Pierce R H, Campbell J S, Stephenson A B, Franklin C C, Chaisson M, Poot M, Kavanagh T J, Rabinovitch P S, Fausto N
Department of Pathology, the University of Washington, Seattle 98195, USA.
Am J Pathol. 2000 Jul;157(1):221-36. doi: 10.1016/S0002-9440(10)64533-6.
Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours approximately 50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFkappaB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant alpha-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses.
肿瘤坏死因子（TNF）是肝脏急性期反应的介质，可引发肝细胞增殖并导致细胞死亡。我们研究了TNF导致肝细胞凋亡的机制，重点关注氧化应激、抗氧化防御和线粒体损伤的作用。研究在培养的AML12细胞（一种分化的小鼠肝细胞系）中进行。与体内肝细胞的情况一样，AML12细胞对单独的TNF诱导的细胞死亡不敏感，但在暴露于TNF和小剂量放线菌素D（Act D）时会发生凋亡。直到处理后6小时才检测到凋亡的形态学迹象，到18小时时约50%的细胞已经死亡。细胞暴露于TNF+Act D并未阻断NFκB的核转位、DNA结合或其总体反式激活能力。凋亡的诱导以氧化应激为特征，表现为NAD(P)H和谷胱甘肽的丧失，随后是线粒体损伤，包括线粒体膜电位丧失、内膜结构损伤和线粒体凝聚。这些变化与细胞色素C释放以及半胱天冬酶-8、-9和-3的激活同时发生。TNF诱导的凋亡依赖于谷胱甘肽水平。在谷胱甘肽水平降低的细胞中，在没有转录阻断的情况下，TNF本身就可作为凋亡剂。相反，抗氧化剂α-硫辛酸可防止暴露于TNF+Act D的细胞中谷胱甘肽的丧失，完全阻止了线粒体损伤、半胱天冬酶激活、细胞色素C释放和凋亡。结果表明，TNF+Act D在AML12细胞中诱导的凋亡涉及氧化损伤和线粒体损伤。由于损伤在很大程度上受细胞谷胱甘肽含量的调节，我们认为TNF+Act D的组合导致凋亡是因为Act D阻断了抗氧化防御所需基因的转录。
