免疫沉淀分析抗人DR5单克隆抗体激活白血病细胞caspase8/10

The activation of caspase-8,caspase-10 in Jurkat and U937 cells treated by anti-human DR5 monoclonal antibody-mDRA-6

目的:分析抗人死亡受体5(death receptor 5,DR5)单克隆抗体mDRA-6对白血病细胞系Jurkat及U937细胞caspase8、caspase10激活变化。方法:采用Western blot方法检测mDRA-6对Jurkat及U937细胞caspase-8、caspase-10、caspase-3的激活改变;免疫沉淀方法分析mDRA-6诱导的Jurkat及U937细胞DISC的caspase-8、caspase-10分子;定量-PCR检测Jurkat、U937细胞caspase-8、caspase-10 mRNA表达。MTT法检测caspase-8、caspase-10和caspase-3抑制剂对mDRA-6抑制Jurkat及U937细胞生长增殖的影响;AnnexinⅤ/PI双染流式细胞术检测caspase-8、caspase-10抑制剂对mDRA-6诱导细胞凋亡作用的影响;结果:Western blot检测显示,mDRA-6作用Jurkat细胞,导致细胞caspase-8分子激活降解,但对caspase-10无激活改变;mDRA-6作用U937细胞,激活细胞caspase-10分子,但对caspase-8无激活表现;mDRA-6作用Jurkat和U937细胞,caspase-3及PARP均表现出激活降解。免疫沉淀检测发现,mDRA-6作用白血病细胞4h,Jurkat细胞DISC的caspase-8条带明显,caspase-10条带无明显显示;相反,U937细胞DISC的caspase-10条带出现,而caspase-8无明显条带显示。定量-PCR检测发现,Jurkat和U937细胞caspase-8 mRNA表达无明显差异,但U937细胞caspase-10 mRNA表达水平明显高于Jurkat细胞(P<0.01)。预先使用caspase-8、caspase-10、caspase-3抑制剂孵育细胞1小时,mDRA-6所致的Jurkat细胞生长抑制率分别降低了72.94%(t=20.27,P<0.01)、8.09%(t=1.82,P>0.05)和31.20%(t=8.06,P<0.01);mDRA-6所致U937细胞生长抑制率分别降低了4.53%(t=0.90,P>0.05)、69.09%(t=17.25,P<0.01)和50.20%(t=13.06,P<0.01)。AnnexinⅤ/PI双染流式细胞术检测发现,预先使用caspase-8、10抑制剂孵育细胞1小时,mDRA-6诱导的Jurkat细胞凋亡率分别降低了80.82%和8.34%;mDRA-6诱导的U937细胞凋亡率分别降低2.50%和48.47%。结论:mDRA-6激活不同起始caspase诱导细胞凋亡,激活caspase-8启动Jurkat细胞凋亡,激活caspase-10启动U937细胞凋亡;U937细胞caspase-10激活可能与细胞caspase-10mRNA高表达有关。

Objective：To analyze the activation of caspase-8, caspase-10 in Jurkat and U937 cells treated by anti-human DR5 monoclonal antibody-mDRA-6. Methods： Tile activation of caspase-8, caspase-10 ,caspase-3 and PARP in Jurkat and U937 cells trea- ted by mDRA-6 were detected by Western blot, respectively. The caspase-8 and easpase-10 molecules of death-inducing signaling com- plex （DISC） in Jurkat and U937 cells treated by mDRA-6 were detected by immunoprecipitation. The mRNA expression of caspase-8 and easpase-10 in Jurkat and U937 cells were detected by real-time PCR. The inhibition and apoptosis of Jurkat and U937 cells after treating with mDRA-6 or/and caspases inhibitors were analyzed using MTT and FITC- Annexin V/PI staining, Results： When the cells were treated with mDRA-6, analysis by immunoblotting indicated that caspase-8, but not caspase-10 was activated in Jurkat cells; while caspase-10, but not caspase-8, was activated in U937 cells; caspases-3 and PARP were activated in both cells. Immunoprecipi- tation revealed that caspase-8 was a participator of DISC in Jurkat cells treated with mDRA-6, while caspase-10 was a participator of DISC in U937 cells treated with mDRA-6, caspase-8 mRNA expression was not difference in Jurkat and U937 cells, but caspase-10 mRNA expression in U937 cells was higher than that in Jurkat cells. When Jurkat and U937 cells were incubated with the inhibitors ofcaspase-8,caspase-10 and caspase-3 for one hour, Jurkat cells inhibition induced by mDRA-6 were reduced by 72.94% （t = 20.27, P 〈0.01 ）, 8.09% （t = 1.82, P 〉0.05） and 31.20% （t =8.06, P 〈0.01 ）, U937 cells inhibition induced by mDRA-6 were reduced by 4.53% （ t = 0.90, P 〉 0.05 ）, 69.09% （ t = 17.25, P 〈 0.01 ） and 50.20% （ t = 13.06, P 〈 0.01 ）, respectively. While the Jurkat and U937 cells were incubated with the inhibitors of caspase-8 and caspase-10 for one hour, Jurkat cells apoptosis induced by mDRA- 6 were reduced by 80.82% and 8.34%, U937 cells apoptosis induced by mDRA-6 were reduced by 2.50% and 48.47%, respective- ly. Conclusion： mDRA-6 can induce apoptosis of both cell lines through activating the caspase signal pathway, but apoptotic signal pathways underlying are cell type dependent, caspase-8 is an initiator caspase for Jurkat cell, caspase-10 is an initiator caspase for U937 cell. The activation of caspase-10 in U937 cells may be correlate with its higher mRNA expression.