Oxidant insults can lead to apoptotic and nonapoptotic cell death. Lung epithelial cells exposed to high levels of oxygen do not die via apoptosis, but through a much slower, morphologically distinct process involving cell and nuclear swelling. In contrast, H2O2 induces a rapid apoptotic cell death. We first assessed the effect of oxidant exposure on activator protein-1 (c-Jun and Fos) and c-Jun N-terminal kinase (JNK) regulation in MLE12 cells. Both oxidants induced c-Jun and Fos expression, albeit with a different pattern of regulation-hyperoxia (95% O2) induced a biphasic response, whereas H2O2 (500 microM) induced a sustained response. We then examined the role of JNK by Western blot, JNK activity assay, and a pull-down assay and observed an identical pattern of regulation. To assess whether JNK functions in a pro-death or pro-survival capacity, we generated stable cell lines that constitutively express a dominant-negative mutation of JNK resulting in significant inhibition of JNK activity. Inhibition of the JNK pathway in this manner prevented hyperoxic and H2O2-induced cell death. These results demonstrate that hyperoxic cell death is pathway-driven and that both modes of death involve the JNK signaling pathway.

It has previously been shown that hyperoxia induces nonapoptotic cell death in cultured lung epithelial cells, whereas hydrogen peroxide (H 2 O 2 ) and paraquat cause apoptosis. To test whether pathways leading to oxidative apoptosis in epithelial cells are sensitive to molecular O 2 , A549 cells were exposed to 95% O 2 prior to exposure to lethal concentrations of H 2 O 2 . The extent of H 2 O 2 -induced apoptosis was significantly reduced in cells preexposed to hyperoxia compared with room-air controls. Preexposure of the hyperoxia-resistant HeLa-80 cell line to 80% O 2 also inhibited oxidant-induced apoptosis, suggesting that this inhibition is not due to O 2 toxicity. Because hyperoxia generates reactive oxygen species and activates the redox-sensitive transcription factor nuclear factor B (NF-B), the role of antioxidant enzymes and NF-B were examined in this inhibitory process. The onset of inhibition appeared to be directly related to the degradation of IB and subsequent activation of NF-B (either by hyperoxia or TNF-␣), whereas no significant up-regulation of endogenous antioxidant enzyme activities was found. In addition, suppression of NF-B activities by transfecting A549 cells with a dominant-negative mutant construct of IB significantly augmented the extent of H 2 O 2 -induced apoptosis. These data suggest that hyperoxia inhibits oxidant-induced apoptosis and that this inhibition is mediated by NF-B.Pulmonary epithelial cell injury is an unfortunate consequence of therapy with supraphysiological concentrations of oxygen (hyperoxia) and a prominent feature of acute inflammatory lung injury (1, 2). Oxidant-induced cell injury and death are generally thought to occur via reactive oxygen species. Although exogenous hydrogen peroxide (H 2 O 2 ) and paraquat induce apoptosis in cultured alveolar epithelial (A549) cells, hyperoxia kills these cells via a mode of cell death that is distinct from apoptosis both morphologically and biochemically (3-5). These observations raise the possibility that hyperoxia actually inhibits apoptosis in pulmonary epithelial cells.There are several possible ways that hyperoxia might inhibit apoptosis in pulmonary epithelial cells. First, one or more enzymes in the apoptotic pathway might be sensitive to direct oxidation by high levels of molecular O 2 , resulting in an irreversible abrogation of the apoptotic pathway in these cells. Alternatively, hyperoxia may activate pathways that inhibit apoptosis. Several laboratories have shown that activation of the transcription factor NF-B can prevent apoptosis induced by chemotherapeutic agents and ionizing radiation in cultured cells (6 -9). In addition, activation of NF-B may be responsible for the protective effect of low-dose amyloid ␤ in neuronal cell death (10) and in the survival and function of hematopoietic stem and progenitor cells (11). We have previously demonstrated that hyperoxia activates NF-B in A549 cells (5). In this report, we examined whether preexposure of cultured epithelial cells to hyperoxia prevents subsequent...

To determine whether overexpression of antioxidant enzymes in lung epithelial cells prevents damage from oxidant injury, stable cell lines were generated with complementary DNAs encoding manganese superoxide dismutase (MnSOD) and/or catalase (CAT). Cell lines overexpressing MnSOD, CAT, or MnSOD + CAT were assessed for tolerance to hyperoxia or paraquat. After exposure to 95% O(2) for 10 d, 44 to 57% of cells overexpressing both MnSOD and CAT and 37 to 47% of cells overexpressing MnSOD alone were viable compared with 7 to 12% of empty vector or parental cells (P < 0.05). To assess if viable cells were capable of cell division after hyperoxic exposures (up to 5 d), a clonogenicity assay was performed. The clonogenic potential of cells overexpressing MnSOD + CAT and MnSOD alone were significantly better than those expressing CAT alone or empty vector controls. In addition, 54 to 72% of cells overexpressing both MnSOD and CAT survived in 1 mM paraquat compared with 58 to 73% with MnSOD alone and 27% with control cells. Overexpression of CAT alone did not improve survival in hyperoxia or paraquat. The combination of MnSOD + CAT did not provide additional protection from paraquat. Data demonstrate that overexpression of MnSOD protects cells from oxidant injury and CAT offers additional protection from hyperoxic injury when co-expressed with MnSOD.