Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide(GSSG) to glutathione (GSH). To evaluate the importance of GR as an antioxidant enzyme, we generated cell lines that overexpressed human GR (hGR), and studied the sensitivities of these cell lines to oxidant stress. We previously reported that the human GR cDNA might not have a mitochondrial targeting signal (MTS). For that reason, we ligated a MTS of human manganese superoxide dismutase 5' to the hGR cDNA, transfected Chinese hamster ovary(CHO) cells with hGR cDNA with/without MST, selected clones with neomycin and hygromycin B, and measured cellular GR activities. After the selection, clones G20 and GL13 showed higher GR activities (G20, 213.1±9.1 mU/mg pro; GL13; 298.8±15.7) than CHO cells (28.3±1.1). GL13 cells, which had been transfected with hGR with MTS cDNA had higher mitochondrial GR activities (819.1±39.7) than G20 cells, transfected with only hGR cDNA(47.1±6.2) or CHO cells (39.9±5.2). Southern analysis of these cell lines revealed hGR cDNA integrated in the genomes, and Northern analysis showed marked increases of transcripts for hGR, along with CHO GR mRNA. GSH concentrations increased in these cell lines (G20, 32.6±1.9 nmol/mg pro; GL13, 33.2±1.2 vs. CHO, 20.6±0.4), while GSSG did not change significantly. We exposed these stable cell lines to t-butyl hydroperoxide (t-BuOOH). After 16 hr exposure in 0.5 mM t-BuOOH, CHO cells released more cellular LDH to media (62.5±7.3%) than stable cell lines(G20, 15.0±3.2%; GL13, 0.8±0.5%). Whereas G20 in 1 mM t-BuOOH released 79.6±3.7% of cellular LDH, GL13 released only 5.8±1.3%, suggesting elevated GR protected cells from oxidant injury and mitochondrial GR appeared to be particularly critical for the protection.
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Tamura, T., McMicken, H., Smith, C. et al. OVEREXPRESSION OF HUMAN GLUTATHIONE REDUCTASE IN CHINESE HAMSTER OVARY CELLS PROTECTS CELLS FROM OXIDANT INJURY. ▴ 1474. Pediatr Res 39, 248 (1996). https://doi.org/10.1203/00006450-199604001-01497