Zhivotovsky B, Gahm A, Ankarcrona M, Nicotera P, Orrenius S
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Exp Cell Res. 1995 Dec;221(2):404-12. doi: 10.1006/excr.1995.1391.
To investigate the involvement of proteases in apoptosis, rat thymocytes were treated with the glucocorticoid hormone methylprednisolone or the topoisomerase II inhibitor etoposide in the presence of selective substrate inhibitors of either interleukin-1 beta-converting enzyme (ICE), (Z-Val-Ala-Asp-chloromethylketone, VADcmk) or Ca(2+)-regulated serine protease (Suc-Ala-Ala-Pro-Phe-chloromethylketone, AAPFcmk). VADcmk protected from lamin proteolysis, chromatin fragmentation, cell shrinkage, and formation of apoptotic nuclei in both methylprednisolone- and etoposide-treated thymocytes when present during the initiation of the apoptotic process. AAPFcmk prevented lamin breakdown, chromatin fragmentation, and apoptotic morphological changes in thymocytes treated with methylprednisolone, but not with etoposide. Both MPS- and etoposide-treated thymocytes exhibited enhanced ICE-like protease activity which was maximal 1 h after treatment. This increase in proteolytic activity was blocked by VADcmk, but not AAPFcmk. Our findings suggest that ICE-like protease activity is critically involved in the early phase of both methylprednisolone- and etoposide-induced apoptosis in thymocytes, whereas the Ca(2+)-regulated serine protease is an obligatory component of the proteolytic cascade in methylprednisolone-induced apoptosis.
为了研究蛋白酶在细胞凋亡中的作用，在存在白细胞介素-1β转换酶（ICE）的选择性底物抑制剂（Z-缬氨酸-丙氨酸-天冬氨酸-氯甲基酮，VADcmk）或钙调节丝氨酸蛋白酶（琥珀酰-丙氨酸-丙氨酸-脯氨酸-苯丙氨酸-氯甲基酮，AAPFcmk）的情况下，用糖皮质激素甲基强的松龙或拓扑异构酶II抑制剂依托泊苷处理大鼠胸腺细胞。当在凋亡过程开始时存在时，VADcmk可保护甲基强的松龙和依托泊苷处理的胸腺细胞免受核纤层蛋白水解、染色质片段化、细胞收缩和凋亡细胞核的形成。AAPFcmk可防止甲基强的松龙处理的胸腺细胞中的核纤层蛋白降解、染色质片段化和凋亡形态变化，但对依托泊苷处理的胸腺细胞无效。甲基强的松龙和依托泊苷处理的胸腺细胞均表现出增强的ICE样蛋白酶活性，在处理后1小时达到最大值。这种蛋白水解活性的增加被VADcmk阻断，但未被AAPFcmk阻断。我们的研究结果表明，ICE样蛋白酶活性在甲基强的松龙和依托泊苷诱导的胸腺细胞凋亡的早期阶段起关键作用，而钙调节丝氨酸蛋白酶是甲基强的松龙诱导凋亡的蛋白水解级联反应的必需成分。
