Effects of transgene expression of superoxide dismutase and glutathione peroxidase on pulmonary epithelial cell growth in hyperoxia

Hshi-chi Koo,1,2 Jonathan M. Davis,1,2 Yuchi Li,1 Dimitrios Hatzis,1,2 Harry Opsimos,1,2 Simcha Pollack,4,5 Marlene S. Strayer,6 Philip L. Ballard,7 and Jeffrey A. Kazzaz1,3

1CardioPulmonary Research Institute and the Departments of 2Pediatrics, 3Medicine, and 4Biostatistics, Winthrop University Hospital, SUNY Stony Brook School of Medicine, Mineola, New York; 5Department of Computer Information Systems and Decision Sciences, St. John's University, Jamaica, New York; 6Department of Pathology, Thomas Jefferson University Medical Center, and 7Joseph Stokes Jr. Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Submitted 31 December 2003 ; accepted in final form 29 November 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Prolonged exposure to supraphysiological oxygen concentrations results in the generation of reactive oxygen species, which can cause significant lung injury in critically ill patients. Supplementation with human recombinant antioxidant enzymes (AOE) may mitigate hyperoxic lung injury, but it is unclear which combination and concentration will optimally protect pulmonary epithelial cells. First, stable cell lines were generated in alveolar epithelial cells (MLE12) overexpressing one or more of the following AOE: Mn superoxide dismutase (MnSOD), CuZnSOD, or glutathione peroxidase 1. Next, A549 cells were transduced with 50–300 particles/cell of recombinant adenovirus containing either LacZ or each of the three AOE (alone or in combination). Cells were then exposed to 95% O2 for up to 3 days, with cell number and viability determined daily. Overexpression of either MnSOD (primarily mitochondrial) or CuZnSOD (primarily cytosolic) reversed the growth inhibitory effects of hyperoxia within the first 48 h of exposure, resulting in a significant increase in viable cells (P < 0.05), with 1.5- to 3-fold increases in activity providing optimal protection. Protection from mitochondrial oxidation was confirmed by assessing aconitase activity, which was significantly improved in cells overexpressing MnSOD (P < 0.05). Data indicate that optimal protection from hyperoxic injury occurs in cells coexpressing MnSOD and glutathione peroxidase 1, with prevention of mitochondrial oxidation being a critical factor. This has important implications for clinical trials in preterm infants receiving SOD supplementation to prevent acute and chronic lung injury.

viral transduction; gene therapy; adenovirus; antioxidants


SUPRAPHYSIOLOGICAL CONCENTRATIONS of O2 (hyperoxia) are often used to treat critically ill patients with underlying respiratory failure. However, hyperoxia is toxic to cells and tissues due to the accumulation of toxic reactive oxygen species (ROS). Families of enzymatic and nonenzymatic antioxidants have evolved to scavenge excessive ROS. However, cellular antioxidants can become overwhelmed by oxidative insults, such as prolonged exposure to hyperoxia. Because the lung is exposed to the highest oxygen concentrations, one strategy to reduce damage is to specifically target increased expression of antioxidant enzymes (AOE) to lung epithelium.

Animal and human studies have suggested that acute and chronic lung injury from hyperoxia and mechanical ventilation may be ameliorated by the administration of one of these antioxidants, specifically superoxide dismutase (SOD) (6, 7, 11, 28, 29). There are three isoforms of SOD that have been found in mammalian cells: CuZnSOD, MnSOD, and extracellular CuZnSOD (ecSOD), which are encoded by three separate genes (sod1, sod2, and sod3, respectively). These enzymes catalyze the conversion of superoxide anions to hydrogen peroxide and water. The isoforms differ in their subcellular distribution, with SOD1/CuZnSOD found primarily in the cytosol and peroxisomes, SOD2/MnSOD in mitochondria, and SOD3/ecSOD in extracellular fluids. All three isoforms have been tested in transgenic animals for their ability to protect from hyperoxic injury. Overexpression of ecSOD, which is not secreted early in development, protects newborn mice from hyperoxia-induced injury (1). In adult mice, overexpression of CuZnSOD in transgenic mice does not prevent hyperoxic injury, whereas targeting MnSOD overexpression specifically to lung epithelium does improve survival in 95% O2, suggesting that protection of the mitochondria from ROS may be an important consideration in strategies designed to prevent hyperoxic lung damage.

