Potentiation of murine astrocyte antioxidant defence by bcl-2: protection in part reflects elevated glutathione levels


Correspondence: R. G. Giffard, as above. E-mail: rona.giffard@stanford.edu


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 β-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 β-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 β-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.