RAPID COMMUNICATION
Increased expression of heat shock protein-70 protects A549 cells against hyperoxia

Hector R. Wong, Ingrid Y. Menendez, Marnie A. Ryan, Alvin G. Denenberg, and Jonathan R. Wispé

Division of Critical Care Medicine and Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Acute and chronic lung injury secondary to hyperoxia remains an important complication in critically ill patients, and, consequently, there is interest in developing strategies to protect the lung against hyperoxia. Heat shock proteins (HSPs) confer protection against a broad array of cytotoxic agents. In this study, we tested the hypothesis that increased expression of the 70-kDa HSP (HSP70) would protect cultured human respiratory epithelium against hyperoxia. Recombinant A549 cells were generated in which human HSP70 was increased by stable transfection with a plasmid containing human HSP70 cDNA under control of the cytomegalovirus promoter (A549-HSP70 cells). A549-HSP70 cells exposed to hyperoxia had greater acute survival rates and clonogenic capacity compared with wild-type A549 cells and with control cells stably transfected with the empty expression plasmid. Hyperoxia-mediated lipid peroxidation and ATP depletion were also attenuated in A549-HSP70 cells exposed to hyperoxia. Increased expression of HSP70 did not detectably alter mRNA levels of the intracellular antioxidants manganese superoxide dismutase, catalase, and glutathione peroxidase. Collectively, these data demonstrate a specific in vitro protective role for HSP70 against hyperoxia and suggest that potential mechanisms of protection involve attenuation of hyperoxia-mediated lipid peroxidation and ATP depletion.

oxidants; epithelium; stress proteins; lung; isoprostanes; adenosine 5'-triphosphate

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

DELIVERY OF SUPPLEMENTAL OXYGEN is frequently life sustaining in critically ill patients with lung disease. Paradoxically, high concentrations of oxygen (hyperoxia) can directly injure the lung and worsen the primary lung disease (reviewed in Ref. 2). Hyperoxia can cause profound cellular injury, in large part, by the generation of oxygen radicals such as superoxide anion (O-2), hydrogen peroxide (H2O2), and hydroxyl radical (· OH) (reviewed in Ref. 15). These oxygen radicals cause cellular injury by several mechanisms, including mitochondrial damage and ATP depletion, oxidation of membrane lipids and intracellular proteins, and DNA damage (reviewed in Ref. 15).

There is much interest in designing novel therapies to protect the lung against oxygen-induced injury. In a broad sense, hyperoxic lung injury involves an imbalance between reactive oxygen species (oxidants) and endogenous antioxidant systems. Many experimental strategies to protect the lung against oxygen-induced injury have focused on increasing intracellular antioxidants such as superoxide dismutase (14, 16) and metallothionein (9, 11). Indeed, recent studies (9, 11, 14, 16) demonstrated that increased intracellular expression of these antioxidants conferred partial protection against oxidant-mediated cellular injury.

Induction of heat shock proteins (HSPs) may be another approach to protect the lung against oxygen-induced injury (reviewed in Ref. 20). Among the various HSPs, 70-kDa HSP (HSP70) appears to serve broad cytoprotective functions. For example, specific increased expression of HSP70 protected against lethal hyperthermia (5), endotoxin (17), ultraviolet radiation (12), nitric oxide (18), and cardiac ischemia (6). The role of HSP70 in protection against hyperoxia has not been directly examined.

This study was designed to test the hypothesis that increased expression of HSP70 protects cultured human respiratory epithelium against hyperoxic injury. Using recombinant respiratory epithelial cells having increased expression of HSP70, we demonstrate that increased expression of HSP70 confers a survival advantage in cells exposed to hyperoxia. We further demonstrate that potential mechanisms of protection may involve attenuation of hyperoxia-mediated lipid peroxidation and ATP depletion.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture and hyperoxic protocols. A human lung adenocarcinoma cell line (A549 cells; American Type Culture Collection, Manassas, VA), representative of distal respiratory epithelium, was used in all experiments. Cells were maintained as previously described (19).