A previous study from our laboratory has demonstrated that stable coexpression of MnSOD with catalase protected lung epithelial cells from prolonged hyperoxia. In the present study, both stable expression and transient expression of MnSOD, CuZnSOD, and glutathione peroxidase 1 (GPx1) were utilized to determine which combination and concentration of AOE best prevents ROS-induced injury. This report demonstrates that expression of MnSOD in combination with glutathione peroxidase (GPx; which detoxifies H2O2 in the corresponding subcellular compartment) confers optimal protection from the growth inhibitory effects of hyperoxia.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Construction of AOE cDNA plasmids. Cloning of the full-length human MnSOD cDNA was described previously (10). cDNAs corresponding to the entire open reading frames of human CuZnSOD and GPx1 were generated by PCR of plasmids or RT-PCR HeLa mRNA using primers described in Table 1. The resultant fragments were cloned to either pOPRSVI/MCS (Stratagene, La Jolla, CA) or pWE vectors (ATCC, Manassas, VA) (Table 1). The DNA sequences were verified by the sequencing facility at SUNY Stony Brook School of Medicine.


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Table 1. Cloning strategy for generation expression vectors

 
Cell culture and generation and screening of stable cell lines. MLE12 cells were grown in Hite’s medium supplemented with 2% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin and maintained at 95% room air-5% CO2 in a humidified chamber. Cell lines overexpressing AOE in combination with MnSOD (i.e., MnSOD + GPx, MnSOD + CuZnSOD) were isolated as described previously (10). Briefly, cells were transfected with Lipofectamine-plus reagent (Invitrogen, Carlsbad, CA). Stable cell lines were also generated containing vector with no insert. Stable cell lines were initially screened based on resistance to the appropriate antibiotic (200 µg/ml of gentamycin for pOPRSV1 and 100 µg/ml of hygromycin for pWE4). Colonies from each group were isolated and screened for enzymatic activity (Table 2). Two or three overexpressing cell lines from each group were randomly selected for further study. The AOE activities were stable in these cell lines for 10–15 passages.


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Table 2. Enzymatic activity of transgenes in MLE stable cell lines

 
Recombinant adenoviral-mediated expression of AOEs. Human adenocarcinoma alveolar epithelial cells (A549, ATCC) were maintained in F12-K medium supplemented with 10% fetal bovine serum, 1% glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (GIBCO BRL, Rockville, MD) and maintained at 37°C in 5% CO2-95% room air.

Recombinant adenovirus (rAd) was used for the transient induction of AOE expression (transduction). Viral transduction was performed with type 5 replication-deficient adenoviruses. Ad.CMVMnSOD, Ad.CMVCuZnSOD, and Ad.CMVGPx1 were constructed and grown at the University of Iowa (14, 33). These constructs contain the cytomegalovirus (CMV) enhancer/promoter and SV40 polyadenylation site for efficient transgene expression. An rAd harboring the bacterial enzyme {beta}-galactosidase (Ad.CBlacZ), constructed and grown in the Vector Core at the Institute for Human Gene Therapy (Philadelphia, PA) (8), was used for transduction controls. A549 cells were seeded at subconfluence and then transduced with the various rAd particles in complete media. Cells were washed, and fresh media were added after 17 h. Cells were then harvested and seeded at 5.5 x 104/cm2 for hyperoxic exposure. AOE enzymatic activity was assayed at the time of experiment in duplicate cultures.

Exposure to hyperoxia, cell viability assessment, and aconitase activity. Cells were harvested, counted, and seeded at a density of 2.5 x 105 and allowed to adhere overnight. The cells were then exposed to 95% O2-5% CO2 for up to 3 days, with media and gasses refreshed daily. Cell viability was determined by the exclusion of Trypan blue dye and counted with a hemacytometer. Triplicate samples were used, and experiments were performed at least twice.

Aconitase activity was determined as described previously (9). Briefly, cell lysate was combined with 1 ml of aconitase substrate solution [4 mM Tris, pH 7.4, 0.1 mM citric acid, 0.2 mg/ml {beta}-NADP, 2 mM MnCl2, 20 µM (NH4)2Fe(SO4)2] followed by the addition of 1 U of isocitrate dehydrogenase. The reaction was monitored at wavelength 340 nm for 5 min with a Beckman DU64 spectrophotometer equipped with a kinetic module. Enzymatic activity was normalized to protein content.

