Expression of Recombinant Human Glutathione Reductase in Eukaryotic Cells after DNA-Mediated Gene Transfer

Cholin, S.; Tonoki, Hidefumi; Hansen, Thomas N.; Ledley, Fred D.

doi:10.1006/bmmb.1993.1012

Cited by 4 publications

(3 citation statements)

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“…The physiological significance of regenerating GSH from GSSG by GR is indicated by studies demonstrating that cells with attenuated GR activities are more susceptible to oxidative stresses (15)(16)(17)(18). Although it does not necessarily follow that increased GR activities would enhance antioxidant defense function significantly, we have observed greater resistance to oxidant stresses in Chinese hamster ovary (CHO) cells with genetically enhanced GR activities, particularly if the mitochondrial GR activities are augmented (19)(20)(21)(22). As ROS have been implicated as pathogenic agents in many disease states, including oxygen toxicity, determining the specific mechanisms governing the protective effect of GR overexpression may be crucial to understanding cellular responses in pulmonary oxygen toxicity and other diseases and toxicities mediated in part by oxidant stresses.…”

mentioning

confidence: 64%

“…Details on the cloning of human GR cDNA and addition of the mitochondrial targeting signal (MTS) from the human manganese superoxide dismutase (MnSOD) gene have been described previously (19). Using the GR cDNA clone with a functional MTS, we constructed an adenoviral vector from the replication-defective human adenovirus type 5 (Ad5).…”

Section: Vector Constructionmentioning

confidence: 99%

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Gene Transfer of Mitochondrially Targeted Glutathione Reductase Protects H441 Cells from t-Butyl Hydroperoxide–Induced Oxidant Stresses

O'Donovan

Katkin²,

Tamura³

et al. 1999

Am J Respir Cell Mol Biol

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Increased generation of reactive oxygen species (ROS) and low levels of antioxidants may cause morbidity in premature infants on supplemental oxygen. Glutathione (GSH)-dependent antioxidant systems protect against ROS, and regenerating GSH from GSH disulfide (GSSG) by the flavoenzyme GSH reductase (GR) is essential for the optimal function of this system. Previously, we have observed enhanced resistance to t-butyl hydroperoxide (t-BuOOH) in Chinese hamster ovary cells stably transfected with a vector (leader sequence GR [LGR]) for human GR cDNA that contained a functional synthetic mitochondrial targeting signal. The present studies were designed to investigate adenovirus-mediated gene transfer of LGR to H441 cells and resistance of such cells to t-BuOOH. Adenovirus-mediated transfection of H441 cells with LGR increased total GR activities more than 11-fold (mitochondria more than 10-fold and cytosolic more than 7-fold) and protected against t-BuOOH cytotoxicity, as indicated by lower fractional release of cellular lactate dehydrogenase (LDH) than was observed in wild-type untransfected cells (CON) or in cells transfected with a control gene (human manganese superoxide dismutase in the antisense orientation [DOS]) (*LGR 6.6 +/- 1.7; DOS 16 +/- 1.8; CON 16.6 +/- 0.7% LDH release). In addition, cells transfected with LGR retained higher GSH/GSSG ratios (*LGR 66 +/- 0.4; DOS 47 +/- 1; CON 52.6 +/- 2.3) and released less GSH + GSSG to the media in response to challenge with t-BuOOH (*LGR 0.05 +/- 0.01; DOS 0.08 +/- 0.01; CON 0.07 +/- 0.01 nmol/mg of protein) than did wild-type cells or cells transfected with a control vector, indicating an enhanced ability of the LGR cells to reduce GSSG formed in response to exposure to t-BuOOH. In conclusion, adenovirus-mediated gene transfer of LGR enhanced cellular GR activities and protected H441 cells from oxidant stresses.