Hyperoxic exposures were performed by placing cells in sealed modular chambers (Billups-Rothenberg, Del Mar, CA) and flushing the chambers with a gas mixture of 95% O2-5% CO2 at 1 l/min for 1 h. The entry and exit ports of the chambers were subsequently clamped, and the chambers were placed in a 37°C incubator. In experiments involving hyperoxic exposures > 24 h, the chambers were flushed with the 95% O2-5% CO2 gas mixture every 24 h. pH indicators in the cell culture medium ensured that there was no significant CO2 accumulation during the 24-h period between flushes.

Assessment of acute survival. To assess acute survival, an equal number of cells (5 × 104 cells/well) were plated in 12-well tissue culture plates. At 24-h intervals, the number of surviving cells was measured. Medium was removed, and the cells were washed once with cold PBS to remove dead and unattached cells. The mass of attached cells was determined by measuring the total DNA content of each well. Attached cells were lysed in 1 ml of 0.5 M NaOH, and the DNA content was measured spectrophotometrically at 260 nm. Acute survival of hyperoxia-exposed cells is expressed as the percentage of room air-exposed cells: (total DNA content of hyperoxia-exposed cells divided by the total DNA content of the respective room air-exposed cells) × 100.

Assessment of clonogenic survival. The ability of cells to repopulate and divide (clonogenic survival) after hyperoxic exposure was determined (18). Cells were plated in 100-mm2 tissue culture plates at a density of 3 × 105 cells/plate. After a 24- or 48-h exposure to hyperoxia or room air, dead and unattached cells were removed by washing with sterile PBS, and the remaining cells were detached with 0.5% trypsin-EDTA (Sigma, St. Louis, MO) and resuspended in 10 ml of basal growth medium. Cells were manually counted with a hemocytometer and equally replated at a density of 5 × 104 cells/plate. After being replated, room air- and hyperoxia-exposed cells were returned to a room air-5% CO2 incubator. After 72 h, the total cell mass of attached cells was determined by measuring the total DNA content of each plate as described in Assessment of acute survival. Clonogenic survival of hyperoxia-exposed cells is expressed as the percentage of room air-exposed cells: (total DNA content of hyperoxia-exposed cells divided by the total DNA content of the respective room air-exposed cells) × 100.

Generation of recombinant cell lines with increased HSP70 expression. A 2.3-kb cDNA encoding human HSP70 (StressGen, Victoria, BC) was cloned into the BamH I polylinker region of the expression plasmid pcDNA3 (Invitrogen, San Diego, CA). The resulting expression plasmid (pcDNA3-HSP70) contained human HSP70 cDNA under control of the cytomegalovirus promoter. The expression plasmid also contained the bacterial neomycin resistance gene. Sense orientation of HSP70 cDNA was confirmed by restriction digest analysis.

A549 cells were transfected with pcDNA3-HSP70 with cationic liposomes (Lipofectin, GIBCO BRL) as previously described (18). Transfected cells were selected with neomycin (400 µg/ml; GIBCO BRL), and clonal populations were expanded and analyzed for the expression of HSP70 by Western blot analysis as previously described (19). A control population of A549 cells was transfected with a pcDNA3 plasmid containing the neomycin resistance gene but not HSP70 cDNA. Clonal populations of these neomycin-resistant control cells were expanded and analyzed for the expression of HSP70 by Western blot analysis.

Measurement of lipid peroxidation. Lipid peroxidation after hyperoxic exposure was determined by measuring concentrations of the isoprostane 8-isoprostaglandin-F2alpha (8-iso-PGF2alpha ). Isoprostanes are stable prostaglandin-like compounds that are formed in vivo by oxidant-mediated peroxidation of phospholipids (reviewed in Ref. 7). Generation of isoprostanes occurs independently of cyclooxygenase. Equal numbers of cells were plated in 12-well tissue culture plates and exposed to room air or hyperoxia for 24 or 48 h. 8-iso-PGF2alpha concentrations in the medium of treated cells were measured with a competitive enzyme-linked immunoassay (Oxford Biomedical Research, Oxford, MI). Because serum can interfere with detection of 8-iso-PGF2alpha with this assay, serum was removed from the culture medium during experiments. All assay procedures were performed with freshly obtained specimens and according to the manufacturer's instructions.

