Grant S, Turner A J, Freemerman A J, Wang Z, Kramer L, Jarvis W D
Department of Medicine, Medical College of Virginia, Richmond 23298, USA.
Exp Cell Res. 1996 Oct 10;228(1):65-75. doi: 10.1006/excr.1996.0300.
Previous studies have shown that pretreatment of human myeloid leukemia cells (HL-60) with the protein kinase C (PKC) activator bryostatin 1 potentiates ara-C-induced apoptosis. To test the hypothesis that this capacity stems from down-regulation of PKC activity and/or Ca2+-dependent (group-I; cPKC) isoform expression, comparisons were made between the effects of this agent and the stage-2 tumor promoter mezerein under conditions favoring either cellular differentiation or drug-induced apoptosis. Twenty-four-hour pretreatment of HL-60 cells with 10 nM bryostatin 1, which does not induce differentiation in this cell line, led to a profound reduction in membrane and cytosolic PKC activity, decreased expression of cPKC isoforms (alpha, betaI, betaII, gamma), and a marked increase in ara-C induced apoptosis. In contrast, 10 nM mezerein, which induces HL-60 cell differentiation, was less effective in down-regulating membrane and cytosolic PKC activity as well as alpha, betaI, and gamma cPKC isoform expression, and failed to potentiate ara-C-related apoptosis. The effects of bryostatin 1 were dominant to those of mezerein, in that the combination resulted in down-regulation of PKC activity and expression and potentiation of ara-C-induced apoptosis, but not cellular maturation. However, coadministration of the Ca2+ ionophore A23187 (250 nM) restored bryostatin 1's differentiating ability while antagonizing its capacity to augment apoptosis, despite failing to reverse bryostatin 1-induced down-regulation of PKC activity and cPKC isoform expression. Furthermore, pretreatment of differentiation-responsive monocytic leukemia cells (U937) with bryostatin 1 substantially reduced PKC activity and cPKC isoform expression, but exerted minimal effects on ara-C-related apoptosis. In contrast, exposure of U937 cells to bryostatin 1 after ara-C dramatically increased apoptosis, a phenomenon that did not occur in differentiation-unresponsive HL-60 cells. Collectively, these observations indicate that down-regulation of total assayable PKC activity and cPKC expression by bryostatin 1 are insufficient, by themselves, to account for potentiation of leukemic cell apoptosis, at least under conditions in which differentiation occurs. They also provide further evidence that a reciprocal and highly schedule-dependent relationship exists between leukemic cell differentiation and drug-induced apoptosis.
先前的研究表明，用蛋白激酶C（PKC）激活剂苔藓抑素1预处理人髓系白血病细胞（HL-60）可增强阿糖胞苷诱导的细胞凋亡。为了验证这种能力源于PKC活性下调和/或Ca2+依赖性（I组；cPKC）亚型表达下调这一假说，在有利于细胞分化或药物诱导凋亡的条件下，对该试剂与2期肿瘤启动子芫花酯素的作用进行了比较。用10 nM苔藓抑素1对HL-60细胞进行24小时预处理，该浓度在该细胞系中不诱导分化，导致膜和胞质PKC活性显著降低，cPKC亚型（α、βI、βII、γ）表达减少，阿糖胞苷诱导的细胞凋亡显著增加。相比之下，10 nM芫花酯素可诱导HL-60细胞分化，但在下调膜和胞质PKC活性以及α、βI和γ cPKC亚型表达方面效果较差，且未能增强阿糖胞苷相关的细胞凋亡。苔藓抑素1的作用强于芫花酯素，因为两者联合使用导致PKC活性和表达下调以及阿糖胞苷诱导的细胞凋亡增强，但未导致细胞成熟。然而，同时给予Ca2+离子载体A23187（250 nM）可恢复苔藓抑素1的分化能力，同时拮抗其增强细胞凋亡的能力，尽管未能逆转苔藓抑素1诱导的PKC活性和cPKC亚型表达下调。此外，用苔藓抑素1预处理对分化有反应的单核细胞白血病细胞（U937）可显著降低PKC活性和cPKC亚型表达，但对阿糖胞苷相关的细胞凋亡影响最小。相比之下，在阿糖胞苷处理后将U937细胞暴露于苔藓抑素1可显著增加细胞凋亡，而在对分化无反应的HL-60细胞中未出现这种现象。总体而言，这些观察结果表明，苔藓抑素1对可检测的总PKC活性和cPKC表达的下调本身不足以解释白血病细胞凋亡的增强，至少在发生分化的条件下是如此。它们还提供了进一步的证据，表明白血病细胞分化与药物诱导的细胞凋亡之间存在相互且高度依赖时间安排的关系。