Mitochondria isolation and localization of AOE expression and activity. We performed mitochondrial/cytosol fractionation using a commercially available kit (BioVision, Mountain View, CA). Briefly, cells (0.5–1.0 x 108) were harvested and washed once with ice-cold 1x PBS and once with 1x cytosol extraction buffer and then resuspended in 1x cytosol extraction buffer and incubated on ice for 10 min. Cells were then homogenized in an ice-cold dounce for 50 passes until more than 50% of the population were lysed. Lysis was monitored using phase microscopy. The lysates were centrifuged at 700 g for 10 min at 4°C, and pellets were discarded. The mitochondria were collected by centrifugation at 10,000 g for 30 min at 4°C. The cytosolic fractions (supernatants) were decanted and stored at –70°C. The mitochondrial pellet was washed with cold fractionation buffer, resuspended in PBS, and stored at –70°C for subsequent analysis.

The protein yield was determined by the BCA assay (Pierce Biochemical, Rockford, IL). For Western blot analysis, 10 µg of protein were resuspended in 1x SDS-denaturing buffer and resolved by SDS-PAGE. Proteins were blotted onto polyvinylidene difluoride membrane and probed with antibodies directed against MnSOD (Stress Gene Biotech, Victoria, BC, Canada) or cytochrome c (BD Pharmingen, San Diego, CA) followed by the appropriate horseradish peroxidase-conjugated secondary antibody. The c-myc-tagged GPx1 was detected with a horseradish peroxidase-conjugated anti-c-myc antibody (Invitrogen). We visualized bands using enhanced chemiluminescence reagents (Cell Signaling, Beverly, MA) followed by exposure to Hyperfilm. SOD (both MnSOD and CuZnSOD) and GPx activities were measured spectrophotometrically as previously described (5, 23). A unit of SOD activity was defined as the amount of SOD required to inhibit the reduction of cytochrome c at 25°C and pH 7.8 by 50%. A unit of GPx activity was defined as the amount required for the oxidation of 1 µmol glutathione by H2O2 per minute at 25°C and pH 7.0. Enzymatic activity was normalized to total protein concentration.

Statistical analyses. Differential pulmonary epithelial cell survival rates were compared with one-way ANOVA with a Fisher- or Bonferroni-adjusted post hoc comparison of means between genes. To protect against type I errors, only P values of <0.01 were accepted as statistically significant. All analyses were performed with the SAS software package version 8.1 (SAS Institute, Cary, NC). Results are reported as means ± SD. To integrate the survival results over the 3-day experiments, an area under the curve (AUC) was computed by the trapezoidal rule for the stable cell lines for each clonal isolate combination. For viral-transduced cells, only one AUC was available for each transgene in each experiment, which did not allow for the computation of standard deviations or P values. However, because each measurement was performed in triplicate (which is assumed to be a random sample from the distribution of cell survival for that day), 3 x 3 x 3 or 27 AUCs could be calculated for a 3-day survival for each experimental group; each can be summarized as an AUC. Note that the average AUC using this technique does not change the value if calculated without adding the permutations. This method only provided a means to generate permutations that would allow the computation of standard deviations or P values.


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Transgene expression of GPx improves survival of MnSOD stable cell lines in hyperoxia. Our previous studies had demonstrated that MnSOD protects cells from hyperoxic injury, whereas coexpression with catalase provided additional protection (10). Because mitochondrial damage is one of the earliest signs of oxygen toxicity, we reasoned that using exogenous expression of the cytosolic enzyme GPx1 (which also localizes in part to mitochondria) would be a logical approach for improving cell survival. To test this hypothesis, exogenous copies of both MnSOD and GPx1 were introduced into MLE12 cells by stable transfection. The resultant MnSOD + GPx1 cells had increased expression of both gene products (Table 2) and were exposed to 95% O2 for up to 3 days. As illustrated in Fig. 1, cells with increased expression of MnSOD in combination with GPx1 were able to replicate through 48 h, at which point cells began to die. In controls (both parental MLE12 and empty vector MLV-0), a typical pattern of oxygen toxicity was observed (12): growth arrest within 24 h followed by cell death. Overexpression of MnSOD alone significantly protected the cells during this time course; however, prevention of the growth inhibitory effects of hyperoxia was not as dramatic (Fig. 1B). Numerous efforts to isolate cells that constitutively overexpressed GPx (alone) over several passages were unsuccessful. Therefore, we utilized viral transduction to further assess the relative contributions of MnSOD and GPx.



