hSMG-1 and ATM sequentially and independently regulate the G1 checkpoint during oxidative stress.

Genotoxic stress activates the phosphatidylinositol 3-kinase-like kinases (PIKKs) that phosphorylate proteins involved in cell cycle arrest, DNA repair and apoptosis. Previous work showed that the PIKK ataxia telangiectasia mutated (ATM) but not ATM and Rad3 related phosphorylates p53 (Ser15) during hyperoxia, a model of prolonged oxidative stress and DNA damage. Here, we show hSMG-1 is responsible for the rapid and early phosphorylation of p53 (Ser15) and that ATM helps maintain phosphorylation after 24 h. Despite reduced p53 phosphorylation and abundance in cells depleted of hSMG-1 or ATM, levels of the p53 target p21 were still elevated and the G(1) checkpoint remained intact. Conditional overexpression of p21 in p53-deficient cells revealed that hyperoxia also stimulates wortmannin-sensitive degradation of p21. siRNA depletion of hSMG-1 or ATM restored p21 stability and the G(1) checkpoint during hyperoxia. These findings establish hSMG-1 as a proximal regulator of DNA damage signaling and reveal that the G(1) checkpoint is tightly regulated during prolonged oxidative stress by both PIKK-dependent synthesis and proteolysis of p21.

p53-dependent induction of p21(Cip1/WAF1/Sdi1) protects against oxygen-induced toxicity.

The beneficial effects of supplemental oxygen delivered to patients suffering from acute respiratory distress is offset by its reduction to genotoxic reactive oxygen species (ROS) that inhibit proliferation and kill pulmonary cells. Cells respond to oxygen-induced damage by expressing the tumor suppressor p53 and the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), which limits proliferation by blocking entry into S phase. Since preventing DNA synthesis during genotoxic stress may enhance survival, the current study examines whether hyperoxia induces p21 through a p53-dependent pathway and whether p21 protects cells from the toxic effects of oxygen. HCT116 colon carcinoma cells and clonal lines lacking p53 or p21were used in this study because they allow direct cytotoxic comparisons between isogenic cells, without complications arising from unknown genetic differences between nonhomologous cell lines. Hyperoxia (95% O2, 5% CO2) increased p53 abundance, phosphorylation of p53 on serine 15, and p21 mRNA and protein in parental HCT116 cells that ceased proliferation. In contrast, p21 was not detected in either p53- or p21-deficient HCT116 cells, which exited the G1 compartment and were arrested in S and G2/M phases during hyperoxia. Trypan blue-dye exclusion revealed that induction of p21 markedly enhanced survival during exposure and colony survival assays showed that p21 enhanced the ability to resume proliferation during recovery in room air. The observation that p53-dependent induction of p21 prevents exit from G1 and promotes survival during hyperoxia is consistent with the importance of limiting DNA replication during genotoxic stress caused by oxygen exposure.

The cyclin-dependent kinase inhibitor p21 protects the lung from oxidative stress.

The lung is a major target tissue for oxidative stress, including hyperoxia used to relieve tissue hypoxia. Unfortunately, severe hyperoxia damages DNA, inhibits proliferation, and kills cells, resulting in morbidity and mortality. Although hyperoxia induces the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), their role in pulmonary injury remains unknown. Using p53- and p21-deficient mice we demonstrate that hyperoxia induces p21 in the absence of p53, suggesting that previous conclusions that p53 does not modify hyperoxic lung injury cannot be extrapolated to p21. In fact, mean survival of p21-deficient mice decreased by 40% and was associated with terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining of alveolar debris, indicative of DNA fragmentation and cell death. Ultrastructural analyses revealed that alveolar endothelial and type I epithelial cells died rapidly by necrosis. Although hyperoxia decreased DNA replication in p21-wild-type lungs, it had no effect on replication in p21-deficient lungs. Our findings suggest that p21 protects the lung from oxidative stress, in part, by inhibiting DNA replication and thereby allowing additional time to repair damaged DNA. Our findings have implications for patients suffering from the toxic effects of supplemental oxygen therapies.

p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia.

Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.

Tumor necrosis factor-alpha-induced lung cell expression of antiapoptotic genes TRAF1 and cIAP2.

