Lin K T, Xue J Y, Lin M C, Spokas E G, Sun F F, Wong P Y
Department of Cell Biology, School of Osteopathic Medicine, University of Medicine and Dentistry of New Jersey, Stratford 08084, USA.
Am J Physiol. 1998 Apr;274(4):C855-60. doi: 10.1152/ajpcell.1998.274.4.C855.
Apoptosis is an active process critical for the homeostasis of organisms. Enzymes of the caspase family are responsible for executing this process. We have previously shown that peroxynitrite (ONOO-), a biological product generated from the interaction of nitric oxide and superoxide, induces apoptosis of HL-60 cells. The aim of this study was to elucidate the mechanisms involved in the execution process of peroxynitrite-induced apoptosis. Proteolytic cleavage of poly(ADP-ribose) polymerase, an indication of caspase-3 family protease activation and an early biochemical event accompanying apoptosis, was observed in a time-dependent manner during peroxynitrite-induced apoptosis of HL-60 cells. Activation of caspase-3 during peroxynitrite-induced apoptosis was substantiated by monitoring proteolysis of the caspase-3 proenzyme and by measuring caspase-3 activity with a fluorogenic substrate. Furthermore, pretreatment of HL-60 cells with N-acetyl-Asp-Glu-Val-Asp-aldehyde, a specific inhibitor of caspase-3, but not N-acetyl-Tyr-Val-Ala-Asp-aldehyde, a specific inhibitor of caspase-1, decreased peroxynitrite-induced apoptosis. These results suggest that the activation of a caspase-3 family protease is essential for initiating the execution process of peroxynitrite-induced apoptosis of HL-60 cells.
细胞凋亡是一个对生物体稳态至关重要的主动过程。半胱天冬酶家族的酶负责执行这一过程。我们之前已经表明，过氧亚硝酸盐（ONOO-）是一氧化氮和超氧化物相互作用产生的一种生物产物，可诱导HL-60细胞凋亡。本研究的目的是阐明过氧亚硝酸盐诱导细胞凋亡的执行过程中涉及的机制。在过氧亚硝酸盐诱导HL-60细胞凋亡的过程中，观察到聚（ADP-核糖）聚合酶的蛋白水解切割，这是半胱天冬酶-3家族蛋白酶激活的一个指标，也是伴随细胞凋亡的早期生化事件，且呈时间依赖性。通过监测半胱天冬酶-3酶原的蛋白水解以及用荧光底物测量半胱天冬酶-3活性，证实了过氧亚硝酸盐诱导细胞凋亡过程中半胱天冬酶-3的激活。此外，用半胱天冬酶-3的特异性抑制剂N-乙酰-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-醛预处理HL-60细胞，但不用半胱天冬酶-1的特异性抑制剂N-乙酰-酪氨酸-缬氨酸-丙氨酸-天冬氨酸-醛预处理，可减少过氧亚硝酸盐诱导的细胞凋亡。这些结果表明，半胱天冬酶-3家族蛋白酶的激活对于启动过氧亚硝酸盐诱导HL-60细胞凋亡的执行过程至关重要。
