Lin K I, Lee S H, Narayanan R, Baraban J M, Hardwick J M, Ratan R R
Department of Molecular Microbiology and Immunology, Johns Hopkins School of Hygiene and Public Health, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Cell Biol. 1995 Dec;131(5):1149-61. doi: 10.1083/jcb.131.5.1149.
Oxidative stress has been proposed as a common mediator of apoptotic death. To investigate further the role of oxidants in this process we have studied the effects of antioxidants on Sindbis virus (SV)-induced apoptosis in two cell lines, AT-3 (a prostate carcinoma line) and N18 (a neuroblastoma line). The thiol antioxidant, N-acetylcysteine (NAC), at concentrations above 30 mM, completely abrogates SV-induced apoptosis in AT-3 and N18 cells. The effects of NAC cannot be attributed to inhibition of viral entry or viral replication, changes in extracellular osmolarity or to increases in cellular glutathione levels, nor can they be mimicked by chelators of trace metals, inhibitors of lipid peroxidation or peroxide scavengers. In contrast, other thiol agents including pyrrolidine dithiocarbamate (PDTC, 75 microM) are protective. Because NAC and PDTC are among the most effective inhibitors of the transcription factor NF-kappa B, we examined SV's ability to activate NF-kappa B before the onset of morphologic or biochemical evidence of apoptosis. Within hours of infection, SV induced a robust increase in nuclear NF-kappa B activity in AT-3 and N18 cells; this activation was suppressible by NAC and PDTC. Over-expression of bcl-2 in AT-3 cells, which has been shown to inhibit SV-induced apoptosis, also inhibits SV-induced NF-kappa B activation. To determine if NF-kappa B activation is necessary for SV-induced apoptosis in these cells, we used double stranded oligonucleotides with consensus NF-kappa B sequences as transcription factor decoys (TFDs) to inhibit NF-kappa B binding to native DNA sites. Wild-type, but not mutant, TFDs inhibit SV-induced apoptosis in AT-3 cells. In contrast, TFD inhibition of NF-kappa B nuclear activity in N18 cells did not prevent SV-induced apoptosis. Taken together, these observations define a cell type-specific, transcription factor signaling pathway necessary for SV-induced apoptosis. Understanding the precise mechanism by which Bcl-2 and thiol agents inhibit SV-induced nuclear NF-kappa B activity in AT-3 cells may provide insights into the pluripotent antiapoptotic actions of these agents.
氧化应激被认为是凋亡性死亡的一个常见介质。为了进一步研究氧化剂在这个过程中的作用，我们研究了抗氧化剂对辛德毕斯病毒（SV）诱导的两种细胞系（AT-3，一种前列腺癌细胞系；N18，一种神经母细胞瘤细胞系）凋亡的影响。硫醇抗氧化剂N-乙酰半胱氨酸（NAC），在浓度高于30 mM时，能完全消除SV在AT-3和N18细胞中诱导的凋亡。NAC的作用不能归因于对病毒进入或病毒复制的抑制、细胞外渗透压的改变或细胞内谷胱甘肽水平的升高，也不能被微量金属螯合剂、脂质过氧化抑制剂或过氧化物清除剂模拟。相反，其他硫醇试剂，包括吡咯烷二硫代氨基甲酸盐（PDTC，75 microM）具有保护作用。因为NAC和PDTC是转录因子NF-κB最有效的抑制剂之一，我们在凋亡的形态学或生化证据出现之前，检测了SV激活NF-κB的能力。在感染后的数小时内，SV在AT-3和N18细胞中诱导核NF-κB活性显著增加；这种激活可被NAC和PDTC抑制。在AT-3细胞中过表达bcl-2，已证明其能抑制SV诱导的凋亡，同时也抑制SV诱导的NF-κB激活。为了确定NF-κB激活对于这些细胞中SV诱导的凋亡是否必要，我们使用具有一致NF-κB序列的双链寡核苷酸作为转录因子诱饵（TFD）来抑制NF-κB与天然DNA位点的结合。野生型而非突变型TFD抑制AT-3细胞中SV诱导的凋亡。相反，TFD对N18细胞中NF-κB核活性的抑制并不能阻止SV诱导的凋亡。综上所述，这些观察结果定义了一种细胞类型特异性的、转录因子信号通路，它是SV诱导凋亡所必需的。了解Bcl-2和硫醇试剂在AT-3细胞中抑制SV诱导的核NF-κB活性的确切机制，可能会为这些试剂的多能抗凋亡作用提供见解。
