Effect of protease inhibitors on early events of apoptosis.
Effect of protease inhibitors on early events of apoptosis.
复制标题
蛋白酶抑制剂对细胞凋亡早期事件的影响。
DOI：
10.1006/excr.1996.0092
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Darzynkiewicz,Z
中科院分区：
文献类型：
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作者：
Hara,S;Halicka,HD;Bruno,S;Gong,J;Traganos,F;Darzynkiewicz,Z
蛋白降解是细胞凋亡的早期事件，它似乎与负责核小体间DNA切割的核酸内切酶的激活有关。本研究旨在揭示蛋白质降解在其他早期事件中的可能作用，如染色质凝集、核破裂和情况DNA双链结构中的不稳定。用不同的药物诱导人白血病HL-60细胞和大鼠胸腺细胞的凋亡，包括DNA拓扑异构酶抑制剂，一种RNA抗代谢药物，以及糖皮质激素，强的松龙。用脉冲场和常规凝胶电泳法以及原位DNA断链的存在来评价DNA降解。通过测定DNA对变性的敏感性来评价DNA的稳定性。用DNA特异性荧光染料4‘，6-二氨基-2-苯基吲哚染色后，通过干涉对比和紫外显微镜观察染色质凝聚、核破裂和其他形态变化。几种不可逆或可逆的丝氨酸蛋白酶抑制剂可防止核小体间DNA降解、核破裂和DNA双链结构的不稳定。然而，有效的抑制剂不能阻止染色质凝聚的开始，也不能阻止精细结构框架的丧失，也不能阻止DNA裂解的初始步骤，产生大小为50kb的≥片段。数据表明，在这两个细胞系统中，需要对所测试的抑制剂敏感的蛋白酶的活性才能发生核小体间DNA切割。数据还表明，这些蛋白水解酶可能参与了核膜的溶解。由于核基质蛋白和组蛋白在原位稳定DNA，而在凋亡过程中DNA稳定性的下降被抑制剂阻止，所以丝氨酸蛋白酶可能会降解DNA稳定蛋白。然而，这些蛋白酶的活性似乎既不是dna裂解成≥50kb片段所必需的，也不是与核结构框架溶解相关的染色质凝聚开始所必需的。
Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.