Radiosensitivity of mammalian cell lines engineered to overexpress cytosolic glutathione peroxidase.
Reactive oxygen species are believed to be involved in radiation lethality. Glutathione peroxidase is an intracellular enzyme with antioxidant functions. To determine whether increasing the cellular antioxidant capacity can confer radiation resistance, the effect of overexpression of glutathione peroxidase on radiosensitivity was determined in two different cell types. An expression construct including the bovine cytosolic glutathione peroxidase cDNA was used to overexpress this enzyme in cells of the human lymphoblast cell line Sup-T1 as well as the Chinese hamster ovary cell line AA8. Supplementation of the culture media with 30 nM sodium selenite was included to obtain optimal glutathione peroxidase activity. Northern blot analysis confirmed the presence of the construct mRNA, and a standard coupled spectrophotometric assay demonstrated significantly increased glutathione peroxidase activity in the transfected cell lines. An approximately 8-fold increase was found in the Sup-T1 cells, and an approximately 30-fold increase was obtained in the Chinese hamster ovary AA8 cells. Clonogenic survival was assayed in the overexpressing cells and compared to that in control cells transfected with vector alone. Despite significantly increased glutathione peroxidase activity, no observable radioprotection was conferred in either of the two cell lines studied, indicating that increased glutathione peroxidase activity is insufficient to confer radioresistance in the two cell types examined. These data are discussed in the context of using antioxidants as adjuncts to clinical radiotherapy.
Mansur
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DB
Radiation Oncology Center, Mallinckrodt Institute of Radiology, Washington University Medical School, Box 8224-21, 4939 Children's Place, Suite 5500, St. Louis, MO 63110, USA.
Kataoka
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Y
Grdina
D J
DJ
Diamond
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AM
D000818:Animals; D016466:CHO Cells / Q000201:enzymology / Q000528:radiation effects; D002417:Cattle; D002460:Cell Line / Q000201:enzymology / Q000528:radiation effects; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D005979:Glutathione Peroxidase / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology / Q000528:radiation effects; D018384:Oxidative Stress; D011836:Radiation Tolerance; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000502:physiology; D015398:Signal Transduction / Q000528:radiation effects; D014162:Transfection
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005979:Glutathione Peroxidase