title: Bcl-2 attenuation of oxidative cell death is associated with up-regulation of gamma-glutamylcysteine ligase via constitutive NF-kappaB activation.
abstract: Oxidative stress induced by reactive oxygen intermediates often causes cell death via apoptosis, which is regulated by many functional genes and their protein products. The evolutionarily conserved protein Bcl-2 blocks apoptosis induced by a wide array of death signals. Despite extensive research, the molecular milieu that characterizes the anti-apoptotic function of Bcl-2 has not been fully clarified. In this work, we have investigated the role of bcl-2 in protecting against oxidative death induced by H(2)O(2) in cultured rat pheochromocytoma PC12 cells. Transfection with the bcl-2 gene rescued PC12 cells from apoptotic death caused by H(2)O(2). Addition of NF-kappaB inhibitors such as pyrrolidine dithiocarbamate and N-tosyl-l-phenylalanine chloromethyl ketone to the medium aggravated oxidative cell death. PC12 cells overexpressing bcl-2 exhibited relatively high constitutive DNA binding and transcriptional activities of NF-kappaB compared with vector-transfected control cells. Western blot analysis and immunocytochemistry revealed that bcl-2-transfected PC12 cells retained a higher level of p65 (the functionally active subunit of NF-kappaB) in the nucleus compared with vector-transfected controls. In addition, sustained activation of ERK1/2 (upstream of NF-kappaB) was observed in bcl-2-overexpressing cells. In contrast, the cytoplasmic inhibitor IkappaBalpha was present in lower amounts in cells overexpressing bcl-2. The ectopic expression of bcl-2 increased the cellular glutathione level and gamma-glutamylcysteine ligase expression, which were attenuated by NF-kappaB inhibitors. These results suggest that NF-kappaB plays a role in bcl-2-mediated protection against H(2)O(2)-induced apoptosis in PC12 cells through augmentation of antioxidant capacity.
authors.affiliation: Laboratory of Biochemistry and Molecular Toxicology, College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-ku, Seoul 151-742, South Korea.
authors.forename: Jung-Hee
authors.initials: JH
authors.lastname: Jang
authors.forename: Young-Joon
authors.initials: YJ
authors.lastname: Surh
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D016923:Cell Death; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D020672:I-kappa B Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008156:Luciferases / Q000378:metabolism; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D015854:Up-Regulation
chemical_list: D000975:Antioxidants; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D019253:Proto-Oncogene Proteins c-bcl-2; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA; D006861:Hydrogen Peroxide; D008156:Luciferases; D020928:Mitogen-Activated Protein Kinases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D010100:Oxygen