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.

• Authors: D J O'Donovan, J P Katkin, T Tamura, C V Smith, S E Welty
• Journal: American journal of respiratory cell and molecular biology (Am J Respir Cell Mol Biol) Vol. 22 Issue 6 Pg. 732-8 (Jun 2000) ISSN: 1044-1549 [Print] United States
• PMID: 10837371 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Growth Inhibitors
• Reactive Oxygen Species
• L-Lactate Dehydrogenase
• Glutathione Reductase
• Glutathione
• Oxygen
• Glutathione Disulfide

Topics

• Adenoviridae (genetics)
• Cell Division (drug effects, physiology)
• Cell Line, Transformed
• Gene Expression Regulation, Enzymologic
• Gene Transfer Techniques
• Glutathione (metabolism)
• Glutathione Disulfide (metabolism)
• Glutathione Reductase (genetics, metabolism)
• Growth Inhibitors (genetics)
• Humans
• Hyperoxia (metabolism)
• L-Lactate Dehydrogenase (metabolism)
• Lung (cytology)
• Mitochondria (enzymology)
• Oxygen (pharmacology)
• Reactive Oxygen Species (metabolism)
• Regulatory Sequences, Nucleic Acid
• Transfection