Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases
Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases
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DOI：
10.1038/sj.leu.2400734
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发表时间：
1997-08
期刊：
影响因子：
11.4
通讯作者：
T. Shimizu;Y. Pommier
中科院分区：
文献类型：
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作者：
T. Shimizu;Y. Pommier
人白血病细胞系HL60对多种凋亡刺激和p53-null非常敏感。caspases家族中与死亡相关的半胱氨酸蛋白酶在凋亡的执行阶段起着核心作用，我们最近报道了丝氨酸蛋白酶激活在喜树碱诱导的HL60细胞凋亡内切酶激活中的重要性。在本研究中，我们研究了半胱天半酶（ICE/ ced -3相关半胱氨酸蛋白酶）和丝氨酸蛋白酶在拓扑异构酶I抑制剂喜树碱诱导的HL60细胞和无细胞系统中细胞死亡中的作用。我们发现CPP32在喜树碱诱导的细胞凋亡过程中被激活，并且细胞可渗透的半胱天蛋白酶抑制剂n-苄基氧羰基- val - ala - asp （o -甲基）-氟甲基酮（Z-VAD-fmk）阻断细胞凋亡的所有特征：形态学改变、半胱天蛋白酶3 （CPP32/Yama/Apopain）和聚（adp -核糖）聚合酶的裂解、层析蛋白B的降解和DNA的断裂。然而，Z-VAD-fmk和另外两种ICE/ ce -3抑制剂YVAD-CHO和DEVD-CHO在由未处理的HL60细胞的细胞核和喜树碱处理的细胞的细胞质重建的无细胞系统中是无活性的，这表明在无细胞系统中，内切酶激活或层粘胶蛋白B切割并不需要半胱天蛋白酶。相比之下，丝氨酸蛋白酶抑制剂3,4 -二氯异香豆素（DCI）和l- 1-氯-3-(4-tosylamido)-4-苯基-2-丁酮toyl - l-苯丙氨酸氯甲基酮（TPCK）在全细胞和无细胞系统中都能消除喜树碱诱导的凋亡相关生化变化。DCI还能抑制CPP32的裂解。综上所述，这些结果表明，在HL60细胞中，CPP32和丝氨酸蛋白酶在喜树碱诱导的凋亡中都被激活。
The human leukemia cell line, HL60 is very sensitive to various apoptotic stimuli and p53-null. The death-related cysteine proteases of the caspases family play a central role in the execution phase of apoptosis, and we recently reported the importance of serine protease activation in camptothecin-induced apoptotic endonuclease activation in HL60 cells. In the present study, we investigated the role of caspases (ICE/CED-3-related cysteine proteases) and serine proteases in cell death induced by the topoisomerase I inhibitor, camptothecin, in HL60 cells and in a cell-free system. We found that CPP32 is activated during camptothecin-induced apoptosis, and that N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethyketone (Z-VAD-fmk), a cell permeable caspase inhibitor blocks all features of apoptosis: morphological changes, cleavage of caspase 3 (CPP32/Yama/Apopain) and poly (ADP-ribose) polymerase, lamin B degradation and DNA fragmentation. However, Z-VAD-fmk and two other ICE/CED-3 inhibitors, YVAD-CHO and DEVD-CHO, were inactive in a cell-free system reconstituted from nuclei of untreated HL60 cells and cytosol from camptothecin-treated cells, suggesting that caspases are not required for endonuclease activation or lamin B cleavage in the cell-free system. By contrast, the serine protease inhibitors, 3, 4-dichloroisocoumarin (DCI) and L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone tosyl-L-phenylalanine chloromethyl ketone (TPCK), abolished the apoptosis-associated biochemical changes induced by camptothecin both in whole cells and in a cell-free system. DCI also inhibited CPP32 cleavage. Taken together, these results suggest that in HL60 cells, both CPP32 and serine proteases are activated in camptothecin-induced apoptosis.