Tumor necrosis factor (TNF) receptor (TNFR)-associated factors 1 and 2 (TRAF1 and TRAF2) and inhibitor of apoptosis proteins cIAP1 (MIHB) and cIAP2 (MIHC) were recently identified as proteins that associate with the TNF-alpha receptors TNFRI (p55) and TNFRII (p75) and inhibit TNF-alpha-induced programmed cell death or apoptosis. In the original reports, TRAF1 expression, unlike the ubiquitous TRAF2, was restricted to specific tissues in the lung, spleen, and testis. TNF-alpha is increased in the lung in many forms of pulmonary disease. In the current study, Western analysis, immunohistochemistry, and ribonuclease protection assays were used to determine whether TNF-alpha regulates the expression of these TNFR-associated proteins in lung cells. We demonstrate for the first time TNF-alpha dose-dependent induction of TRAF1 protein and messenger RNA (mRNA) in human H441 and A549 pulmonary adenocarcinoma cell lines, as well as in lung cells of C57BL/6J mice after intratracheal administration of TNF-alpha. In contrast to the epithelial cells, TRAF1 was not induced by TNF-alpha in U937 cells, a human monocytic cell line, suggesting cell type-specific regulation. Similarly, cIAP2 mRNA was induced by TNF-alpha in both H441 and A549 pulmonary epithelial cells but not in U937 cells. TNF-alpha is a primary mediator of acute pulmonary inflammation and contributes to the pathophysiology of chronic lung diseases such as bronchopulmonary dysplasia (BPD), a fibrotic disease of prematurely born infants. Immunohistochemical staining of human neonatal lung tissue demonstrated increased TRAF1 in lungs of infants dying of pneumonia or BPD in comparison with those dying of congenital malformation. These studies support the hypothesis that the TRAF1 and cIAP2 genes are highly regulated in pulmonary cells and may play a role in human lung disease.

Bcl-2 family gene expression during severe hyperoxia induced lung injury.

Exposure of the lung to severe hyperoxia induces terminal transferase dUTP end-labeling (TUNEL) indicative of DNA damage or apoptosis and increases expression of the tumor suppressor p53 and of members of the Bcl-2 gene family. Because cell survival and apoptosis are regulated, in part, by the relative abundance of proteins of the Bcl-2 family, we hypothesized that lung cells dying during exposure would show increased expression of pro-apoptotic members, such as Bax, whereas surviving cells would have increased expression of anti-apoptotic members, such as Bcl-X(L). The hypothesis is tested in the current study by determining which Bcl-2 genes are regulated by hyperoxia, with specific focus on correlating expression of Bax and Bcl-X(L) with morphologic evidence of apoptosis or necrosis. Adult mice exposed to greater than 95% oxygen concentrations for 48 to 88 hours had increased whole-lung mRNA levels of Bax and Bcl-X(L), no change in Bak, Bad, or Bcl-2, and decreased levels of Bcl-w and Bfl-1. In situ hybridization revealed that hyperoxia induced Bax and Bcl-X(L) mRNA in uniform and overlapping patterns of expression throughout terminal bronchioles and parenchyma, coinciding with TUNEL staining. Electron microscopy and DNA electrophoresis, however, suggested relatively little classical apoptosis. Unexpectedly, Western analysis demonstrated increased Bcl-X(L), but not Bax, protein in response to hyperoxia. Bax and Bfl-1 were not altered by hyperoxia in p53 null mice; however, oxygen toxicity was not lessened by p53 deficiency. These findings suggest that oxygen-induced lung injury does not depend on the relative expression of these Bcl-2 members.

Hyperoxia inhibits proliferation of Mv1Lu epithelial cells independent of TGF-beta signaling.

High concentrations of O(2) inhibit epithelial cell proliferation that resumes on recovery in room air. To determine whether growth arrest is mediated by transforming growth factor-beta (TGF-beta), changes in cell proliferation during exposure to hyperoxia were assessed in the mink lung epithelial cell line Mv1Lu and the clonal variant R1B, which is deficient for the type I TGF-beta receptor. Mv1Lu cells treated with TGF-beta accumulated in the G(1) phase of the cell cycle as determined by propidium iodide staining, whereas proliferation of R1B cells was unaffected by TGF-beta. In contrast, hyperoxia inhibited proliferation of both cell lines within 24 h of exposure through an accumulation in the S phase. Mv1Lu cells treated with TGF-beta and exposed to hyperoxia accumulated in the G(1) phase, suggesting that TGF-beta can inhibit the S phase accumulation observed with hyperoxia alone. Cyclin A was detected in cultures exposed to room air or growth arrested by hyperoxia while decreasing in cells growth arrested in the G(1) phase by TGF-beta. Finally, hyperoxia failed to activate a TGF-beta-dependent transcriptional reporter in both Mv1Lu and R1B cells. These findings reveal that simple growth arrest by hyperoxia involves a defect in S phase progression that is independent of TGF-beta signaling.

