Am. J. Respir. Cell Mol. Biol.,
Volume 22, Number 6, June, 2000 732-738
Attenuation of Hyperoxia-Induced Growth Inhibition in H441 Cells by
Gene Transfer of Mitochondrially Targeted Glutathione Reductase
Donough J.
O'Donovan,
Julie P.
Katkin,
Toshiya
Tamura,
Charles V.
Smith,
and
Stephen E.
Welty
Department of Pediatrics, Baylor College of Medicine, Houston, Texas
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 × 106 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 × 106 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 × 106 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.
