Potentiation of murine astrocyte antioxidant defence by bcl-2: protection in part reflects elevated glutathione levels.
Overexpression of the proto-oncogene bcl-2 has been shown to protect a variety of cell types from oxidative and non-oxidative injury, blocking apoptotic and necrotic types of cell death. Retroviral vectors were used to stably overexpress bcl-2 in primary murine astrocyte cultures with more than 95% efficiency. Compared to beta-galactosidase-expressing and uninfected control cells, bcl-2 overexpressing astrocytes suffered < 40% injury after 24 h glucose deprivation, while controls were essentially completely injured. After exposure to 0.2 mM hydrogen peroxide, the bcl-2 overexpressing astrocytes suffered < 40% the injury seen in controls. In contrast, when the cultures were injured by combined oxygen-glucose deprivation, no difference in the extent or time course of injury was found between cells overexpressing bcl-2 and those expressing beta-galactosidase. To investigate one possible mechanism of bcl-2 protection, we measured the levels of glutathione and three antioxidant enzymes. Astrocytes overexpressing bcl-2 had elevated glutathione levels (130-200%), increased superoxide dismutase (170%) and glutathione peroxidase (140%) activities compared with beta-galactosidase-expressing controls. Bcl-2 overexpressing astrocytes suffered less lipid peroxidation after glucose deprivation, as assessed by cis-parinaric acid fluorescence, and demonstrated more rapid removal of hydrogen peroxide from the medium. When glutathione levels were decreased 80% by pretreatment with buthionine sulfoximine, the extent of protection from glucose deprivation of bcl-2 overexpressing cells was decreased by about half. Increased antioxidant defence contributes to protection from glucose deprivation in bcl-2 overexpressing astrocytes.
Papadopoulos
M C
MC
Department of Anaesthesia, Stanford University School of Medicine, CA 94305, USA.
Koumenis
I L
IL
Xu
L
L
Giffard
R G
RG
D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D001253:Astrocytes / Q000502:physiology*; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D005822:Genetic Vectors; D005978:Glutathione / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis*; D012190:Retroviridae / Q000235:genetics; D013482:Superoxide Dismutase / Q000378:metabolism
D000975:Antioxidants; D009419:Nerve Tissue Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D002374:Catalase; D013482:Superoxide Dismutase; D005978:Glutathione