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

Abstract

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.

CoASH and CoASSG Levels in Lungs of Hyperoxic Rats as Potential Biomarkers of Intramitochondrial Oxidant Stresses

“…We have observed protection against oxidant stresses in Chinese hamster ovary cells stably transfected with the cDNA for human glutathione reductase, with a most dramatic protection when the glutathione reductase activities were increased selectively in the mitochondria (27). In other studies, we observed that H441 cells, which most closely resemble lung Clara cells, were protected from tert-butyl-hydroperoxideinduced cell death (28) or from the cytostatic effects of hyperoxia in vitro (29) by adenovirus-mediated gene transfer of glutathione reductase similarly targeted for expression in the mitochondria. Mitochondrial GSSG, acting through thioldisulfide exchange reactions with PSH in processes reflected by GSH/GSSG ratios, could affect cell viability and function through alterations in protein structure (Eq.…”

Regression of A549 lung cancer tumors by anti-miR-150 vector

“…After cells were tranfected with PR-ASO-150 for 72 h, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) assay was carried as follows: 1x10 4 cells were cultured into each well of 96-well flat-bottom microtiter plates and 10 µl MTT (5 mg/ml) were added 4 h before the end of incubation, the supernatant was removed and 100 µl dimethyl sulfoxide (DMSO) were added to determine the OD value at 570 nm using an enzymelinked immunosorbent assay reader (ELX800). The growth inhibition rate was dectected according to the studies of Ren et al and O'Donovan et al (27,28). The following formula was used: growth inhibition rate = (OD control − OD sample )/ OD control x 100 (%).…”

Redox Regulation of Lung Inflammation by Thioredoxin

“…Tolerance to hyperoxia is associated with increased GSH peroxidase and reductase (66). Gene transfer of mitochondrial GSH reductase (105,106) or GSH peroxidase (10) protects cells against oxidative stress. N-acetyl-L-cysteine (NAC), a precursor of GSH, attenuates apoptotic lung injury in a sepsis model (115).…”