Tang L, Boise L H, Dent P, Grant S
Department of Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA, USA.
Biochem Pharmacol. 2000 Nov 15;60(10):1445-56. doi: 10.1016/s0006-2952(00)00463-9.
Antileukemic interactions between the nucleoside analog 1-beta-D-arabinofuranosylcytosine (ara-C) and the kinase inhibitor 7-hydroxystaurosporine (UCN-01) have been examined in relation to Bcl-2 expression/phosphorylation, mitochondrial damage, caspase activation, and loss of clonogenic potential. Subsequent exposure of ara-C-pretreated U937 cells (1 microM; 6 hr) to UCN-01 (300 nM; 24 hr) resulted in marked potentiation of pro-caspase-3 and -9 cleavage/activation, poly(ADP-ribose)polymerase degradation, diminished mitochondrial membrane potential (Deltapsi(m)), enhanced cytochrome c release, reduction in the S-phase fraction, and induction of classic apoptotic morphologic features. Enforced expression of full-length Bcl-2 significantly protected cells (at 24 hr) from ara-C/UCN-01-induced caspase activation and apoptosis, but was ineffective in preventing loss of Deltapsi(m) and cytochrome c release. Ectopic expression of a Bcl-2 N-terminal phosphorylation loop-deleted protein (Bcl-2Delta(32-80)) was more potent than its full-length counterpart in blocking drug-induced loss of Deltapsi(m, ) caspase activation, and apoptotic morphology, but not cytochrome c release. Examination of cells at later intervals revealed that ectopic expression of Bcl-2 or Bcl-2Delta(32-80) could only delay, but not prevent, mitochondrial damage, caspase activation, and cell death induced by ara-C/UCN-01 treatment. Despite their initial ability to inhibit apoptosis, neither full-length nor truncated Bcl-2 protein restored clonogenic potential to drug-treated cells. These findings indicate that subsequent exposure of ara-C-pretreated human leukemia cells to UCN-01 potently triggers mitochondrial damage and apoptosis, and that these events are postponed but not prevented by ectopic expression of Bcl-2 or its phosphorylation loop-deleted counterpart.
已研究了核苷类似物1-β-D-阿拉伯呋喃糖基胞嘧啶（ara-C）与激酶抑制剂7-羟基星状孢菌素（UCN-01）之间的抗白血病相互作用，涉及Bcl-2表达/磷酸化、线粒体损伤、半胱天冬酶激活和克隆形成潜力丧失。将ara-C预处理的U937细胞（1 microM；6小时）随后暴露于UCN-01（300 nM；24小时）导致前半胱天冬酶-3和-9的切割/激活、聚（ADP-核糖）聚合酶降解显著增强，线粒体膜电位（Δψm）降低，细胞色素c释放增加，S期分数减少，并诱导出典型的凋亡形态特征。全长Bcl-2的强制表达显著保护细胞（在24小时时）免受ara-C/UCN-01诱导的半胱天冬酶激活和凋亡，但在防止Δψm丧失和细胞色素c释放方面无效。Bcl-2 N端磷酸化环缺失蛋白（Bcl-2Δ(32-80)）的异位表达在阻断药物诱导的Δψm丧失、半胱天冬酶激活和凋亡形态方面比其全长对应物更有效，但对细胞色素c释放无效。在后续时间点检查细胞发现，Bcl-2或Bcl-2Δ(32-80)的异位表达只能延迟，但不能阻止ara-C/UCN-01处理诱导的线粒体损伤、半胱天冬酶激活和细胞死亡。尽管它们最初具有抑制凋亡的能力，但全长或截短的Bcl-2蛋白均未恢复药物处理细胞的克隆形成潜力。这些发现表明，将ara-C预处理的人白血病细胞随后暴露于UCN-01会强烈触发线粒体损伤和凋亡，并且这些事件会被Bcl-2或其磷酸化环缺失对应物的异位表达延迟但不会被阻止。
