Overexpression of bcl-xL protects astrocytes from glucose deprivation and is associated with higher glutathione, ferritin, and iron levels.
BACKGROUND
The possibility of altering outcome from ischemia-like injury by overexpressing the anti-cell death gene bcl-xL was studied. Cells are known to die by different pathways including apoptosis, or programmed cell death, and necrosis. The bcl-xL gene is a member of a family of apoptosis regulating genes and often displays the death-inhibiting properties of the prototype of this family, bcl-2. It is of special interest to study bcl-xL for possible brain protection, because, unlike bcl-2, it is important for normal brain development.


METHODS
Overexpression of bcl-xL was achieved in primary astrocyte cultures using a retroviral vector. Cultures of astrocytes overexpressing bcl-xL or a control gene were injured by hydrogen peroxide, glucose deprivation, or combined oxygen and glucose deprivation. Outcome was assessed morphologically and by release of lactate dehydrogenase. We assessed antioxidant effects by measuring glutathione using monochlorobimane, ferritin by immunoblotting, the level of iron spectrophotometrically, and superoxide using iodonitrotetrazolium violet and dihydroethidium.


RESULTS
Protection by bcl-xL was found against glucose deprivation and hydrogen peroxide exposure but not combined oxygen and glucose deprivation. Higher levels of superoxide were found, without increased levels of lipid peroxidation. Overexpression of bcl-xL was associated with elevated glutathione levels, elevated ferritin levels, and increased amounts of iron. The increased glutathione contributed to the protection from glucose deprivation.


CONCLUSIONS
Overexpression of bcl-xL protects astrocytes from oxidative injury with the same spectrum of protection seen previously for bcl-2. The increased antioxidant defense observed should be beneficial against both apoptotic and necrotic cell death. The effects on levels of ferritin and iron are novel and identify a new area of interest for this gene family. Whether this relates to the effects of these genes on mitochondrial function remains to be elucidated.
Xu
L
L
Department of Anesthesia, Stanford University School of Medicine, California 94305-5117, USA.
Koumenis
I L
IL
Tilly
J L
JL
Giffard
R G
RG
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D016923:Cell Death / Q000235:genetics; D015687:Cell Hypoxia / Q000235:genetics; D002478:Cells, Cultured; D005293:Ferritins / Q000378:metabolism*; D005786:Gene Expression Regulation; D005947:Glucose / Q000172:deficiency*; D005978:Glutathione / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000633:toxicity; D007150:Immunohistochemistry; D007501:Iron / Q000378:metabolism*; D015227:Lipid Peroxidation; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D013481:Superoxides / Q000378:metabolism; D014162:Transfection; D051020:bcl-X Protein
C494812:Bcl2l1 protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D013481:Superoxides; D005293:Ferritins; D006861:Hydrogen Peroxide; D007501:Iron; D005978:Glutathione; D005947:Glucose