Thiol agents and Bcl-2 identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor NF-kappa B.

Abstract

Abstract. 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 SVinduced 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 IxM) 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. Overexpression of bcl-2 in AT-3 cells, which has been shown to inhibit SV-induced apoptosis, also inhibits SVinduced NF-kappa B activation. To determine if NFkappa 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.

Sp1 and Sp3 Are Oxidative Stress-Inducible, Antideath Transcription Factors in Cortical Neurons

“…Embryonic cortical neurons were lightly trypsinized, pelleted, and resuspended in cold PBS. All subsequent steps were performed at 4°C as described previously (Lin et al, 1995, Chatterjee et al, 2001. To evaluate the effects of various agents on Sp1 and Sp3 DNA binding, we added deferoxamine mesylate (10 M), D-amino acid oxidase (1-5 mU from red yeast), and D-alanine (20 mM).…”

Nuclear Factor-κB Activation: A Question of Life or Death

“…Helicobacter pylori induces NF-κB-mediated apoptosis in chronic gastritis (Gupta et al, 2001). The apoptosis that is induced by alphavirus was also found to require the activation of NF-κB (Lin et al, 1995), since thiol agents and Bcl-2 blocked both activities. During adenoviral infection, NF-κB mediates apoptosis through transcriptional activation of Fas (CD95) (Kuhnel et al, 2000).…”

Glutathione depletion in fibroblasts is the basis for apoptosis-induction by endogenous reactive oxygen species

“…Activation of apoptosis-related transcription factors represents a very intriguing possible mechanism (Westerndorp et al, 1995;Lin et al, 1995). In addition, ROS are known as very effective and specific signal transducers, which may be involved in a variety of biochemical events.…”

“…This concept is based on several lines of evidence: (1) there are several reports on direct induction of apoptosis by reactive oxygen species (Lennon et al, 1991;Sato et al, 1995), redox cyclers (Dypbukt et al, 1994) or oxidized macromolecules (Escargueil et al, 1992;Sandstrom et al, 1994); (2) several inducers of apoptosis stimulate production of reactive oxygen species in cells (Torres Roca et al, 1995;Yamauchi et al, 1989;Zamzami et al, 1995;Hennet et al, 1993); (3) several ways of apoptosis induction are blocked by antioxidants and radical scavengers (Matthews et al, 1987;Chang et al, 1992;Hockenberry et al, 1993;Kane et al, 1993;Sandstrom et al, 1993;Wolfe et al, 1994;Lin et al, 1995;Slater et al, 1995;Talley et al, 1995;Schaefer et al, 1995;Langer et al, 1996).…”

“…ROS can cause activation of apoptosis-related transcription factors, such as NFkappa B (by inactivating their inhibitory subunit) and leading to activation of apoptosis-relevant genes further on (Lin et al, 1995;Westerndorp et al, 1995). ROS may regulate the activity of cysteine-containing proteases or may influence calcium homeostasis (Briehl and Baker, 1996).…”