Gong J, Li X, Darzynkiewicz Z
Cancer Research Institute, New York Medical College, Valhalla 10595.
J Cell Physiol. 1993 Nov;157(2):263-70. doi: 10.1002/jcp.1041570208.
Cells of the human promyelocytic HL-60 line, when treated with a variety of antitumor agents in the presence of the protein synthesis inhibitor cycloheximide (CHX), or with CHX alone, rapidly undergo apoptosis ("active cell death"). It is presumed, therefore, that such cells are "primed" to apoptosis in that no new protein synthesis is required for induction of their death. We have studied apoptosis of HL-60 cells triggered by the DNA topoisomerase I inhibitor camptothecin (CAM) in the absence and presence of CHX and apoptosis induced by CHX alone. Two different flow cytometric methods were used, each allowing us to relate the apoptosis-associated DNA degradation to the cell cycle position. Apoptosis induced by CAM was limited to S phase cells, e.g., at a CAM concentration of 0.15 microM, nearly 90% of the S phase cells underwent apoptosis after 4 h. In contrast, apoptosis triggered by CHX was indiscriminate, affecting all phases of the cycle: approximately 40% of the cells from each phase the cycle underwent apoptosis at 5 microM CHX concentration. When CAM and CHX were added together, the pattern of apoptosis resembled that of cycloheximide alone, namely, cells in all phases of the cycle in similar proportion were affected. Thus, CHX, while itself inducing apoptosis of a fraction of cells, protected the S phase cells against apoptosis triggered by CAM. Because CHX (5 microM) did not significantly affect the rate of cell progression through S phase, the observed protective effect was most likely directly related to inhibition of protein synthesis, rather than to its possible indirect effect on DNA replication. Furthermore, whereas apoptosis (DNA degradation) triggered by CAM was prevented by the serine protease inhibitor N-tosyl-L-lysylchloromethyl ketone (TLCK), this process was actually potentiated by this inhibitor when induced by CHX. The present data indicate differences in mechanism of apoptosis triggered by CAM (and perhaps other antitumor drugs) as compared with CHX. Apoptosis caused by CHX may be unique in that it may not involve new protein synthesis. These data are compatible with the assumption that the loss of a hypothetical, rapidly turning over suppressor of apoptosis may be the trigger of apoptosis of HL-60 cells treated with CHX, whereas de novo protein synthesis is required when apoptosis is triggered by other agents.
人早幼粒细胞HL - 60系细胞，在蛋白质合成抑制剂环己酰亚胺（CHX）存在的情况下用多种抗肿瘤药物处理，或单独用CHX处理时，会迅速发生凋亡（“主动细胞死亡”）。因此据推测，此类细胞对凋亡处于“致敏”状态，因为诱导其死亡不需要新的蛋白质合成。我们研究了在不存在和存在CHX的情况下，DNA拓扑异构酶I抑制剂喜树碱（CAM）触发的HL - 60细胞凋亡以及单独CHX诱导的凋亡。使用了两种不同的流式细胞术方法，每种方法都能让我们将与凋亡相关的DNA降解与细胞周期位置联系起来。CAM诱导的凋亡仅限于S期细胞，例如，在CAM浓度为0.15微摩尔时，4小时后近90%的S期细胞发生凋亡。相比之下，CHX触发的凋亡是无差别的，影响细胞周期的所有阶段：在5微摩尔CHX浓度下，每个细胞周期阶段约40%的细胞发生凋亡。当CAM和CHX一起添加时，凋亡模式类似于单独使用环己酰亚胺的情况，即细胞周期所有阶段中比例相似的细胞受到影响。因此，CHX虽然本身诱导一部分细胞凋亡，但保护S期细胞免受CAM触发的凋亡。因为CHX（5微摩尔）对细胞通过S期的进程速率没有显著影响，所以观察到的保护作用很可能直接与蛋白质合成的抑制有关，而不是与其对DNA复制可能的间接影响有关。此外，虽然CAM触发的凋亡（DNA降解）被丝氨酸蛋白酶抑制剂N - 甲苯磺酰 - L - 赖氨酰氯甲基酮（TLCK）阻止，但当由CHX诱导时，这个过程实际上被该抑制剂增强。目前的数据表明，与CHX相比，CAM（可能还有其他抗肿瘤药物）触发的凋亡机制存在差异。CHX引起的凋亡可能是独特的，因为它可能不涉及新的蛋白质合成。这些数据与以下假设相符：一种假设的、快速周转的凋亡抑制因子的丧失可能是用CHX处理的HL - 60细胞凋亡的触发因素，而当凋亡由其他因素触发时则需要从头合成蛋白质。