Measurement of intracellular ATP content. The ATP content of the cells was measured with an HPLC-based assay (18). Equal numbers of cells were plated in 12-well tissue culture plates and exposed to room air or hyperoxia for 24 or 48 h. The cells were subsequently scraped into 0.5 ml of iced 0.6 N perchloric acid, sonicated for 10 s, and placed on ice for 60 min. Potassium phosphate (1.0 M) was added, and the cells were centrifuged at 10,000 rpm at 4°C for 15 min. The protein concentration of the pelleted cellular debris was measured with a Bio-Rad (Hercules, CA) protein detection kit, and individual supernatants were filtered through a 0.2-µm syringe filter and frozen at -70°C. ATP levels of thawed lysates were determined spectrophotometrically at 254 nm on a Beckman model 126 HPLC unit (Beckman Instruments, San Ramon, CA) as previously described (18). Sample peak areas were compared with standard peak areas. ATP levels were calculated as nanomoles per milligram of cellular protein and are expressed as a percentage of the respective control levels.

Northern blot analysis. Total cellular RNA was recovered from recombinant and wild-type cells as previously described (19). Membranes containing 15 µg of total RNA from each cell type were hybridized with radiolabeled cDNAs encoding for the intracellular antioxidants manganese superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (16). Hybridized membranes were analyzed with a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) and analyzed with ImageQuant software (Molecular Dynamics). To control for loading differences, the membranes were stripped with boiling 5 mM EDTA and rehybridized with an oligonucleotide 18S rRNA-radiolabeled oligonucleotide probe (19). Relative antioxidant mRNA densitometry values were corrected for respective 18S rRNA densitometry values and are expressed as a percentage of wild-type cells.

Statistical analysis. Differences in acute survival, clonogenic survival, 8-iso-PGF2alpha concentrations, and intracellular ATP levels were evaluated by one-way ANOVA and the Student-Newman-Keuls test. P < 0.05 was considered significant. Experiments were performed with three different clonal populations of A549 cells having increased expression of HSP70 and three different clonal populations of A549 transfected with the empty expression vector.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Increased expression of HSP70 in A549 cells. To directly determine whether HSP70 can protect respiratory epithelium against hyperoxia, we developed a stably transfected A549 cell line having increased expression of human HSP70 (A549-HSP70 cells). A control group of cells was transfected with empty pcDNA3 and selected in neomycin (A549-Neo). Monoclonal populations of A549-HSP70 and A549-Neo cells were expanded and analyzed by Western blot for increased expression of HSP70. A549-HSP70 cells (Fig. 1, lane 3) expressed increased levels of HSP70 compared with wild-type A549 (A549-wt; Fig. 1, lane 1) and A549-Neo cells (Fig. 1, lane 4). The amount of HSP70 expressed by A549-HSP70 cells appeared relatively less than the amount of HSP70 expressed by A549-wt cells 4 h after exposure to heat shock (43°C for 1 h; Fig. 1, lane 2). A549-HSP70 cells continued to express similar levels of HSP70 over several passages, and their basal growth rate was similar to that of A549-wt and A549-Neo cells (data not shown).


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Fig. 1.   Representative Western blot analysis demonstrating increased expression of 70-kDa heat shock protein (HSP70) in stably transfected A549 cells. Lane 1, wild-type A549 (A549-wt) cells; lane 2, A549-wt cells incubated at 43°C for 1 h and harvested for Western blot analysis 4 h later; lane 3, A549 cells having increased expression of HSP70 (A549-HSP70 cells); lane 4: control A549 cells transfected with empty pcDNA3 and selected in neomycin (A549-Neo cells).

