Cartee L, Smith R, Dai Y, Rahmani M, Rosato R, Almenara J, Dent P, Grant S
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia 23298-0230, USA.
Mol Pharmacol. 2002 Jun;61(6):1313-21. doi: 10.1124/mol.61.6.1313.
Previous studies have shown that coexposure to marginally toxic concentrations of phorbol 12-myristate 13-acetate (PMA; 10 nM) and the cyclin-dependent kinase inhibitor flavopiridol (FP; 100-200 nM) synergistically induces apoptosis in human myeloid leukemia cells U937 and HL-60 (i.e., >50% apoptotic at 24 h). Attempts have now been made to characterize the cell death pathway(s) involved in this phenomenon. In contrast to cytochrome c release and caspase-3 activation, which occur within 2.5 h of PMA/FP coexposure, caspase-8 activation and Bid cleavage appeared as later events. Such findings implicate the mitochondria-dependent pathway in the initial induction of apoptosis by PMA/FP. However, U937 cells ectopically expressing CrmA, dominant-negative caspase-8, or dominant-negative Fas-associated death domain that were highly resistant to tumor necrosis factor (TNF)/cycloheximide-induced lethality displayed significant, albeit incomplete, resistance to PMA/FP-induced apoptosis after 24 h. Furthermore, coadministration of TNF soluble receptor significantly attenuated PMA/FP-induced apoptosis in U937 (p < 0.02) and HL-60 (p < 0.03) cells at 24 h. PMA/FP coadministration also triggered substantial increases in TNFalpha mRNA and protein secretion compared with the effects of PMA administered alone. The protein kinase C (PKC) inhibitor bisindolylmaleimide (1 microM) completely blocked PMA/FP-induced TNFalpha secretion in U937 cells and attenuated apoptosis. Taken together, these results suggest that coadministration of PMA with FP in myeloid leukemia cells initially triggers mitochondrial damage, an event followed by the PKC-dependent induction and release of TNFalpha, supporting a model in which the synergistic induction of leukemic cell apoptosis by this drug combination proceeds via both mitochondrial- and TNF receptor-related apoptotic pathways.
先前的研究表明，同时暴露于边缘毒性浓度的佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA；10 nM）和细胞周期蛋白依赖性激酶抑制剂黄酮哌啶醇（FP；100 - 200 nM）可协同诱导人髓系白血病细胞U937和HL - 60凋亡（即24小时时凋亡率>50%）。现在已尝试对参与此现象的细胞死亡途径进行表征。与在PMA/FP共同暴露2.5小时内发生的细胞色素c释放和半胱天冬酶-3激活相反，半胱天冬酶-8激活和Bid裂解出现得较晚。这些发现表明线粒体依赖性途径参与了PMA/FP对凋亡的初始诱导。然而，异位表达CrmA、显性负性半胱天冬酶-8或显性负性Fas相关死亡结构域且对肿瘤坏死因子（TNF）/环己酰亚胺诱导的致死性具有高度抗性的U937细胞，在24小时后对PMA/FP诱导的凋亡表现出显著但不完全的抗性。此外，在24小时时，共给予TNF可溶性受体可显著减弱U937（p < 0.02）和HL - 60（p < 0.03）细胞中PMA/FP诱导的凋亡。与单独给予PMA的效果相比，PMA/FP共同给药还引发了TNFα mRNA和蛋白分泌的大幅增加。蛋白激酶C（PKC）抑制剂双吲哚马来酰亚胺（1 microM）完全阻断了U937细胞中PMA/FP诱导的TNFα分泌并减弱了凋亡。综上所述，这些结果表明，在髓系白血病细胞中PMA与FP共同给药最初会引发线粒体损伤，随后是PKC依赖性的TNFα诱导和释放，支持了这样一种模型，即这种药物组合对白血病细胞凋亡的协同诱导是通过线粒体和TNF受体相关的凋亡途径进行的。
