Toshiya Tamura

Stable transfection of Chinese hamster ovary cells with glutamate-cysteine ligase catalytic subunit cDNA confers increased resistance to tert-butyl hydroperoxide toxicity

Glutathione (GSH) plays vital roles in antioxidant defense mechanisms. To determine whether gene transfection strategies could be used to enhance GSH synthetic capacities and protect mammalian cells against oxidant stresses, we used liposome-mediated transfer of the cDNA for rat glutamate-cysteine ligase (GLCL) catalytic subunit (GLCLC) to transfect Chinese hamster ovary (CHO) cells. CHO cell lines (CHOhi) with stably enhanced GLCL activities (14.61+/-0.82 mU/mg protein) and greater GSH contents (45.7+/-1.37 nmol/mg protein) than observed in wild-type CHO K1 cells (0.26+/-0.01 mU/mg protein and 20.7+/-1.15 nmol/mg protein, respectively) were developed and were confirmed to have integrated the GLCLC cDNA into their genomic DNA and to exhibit increased GLCLC mRNA levels, by Southern and northern analyses, respectively. Similarly treated and selected CHO cell lines that showed no increases in GLCL activities (CHOun) were studied as controls for the effects of GLCLC transgene expression. CHOhi cells showed significantly greater resistance to oxidant stress caused by exposure to tert-butyl hydroperoxide (tBuOOH) than did CHO or CHOun cells. Twenty-four hours after exposure to 400 or 800 microM tBuOOH, wild-type CHO cells had released more cellular lactate dehydrogenase (67.3+/-14.5% and 94.4+/-2%) than had CHOhi cells (5.11+/-0.5% and 46.0+/-5.4%, n=4, P<0.05). The present data demonstrate improved resistance to oxidant injury of CHO cells stably transfected with the GLCLC cDNA. Although additional enhancements in GLCL activities are possible by transfection with cDNAs for both catalytic and regulatory GLCL subunits, our results demonstrate that the increases in GLCL activities that can be attained by transfection of the GLCLC cDNA alone can enhance cellular antioxidant defense function.

OVEREXPRESSION OF HUMAN GLUTATHIONE REDUCTASE IN CHINESE HAMSTER OVARY CELLS PROTECTS CELLS FROM OXIDANT INJURY. ▴ 1474

OVEREXPRESSION OF HUMAN GLUTATHIONE REDUCTASE IN CHINESE HAMSTER OVARY CELLS PROTECTS CELLS FROM OXIDANT INJURY. ▴ 1474

Protection from Hyperoxia-Induced Cytostatis in H441 Cells by Enhancement of Mitochondrial Glutathione Reductase Activities Via Adenoviral Transfection

Protection from Hyperoxia-Induced Cytostatis in H441 Cells by Enhancement of Mitochondrial Glutathione Reductase Activities Via Adenoviral Transfection

Effective Attenuation of Cellular Glutathione Reductase Activities by Transgenic Expression of Dominant Negative Mutants

Effective Attenuation of Cellular Glutathione Reductase Activities by Transgenic Expression of Dominant Negative Mutants

Mitochondrially Targeted Gene Transfer of Glutathione Reductase Protects H441 cells from t-BuOOH Induced Oxidant Stresses • 1713

Mitochondrially Targeted Gene Transfer of Glutathione Reductase Protects H441 cells from t-BuOOH Induced Oxidant Stresses • 1713

OVEREXPRESSION OF HUMAN GLUTATHIONE REDUCTASE (GR) PROTECTS CHINESE HAMSTER OVARY (CHO) CELLS AGAINST INJURY FROM OXIDANTS GENERATED INTRACELLULARLY. |[diams]| 1026

We generated cells with increased activities of GR by transfection of CHO cells with the cDNA for human GR, with and without a mitochondrial targeting signal (MTS). Clones G20 and GL13 showed higher GR activities (G20, 164 mU/mg pro; GL13, 184) than did CHO cells (28). GL13 cells, transfected with hGR with MTS cDNA had higher mitochondrial GR activities (434) than did G20 cells transfected with only hGR cDNA (56) or CHO cells (48). GSH concentrations were increased in both transfected cell lines, while GSSG concentrations did not change. Previous studies revealed that overexpression of GR in mitochondria is more protective against t-butyl hydroperoxide than extramitochondrial GR, but the effects of oxidants added extracellularly might not give an accurate assessment of protection against oxidants generated intracellularly, which we investigated in the present studies. Incubation of CHO and G20 cells with 0.1mM diquat caused increased release of LDH to the media beginning at 50 h and half-maximal release was observed at 60 h. In GL13 cells the effects of diquat were delayed by 20 h, and these cells showed little LDH leakage when the CHO and G20 cells showed almost complete cell death. These studies show that enhanced GR activities in mitochondria protect against the cytotoxic effects of reactive oxygen species generated intracellularly. Supported by GM44263 and a summer research fellowship (to VRR) by the Society for Pediatric Research.

MITOCHONDRIAL AND NUCLEAR DNA ALTERATIONS CAUSED BY HEPATOTOXIC DOSES OF ACETAMINOPHEN (AP) IN MICE. |[dagger]| 970

Reactive metabolites of AP bind to hepatic and renal DNA and hepatotoxic doses initiate oligonucleosomal fragmentation of nuclear DNA. Despite generation of the reactive metabolites of AP primarily in the endoplasmic reticulum, alterations of mitochondrial proteins by reactive metabolites of AP also have been observed; however, the effects of AP metabolites on mitochondrial DNA have not been investigated. Fasted male ICR mice were given 400 mg/kg of AP or equal volumes of saline, killed at 2 h, mitochondria isolated, and DNA extracted. The mitochondrial DNA was amplified by polymerase chain reaction, using sets of primers that collectively span the entire mitochondrial genome and which were selected to amplify the common deletions that have been identified in studies of aging and degenerative diseases as critical targets for damage to mitochondrial DNA. Our studies to date do not indicate that significant increases in common deletions of mitochondrial DNA are associated with acutely hepatotoxic doses of AP, although results indicative of altered PCR amplification of the mitochondrial DNA in the AP-treated mice suggest other forms of damage to the DNA, such as alkylation. The correlation between cumulative ingestion of AP and incidence of end stage renal disease in humans [NEJM 1675, 1994] suggests a chronic injury, which may include DNA damage and mitochondrial dysfunction. Therefore, the investigations of alterations of mitochondrial DNA in relevant models of chronic exposure to AP are needed. Supported by GM44263 and a summer research fellowship (to SAM) from the Society for Pediatric Research.

OVEREXPRESSION OF HEME OXYGENASE BY CALCIUM PHOSPHATE MEDIATED TRANSFECTION IN CULTURED FETAL LUNG CELLS † 1470

OVEREXPRESSION OF HEME OXYGENASE BY CALCIUM PHOSPHATE MEDIATED TRANSFECTION IN CULTURED FETAL LUNG CELLS † 1470

DOMINANT NEGATIVE MUTANTS OF HUMAN GLUTATHIONE REDUCTASE (GR) DECREASE ACTIVITIES IN TRANSFECTED CHINESE HAMSTER OVARY (CHO) CELLS. ▴ 400

DOMINANT NEGATIVE MUTANTS OF HUMAN GLUTATHIONE REDUCTASE (GR) DECREASE ACTIVITIES IN TRANSFECTED CHINESE HAMSTER OVARY (CHO) CELLS. ▴ 400