Increased expression of HSP70 improves survival after exposure to hyperoxia. Kazzaz et al. (3) previously reported that exposing A549 cells to hyperoxia caused cell death by necrosis. To determine whether increased expression of HSP70 confers protection against hyperoxia, A549-HSP70, A549-Neo, and A549-wt cells were exposed to hyperoxia and analyzed for acute and clonogenic survival. After exposure to hyperoxia for 24-72 h, A549-HSP70 cells had significantly greater acute survival rates at all three time points compared with A549-Neo and A549-wt cells (Fig. 2). Clonogenic survival, the ability of cells to repopulate and divide after exposure to hyperoxia, was significantly improved in A549-HSP70 cells compared with A549-Neo and A549-wt cells (Fig. 3). The improvement in clonogenic survival was evident after exposure to either 24 or 48 h of hyperoxia. These data demonstrate that increased expression of HSP70 protects A549 cells against hyperoxia.


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Fig. 2.   Acute survival assay demonstrating that increased expression of HSP70 protects A549 cells against hyperoxia. Cells were exposed to hyperoxia for 24-72 h. Survival is expressed as percent survival relative to respective control cells maintained in room air for 24-72 h. Data are means ± SE from 9 separate experiments. * P < 0.05 vs. A549-wt and A549-Neo cells.


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Fig. 3.   Clonogenic survival assay demonstrating that increased expression of HSP70 protects A549 cells against hyperoxia. After a 24- or 48-h hyperoxic exposure, cells were harvested and replated, then allowed to repopulate and divide for 72 h in room air as described in METHODS. Clonogenic survival of hyperoxia-exposed cells is expressed as percentage of respective control cells exposed to room air for 24 or 48 h. After exposure to room air, respective control cells were replated as described for hyperoxia-exposed cells. Data are means ± SE from 5 separate experiments for each time point. * P < 0.05 vs. A549-wt and A549-Neo cells.

Effect of increased HSP70 expression on hyperoxia-mediated generation of 8-iso-PGF2alpha . Oxidant stress is thought to cause cellular injury, in part, by inducing membrane lipid peroxidation (reviewed in Ref. 15). Isoprostanes are unique prostaglandin-like products of lipid peroxidation. We measured concentrations of 8-iso-PGF2alpha in the medium of cells exposed to hyperoxia. 8-iso-PGF2alpha was not detectable in the medium of cells exposed to room air (data not shown) and was barely detectable after 24 h of hyperoxia (Fig. 4). In contrast, 8-iso-PGF2alpha was readily detectable in the medium of A549-wt and A549-Neo cells exposed to 48 h of hyperoxia (Fig. 4). The concentration of 8-iso-PGF2alpha was significantly less in the medium of A549-HSP70 cells exposed to 48 h of hyperoxia compared with that of A549-Neo and A549-wt cells. These data demonstrate that increased expression of HSP70 is associated with attenuation of hyperoxia-mediated lipid peroxidation.


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Fig. 4.   Isoprostane (8-iso-PGF2alpha ) concentrations in medium of cells exposed to 24 or 48 h of hyperoxia. 8-iso-PGF2alpha was not detectable in medium of cells maintained in room air (data not shown). Conditions were carried out in duplicate for each experiment. Data are means ± SE from 6 separate experiments for each time point. * P < 0.05 vs. A549-wt and A549-Neo cells.