Accumulation of p21(Cip1/WAF1) during hyperoxic lung injury in mice.

Hyperoxic lung injury results in decreased cell proliferation, DNA damage, and cell death. Because the cyclin-dependent kinase inhibitor p21(Cip1/WAF1) (p21) inhibits cell proliferation in G1/S, enhances DNA repair, and regulates apoptosis in some cells, we hypothesized that the expression of p21 would increase in lungs of C57Bl/6J male mice exposed to and recovered from > 95% oxygen. A low level of p21 messenger RNA (mRNA) expression was detected by Northern blot analysis of room air-exposed lungs. Exposure to hyperoxia resulted in a modest increase in p21 mRNA expression by 24 h, followed by a marked induction by 48 to 72 h. In situ hybridization revealed that p21 mRNA abundance increased in bronchiolar epithelium and in resident alveolar cells, but not in smooth-muscle cells or large airway epithelium. Hyperoxia increased the expression of p21 protein by 24 h and continued to increase at 48 and 72 h. Immunohistochemical staining showed that p21 protein accumulated in the bronchiolar epithelium and in alveolar regions that had increased p21 mRNA expression. In contrast, the expression of the cyclin-dependent kinase inhibitor p27(Kip1) was not altered by hyperoxia. To determine whether p21 expression was altered during the repair process, mice were exposed to hyperoxia for 64 h and allowed to recover for up to 4 d in room air. The abundance of p21 mRNA and protein decreased by 1 to 2 d of recovery and returned to room air-exposed levels by 3 to 4 d of recovery. These findings support the concept that bronchiolar epithelial and alveolar cells damaged by hyperoxia express molecules such as p21, which may participate in regulating cell proliferation, DNA repair, and cell death.

Exposure to hyperoxia induces p53 expression in mouse lung epithelium.

Cells that are exposed to free radicals have increased levels of DNA strand breaks with accumulation of the tumor suppressor protein p53, which induces cell cycle arrest and/or apoptosis. Because oxidants injure pulmonary epithelial cells, it was hypothesized that exposure to hyperoxia promotes DNA strand breaks in lung epithelium, resulting in increased expression of p53 and loss of epithelial cell function. Adult male C57Bl/6J mice were exposed to > 95% oxygen for 72 h and DNA integrity was determined in their lungs by terminal transferase immunoreactivity. Both nonimmunoreactive and lightly stained nuclei were observed in cells comprising the airway and parenchyma. Exposure to hyperoxia resulted in a marked increase in the intensity of nuclear staining in distal bronchiolar epithelium and alveolar epithelial and endothelial cells. Airway epithelial cells from control lungs contained detectable levels of p53 protein, which markedly increased in both nuclei and cytoplasm of distal bronchiolar epithelial cells and to a lesser extent in alveolar epithelial cells that were morphologically consistent with type II cells. Western and Northern blot analyses revealed that hyperoxia increased total lung p53 protein expression but not levels of mRNA. Changes in terminal transferase immunoreactivity and p53 expression were not observed in large airway cells, fibroblasts underlying distal airway, or smooth muscle cells. Expression of SP-B mRNA modestly increased and Clara cell secretory protein and cytochrome P-450 2F2 mRNAs decreased, providing additional evidence that hyperoxia injured pulmonary epithelial cells. These findings support the concept that hyperoxia damages DNA of pulmonary epithelial cells, which respond by accumulating p53 and changes in epithelial cell-specific gene expression.

Temporal changes in expression of TGF-beta isoforms in mouse lung exposed to oxygen.

Oxygen-induced pulmonary injury is associated with cell death and a significant inflammatory response. Because transforming growth factor (TGF)-beta is a potent modulator of the immune response, changes in expression of the three TGF-beta (beta 1, beta 2, beta 3) isoforms was determined in lungs of adult mice exposed to > 95% oxygen. TGF-beta 1 immunostaining within cuboidal nonciliated bronchiolar epithelial cells was increased within 3 h of oxygen exposure and continued to increase for 48 h before decreasing to control levels by 72 h. A similar but less marked change that was morphologically consistent with alveolar type II cells was observed in granulated cells. Immunostaining for TGF-beta 2 and TGF-beta 3 revealed a similar change in bronchiolar epithelium with little change observed in the alveolar epithelium. Immunohistochemical changes in TGF-beta expression were not observed in any other pulmonary cells. Northern blot analysis of total lung RNA revealed that expression of the TGF-beta mRNA was not markedly altered over the 72-h exposure period. Exposure to > 95% oxygen resulted in cell type-specific posttranscriptional changes in TGF-beta isoforms in the lung.