突变型IκBα基因转染至HL-60细胞。通过有限稀释法获得稳定表达突变型IκBα的G418抗性克隆。采用电泳迁移率变动分析（EMSA）检测TNF-α诱导的NF-κB激活。将亲本HL-60细胞和转染后的HL-60细胞暴露于不同浓度的TNF-α 4小时后，采用逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测bcl-xL的表达。通过流式细胞术（FCM）评估凋亡白血病细胞的百分比。
蛋白质免疫印迹法证实突变型IκBα蛋白存在。EMSA结果显示，TNF-α诱导HL-60细胞中NF-κB的激活呈剂量依赖性，但在转染细胞中几乎完全被突变型IκBα阻遏蛋白抑制。HL-60细胞暴露于TNF-α后，bcl-xL mRNA和蛋白水平升高，但在转染的HL-60细胞中变化很小。突变型IκBα对NF-κB激活的抑制增强了TNF-α诱导的凋亡。TNF-α的细胞毒性作用呈时间和剂量依赖性增强。
NF-κB激活在抵抗TNF-α诱导的凋亡中起重要作用。突变型IκBα对NF-κB的抑制可能提供一种新方法，可增强TNF-α甚至其他细胞毒性药物的抗白血病作用。