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mentioning

confidence: 64%

Section: Vector Constructionmentioning

confidence: 99%

Gene Transfer of Mitochondrially Targeted Glutathione Reductase Protects H441 Cells from t-Butyl Hydroperoxide–Induced Oxidant Stresses

O'Donovan

Katkin²,

Tamura³

et al. 1999

Am J Respir Cell Mol Biol

View full text Add to dashboard Cite

show abstract

“…Details on the cloning of human GR cDNA and addition of the mitochondrial targeting signal (MTS) from the human manganese superoxide dismutase (MnSOD) gene have been described previously (29). Using a GR cDNA with a functional MTS, we constructed an adenoviral vector from the replication-defective human adenovirus type 5 (Ad5).…”

Section: Vector Constructionmentioning

confidence: 99%

Attenuation of Hyperoxia-Induced Growth Inhibition in H441 Cells by Gene Transfer of Mitochondrially Targeted Glutathione Reductase

O'Donovan

Katkin

Tamura

et al. 2000

Am J Respir Cell Mol Biol

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Reactive oxygen species (ROS) are implicated as agents of cellular damage in pulmonary oxygen toxicity. Glutathione (GSH) and GSH-dependent antioxidant enzymes protect against damage by ROS, and recycling of glutathione disulfide (GSSG) to GSH by glutathione reductase (GR) is essential for the optimum functioning of this system. Exposure to hyperoxia inhibits lung development in newborn animals and humans, and attenuates cell growth in proliferating cell cultures. Considerable evidence supports a role for ROS as growth-altering molecules. Previously, we have observed that gene transfer of GR to mitochondria in H441 cells, using a vector containing a mitochondrial leader sequence (LGR), protected these cells against t-BuOOH-induced cytotoxicity. The present studies tested the hypothesis that gene transfer of LGR would attenuate the cytostatic effects of hyperoxia exposure in H441 cells. H441 cells (0.9 x 10(6) cells/plate) transfected with adenovirus containing LGR or the complementary DNA (cDNA) for manganese superoxide dismutase in reverse orientation (DOS) as a control construct, and untransfected cells (CON) were maintained in 21% oxygen (normoxia) or 95% oxygen (hyperoxia) for 48 h, and cell growth was assessed by cell counts and by reduction of the tetrazolium dye 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan. Cells maintained in normoxia achieved normal growth (CON, 1.98; DOS, 1.91; LGR, 2.0 x 10(6) cells/plate). Hyperoxia inhibited cell growth and the reduction of MTT; however, cells transfected with LGR had greater mitochondrial GR activities (CON, 16+/-2; DOS, 19+/-3; LGR, 322+/-18 mU/mg of protein), sustained more normal growth patterns (CON, 1.25+/-0.12; DOS, 1.24 +/-0.21, LGR, 1.8+/-0.25 x 10(6) cells/plate), and had less inhibition of MTT reduction (CON, 29; DOS, 27; LGR, 16% inhibition, P<0.01) after exposure to hyperoxia for 48 h than was observed in cells transfected with DOS or in control cells not infected with virus. In addition, resistant cells had higher mitochondrial GSH levels and maintained mitochondrial GSH/GSSG ratios in hyperoxia, suggesting that maintaining mitochondrial GSH homeostasis determined critical aspects of cell division in these studies. The mechanisms for sustaining cell growth during hyperoxia in H441 cells with enhanced mitochondrial GR activities are unknown, but similar effects in infants exposed to supplemental oxygen could be highly beneficial.

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Mitochondrial Glutathione and Oxidative Stress: Implications for Pulmonary Oxygen Toxicity in Premature Infants

O'Donovan

Fernandes

2000

Molecular Genetics and Metabolism

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Expression of Recombinant Human Glutathione Reductase in Eukaryotic Cells after DNA-Mediated Gene Transfer

Cited by 4 publications

References 0 publications

Gene Transfer of Mitochondrially Targeted Glutathione Reductase Protects H441 Cells from t-Butyl Hydroperoxide–Induced Oxidant Stresses

Gene Transfer of Mitochondrially Targeted Glutathione Reductase Protects H441 Cells from t-Butyl Hydroperoxide–Induced Oxidant Stresses

Attenuation of Hyperoxia-Induced Growth Inhibition in H441 Cells by Gene Transfer of Mitochondrially Targeted Glutathione Reductase

Mitochondrial Glutathione and Oxidative Stress: Implications for Pulmonary Oxygen Toxicity in Premature Infants

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