Effect of increased HSP70 expression on hyperoxia-mediated decreases in intracellular ATP levels. Mitochondrial injury and subsequent depletion of intracellular ATP levels is thought to be another mechanism by which oxidant stress causes cellular injury (reviewed in Ref. 15). Twenty-four hours of hyperoxia decreased intracellular ATP levels in A549-wt cells to ~75% of the levels in cells incubated in room air (Fig. 5). Relative intracellular ATP levels were higher in A549-HSP70 cells exposed to hyperoxia compared with those of A549-wt cells. However, the difference in relative intracellular ATP levels between A549-HSP70 and A549-Neo cells was not significant after 24 h of hyperoxia. After 48 h of hyperoxia, intracellular ATP levels decreased further in A549-wt and A549-Neo cells. In contrast to the 24-h time point, after 48 h of hyperoxia, relative intracellular ATP levels were significantly higher in A549-HSP70 cells compared with both A549-wt and A549-Neo cells. These data demonstrate that increased expression of HSP70 is associated with the attenuation of hyperoxia-mediated ATP depletion.


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Fig. 5.   Intracellular ATP levels in cells exposed to 24 or 48 h of hyperoxia. Data are expressed as percentage of respective control cells maintained in room air for 24 or 48 h. Conditions were carried out in duplicate for each experiment. Data are means ± SE from 6 separate experiments for each time point. * P < 0.05 vs. A549-wt cells.

Intracellular antioxidant gene mRNA levels in recombinant cell lines. To determine whether increased expression of HSP70 alters intracellular mRNA levels of known antioxidant genes, we performed Northern blot analyses for the antioxidants MnSOD, catalase, and glutathione peroxidase. Steady-state mRNA levels of MnSOD, catalase, and glutathione peroxidase were not substantially changed in A549-HSP70 cells and A549-Neo cells compared with A549-wt cells (Fig. 6).


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Fig. 6.   Relative steady-state antioxidant mRNA levels in A549-wt, A549-HSP70, and A549-Neo cells. MnSOD, manganese superoxide dismutase. Densitometry values of respective antioxidant mRNAs were corrected for respective 18S rRNA densitometry values and are expressed as percentage of A549-wt cells (with 100% as value for A549-wt cells).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The respiratory epithelium is directly exposed to hyperoxia in patients treated with high concentrations of inspired supplemental oxygen and is an early site of oxidant injury. Therefore, there is a need to develop novel strategies to protect the respiratory epithelium against hyperoxia. A549 cells, derived from a human lung adenocarcinoma, are not fully representative of normal human respiratory epithelium but have been a useful in vitro model to study human respiratory epithelial cell biology (1, 3, 4, 19). Our present study demonstrated that increased expression of HSP70 protected A549 cells against hyperoxia. Protection was evident as improvements in acute survival and clonogenic capacity. The observation that A549-Neo cells were not protected against hyperoxia indicates that protection could be attributed to increased expression of HSP70 rather than to a nonspecific effect of clonal selection in neomycin (8, 14). Further data supporting a specific role for HSP70 include 1) the use of several clonal populations of recombinant cell lines and 2) the demonstration that intracellular antioxidant mRNA levels of MnSOD, catalase, and glutathione peroxidase were not substantially altered in the recombinant cell lines. Collectively, these data are consistent with previous data (10, 18) demonstrating that increased expression of HSP70 protected cells against oxidant injury other than hyperoxia. To our knowledge, this is the first demonstration that increased expression of HSP70 protects human respiratory epithelium against oxidant injury secondary to hyperoxia, a unique and clinically relevant form of oxidant injury.

Several mechanisms are involved in the pathophysiology of acute pulmonary oxygen injury (reviewed in Ref. 15). We focused our study on hyperoxia-mediated lipid peroxidation and ATP depletion. Our data demonstrated that increased expression of HSP70 was associated with decreased production of 8-iso-PGF2alpha by cells exposed to hyperoxia. Generation of 8-iso-PGF2alpha is a sensitive marker of cellular oxidant stress and is thought to be a more specific marker of membrane lipid peroxidation than traditional measurements such as thiobarbituric acid-reactive substances and conjugated dienes (reviewed in Ref. 7). These data suggest that HSP70 protects against hyperoxia by decreasing lipid peroxidation. How HSP70, an intracellular protein with no known enzymatic antioxidant activity, attenuates lipid peroxidation remains to be elucidated. We speculate that HSP70 acts as an expendable target, or nonspecific scavenger, for oxygen-derived reactive species and/or toxic intermediate lipid peroxides. This hypothesis was previously proposed by Pitt et al. (9) and Schwarz et al. (11) with regard to metallothionein after they demonstrated that increased expression of metallothionein, a nonenzymatic antioxidant, protected cells against oxidant injury.

