Sané A T, Bertrand R
Hospital Research Center of University of Montreal (CHUM), Notre-Dame Hospital, Montreal, Quebec, Canada.
Cancer Res. 1998 Jul 15;58(14):3066-72.
Monocytic-like leukemia U-937 cells rapidly undergo morphological changes and DNA fragmentation that is typical of apoptosis following treatment with DNA topoisomerase I inhibitor [20-S-camptothecin lactone (CPT)]. The tripeptide derivative benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone blocks Asp-Glu-Val-Asp-ase (DEVDase) activity and prevents the occurrence of high molecular weight and oligonucleosome-sized DNA fragments associated with apoptosis in CPT-treated cells. In contrast, N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) does not prevent DEVDase activity and high molecular weight DNA fragmentation but completely abrogates the appearance of oligonucleosome-sized DNA fragmentation. These results suggest that caspase 3-like activities are involved with high molecular weight DNA fragmentation pathway, whereas TPCK-sensitive activities are involved in oligonucleosome-sized DNA fragmentation pathway in CPT-treated cells. Electron micrographs reveal that caspase inhibition by benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone also abrogates the typical morphological changes associated with apoptosis, whereas TPCK does not delay these morphological changes that are typical of apoptosis. Caspase inhibition slows passage of the cells through G2 and causes a transient accumulation of these cells at the G0/G1 phase of the cell cycle following CPT treatment. In a cell-free system, when purified nuclei are incubated with apoptotic cytosolic extracts obtained from CPT-treated U-937 cells, TPCK causes a similar effect in abrogating the oligonucleosome-sized DNA fragmentation but does not affect DEVDase activity. Addition of either benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group or acetyl-Asp-Glu-Val-Asp-aldehyde completely inhibits DEVDase activity in these extracts. However, acetyl-Asp-Glu-Val-Asp-aldehyde does not affect the occurrence of oligonucleosome-sized DNA fragmentation in the cell-free system, whereas the benzyloxycarbonyl derivatives benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group, benzyloxycarbonyl-Val-Ala-free hydroxyl group, benzyloxycarbonyl-Val-free hydroxyl group, and benzyloxycarbonyl hydrazide abolish it markedly. Taken together, these observations show the pivotal role of DEVDase activity in triggering the apoptotic process and high molecular weight DNA fragmentation, whereas TPCK- and benzyloxycarbonyl-sensitive activities are involved in the oligonucleosome-sized DNA fragmentation pathway induced by CPT.
单核细胞样白血病U - 937细胞在用DNA拓扑异构酶I抑制剂[20 - S -喜树碱内酯（CPT）]处理后，会迅速发生形态变化和DNA片段化，这是典型的凋亡特征。三肽衍生物苄氧羰基 - 缬氨酸 - 丙氨酸 - 天冬氨酸（甲氧基）氟甲基酮可阻断天冬氨酸 - 谷氨酸 - 缬氨酸 - 天冬氨酸酶（DEVDase）的活性，并阻止CPT处理的细胞中出现与凋亡相关的高分子量和寡核小体大小的DNA片段。相比之下，N - 对甲苯磺酰基 - L - 苯丙氨酰氯甲基酮（TPCK）不能阻止DEVDase活性和高分子量DNA片段化，但能完全消除寡核小体大小的DNA片段的出现。这些结果表明，类似半胱天冬酶3的活性参与高分子量DNA片段化途径，而TPCK敏感的活性参与CPT处理细胞中的寡核小体大小的DNA片段化途径。电子显微镜照片显示，苄氧羰基 - 缬氨酸 - 丙氨酸 - 天冬氨酸（甲氧基）氟甲基酮对半胱天冬酶的抑制也消除了与凋亡相关的典型形态变化，而TPCK并不能延迟这些典型的凋亡形态变化。半胱天冬酶抑制减缓细胞通过G2期的进程，并导致CPT处理后这些细胞在细胞周期的G0/G1期短暂积累。在无细胞系统中，当将纯化的细胞核与从CPT处理的U - 937细胞获得的凋亡细胞溶质提取物一起孵育时，TPCK在消除寡核小体大小的DNA片段化方面产生类似的效果，但不影响DEVDase活性。添加苄氧羰基 - 缬氨酸 - 丙氨酸 - 天冬氨酸游离羧基或乙酰天冬氨酸 - 谷氨酸 - 缬氨酸 - 天冬氨酸醛可完全抑制这些提取物中的DEVDase活性。然而，乙酰天冬氨酸 - 谷氨酸 - 缬氨酸 - 天冬氨酸醛不影响无细胞系统中寡核小体大小的DNA片段化的发生，而苄氧羰基衍生物苄氧羰基 - 缬氨酸 - 丙氨酸 - 天冬氨酸游离羧基、苄氧羰基 - 缬氨酸 - 丙氨酸游离羟基、苄氧羰基 - 缬氨酸游离羟基和苄氧羰基酰肼可显著消除它。综上所述，这些观察结果表明DEVDase活性在触发凋亡过程和高分子量DNA片段化中起关键作用，而TPCK和苄氧羰基敏感的活性参与CPT诱导的寡核小体大小的DNA片段化途径。