ATP depletion was modestly attenuated in A549-HSP70 cells exposed to hyperoxia compared with A549-wt cells and A549-Neo cells exposed to hyperoxia, thus suggesting that another potential mechanism of protection may involve relative preservation of intracellular ATP levels. Indeed, Polla et al. (10) recently demonstrated that induction of HSP70 protected mitochondria against oxidant injury secondary to H2O2. How increased HSP70 attenuates oxidant-mediated ATP depletion and mitochondrial injury remains to be elucidated. Based on a recent study by Allen and White (1), one possible mechanism could involve attenuation of hyperoxia-mediated glucose consumption. Allen and White demonstrated that hyperoxia dramatically increased glucose consumption by A549 cells. Moreover, hyperoxia-mediated glucose depletion in the cell culture medium strongly correlated with intracellular ATP depletion and cell death. However, in our hyperoxic model, glucose depletion does not appear to be a contributing factor in the mechanism of hyperoxia-mediated cell death (Wong, unpublished data). The discrepancy between our model and that of Allen and White probably reflects the fact that our cells are maintained and treated in culture medium having a substantially higher glucose concentration.

Increased expression of HSP70 did not completely protect cells against hyperoxia. Hyperoxia-induced cellular injury is a complex process involving multiple cellular compartments and targets, and it is unlikely that single factors will provide complete protection. For example, Warner et al. (14) reported that increased expression of mitochondrial MnSOD partially protected cells against hyperoxia-mediated injury. Lee et al. (4) demonstrated that selective increased expression of cytosolic heme oxygenase-1 partially protected A549 cells against hyperoxia. Collectively, these studies suggest that it will be necessary to increase expression of multiple intracellular antioxidants within various intracellular compartments to completely protect the lung against hyperoxia.

Chronic lung injury secondary to hyperoxia results when a functional respiratory epithelium is not regenerated (reviewed in Ref. 15). This process is difficult to model in vitro, but clonogenic survival assays, which measure the ability of cells to repopulate and divide after injury, have been useful in other systems (14, 18). We used this assay to determine the ability of oxygen-stressed cells to recover and regenerate. We were particularly interested by the improved clonogenic capacity of A549-HSP70 cells exposed to hyperoxia compared with the A549-wt and A549-Neo cells exposed to hyperoxia. These data may have important implications for HSP70-mediated protection against in vivo chronic lung injury after hyperoxia.

Our data provide a rationale to further investigate the potential therapeutic advantages of increased HSP70 expression in the lung exposed to hyperoxia. In contrast, Strand et al. (13) demonstrated that induction of HSPs did not protect cultured rat fibroblasts or whole rat lungs against hyperoxia. There are important methodological differences between the study by Strand et al. and our present work. To directly test the hypothesis that increased HSP70 expression will ameliorate acute and chronic hyperoxic lung injury in vivo, we are developing transgenic mice having increased expression of HSP70 in a lung-specific manner.

    ACKNOWLEDGEMENTS

We thank Caroline A. Wong for technical assistance.

    FOOTNOTES

This work was supported by a Research Grant from the American Lung Association, National Institute of Child Health and Human Development Pediatric Center for Gene Expression and Development Pediatrician/Scientist Career Development Award HD-28827-05, and National Heart, Lung, and Blood Institute Grant 1K08-HL-03725-01 to H. R. Wong.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: H. R. Wong, Division of Critical Care Medicine-OSB5, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229.

Received 5 May 1998; accepted in final form 13 July 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(4):L836-L841
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