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Fig. 1. Growth curves of stable cell lines expressing Mn superoxide dismutase (MnSOD) or MnSOD + glutathione peroxidase 1 (GPx1). Various cell lines were grown in room air, harvested, and plated at subconfluent (~30% confluent) levels and allowed to adhere overnight. Cells were exposed to 95% O2 in the absence of antibiotic and harvested at the days indicated, and cell numbers were assessed by dye exclusion. A: growth curves of nontransfected controls (MLE12) and empty vector controls (MLV-0). B and C: growth curves of cell lines of stable cell lines overexpressing MnSOD and MnSOD + GPx1, respectively. Values represent triplicate samples from at least 2 independent experiments (n = 6).

 
Transient overexpression of MnSOD and GPx. Because hyperoxic exposures occurred for several days, it was necessary to determine the temporal aspects of transduced expression with rAd. A549 cells were used because they are commonly used as a model of differentiated human lung epithelium (17, 27, 31), and the rAd transgenes were derived from the human cDNAs. Use of these cells would avoid any potential altered posttranslational processing or trafficking of proteins due to species-related differences in the localization signals. A549 cells were infected at a multiplicity of infection of 200 with rAd.CMVMnSOD for 24 h and then harvested and reseeded on 100-mm plates and allowed to adhere overnight. Cells were then exposed to 95% O2 for up to 3 days, and SOD activity was determined. Figure 2 demonstrates that the level of MnSOD activity remained elevated, demonstrating that viral transduction enhanced AOE overexpression during the entire exposure. To determine the subcellular distribution of the exogenous genes, mitochondrial extracts were prepared from cells transduced with rAd.CMVMnSOD. The purity of the preparations was determined by Western blot analysis with an antibody directed against the mitochondrial protein cytochrome c. As shown in Fig. 3, bottom, cytochrome c was detected predominantly in mitochondrial fractions. The distribution of the transgenes was then assessed by both Western blot analysis and enzymatic activity. MnSOD was only detected in the mitochondrial fraction of control (mock-infected) cells in a single band. In cells transduced with rAd.CMVMnSOD, a doublet was evident with a clear increase in the level of MnSOD in both the mitochondrial and cytosolic fractions. The more slowly migrating band (26 kDa) coincides with the precursor MnSOD form that is normally cleaved to form the mature 24-kDa band (30). To ensure our ability to measure enzymatic activity of the MnSOD transgene, cells were transduced with a higher multiplicity of infection, and mitochondria were isolated. MnSOD activity was increased over 13-fold in the mitochondrial fraction of MnSOD-transduced cells (93.8 U/108 cells) compared with mock controls (6.9 U/108 cells) (Table 3).



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Fig. 2. Temporal expression of exogenous MnSOD in hyperoxia. A549 cells were mock infected or infected with Ad.CMVMnSOD (where CMV is cytomegalovirus) at a multiplicity of infection (moi) of 200 (particles/cell). Cells were exposed to 95% O2 for the times indicated and harvested, and MnSOD activity was determined and normalized to total protein concentration. Values represent mean activities ± SD of duplicate cultures relative to nonexposed, mock-infected cells.

 


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Fig. 3. Localization of transduced MnSOD and GPx1. Cells were either mock infected or infected with Ad.CMVMnSOD or Ad.CMVGPx1. Cells were harvested after 48 h, and cell fractionation was performed. Ten micrograms of protein from cytosolic (C) or mitochondrial (M) fractions were resolved on 10% (for MnSOD and GPx-1) or 12% [for cytochrome c (cyt c)] SDS-PAGE. Gels were blotted, and proteins were detected with the antibodies indicated. The GPx-1 transgene was detected using an antibody directed against the c-myc epitope-tag.

 

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Table 3. Activity of transgenes in the mitochondria and cytosolic fractions of A549 cells

 
A similar series of experiments was performed with Ad.GPx. Unlike MnSOD activity, which can be differentiated biochemically from CuZnSOD, GPx1 activity cannot be differentiated from activity from other isoforms. When assessed enzymatically, the majority of the GPx activity is found in the cytosolic fraction, and only transduction with Ad.CMVGPx1 increased expression (Table 3). In addition, the transgene is distributed in a similar manner as the endogenous gene. The GPx1 transgene in this vector is epitope (c-myc)-tagged and thus allows easy identification by Western blot. As illustrated in Fig. 3, middle, the transgene was detected in the cytosolic fraction. Levels of transgene were below detectable limits in the mitochondrial fraction. Screening the same extracts with an anti-MnSOD antibody demonstrates that adenovirus did not affect MnSOD activity (Fig. 3, top). These results demonstrate that the transgenes (MnSOD and GPx) are expressed and functional in the mitochondria as well as increases of the corresponding cytosolic activity.

To test the efficacy of MnSOD and GPx1, expression in A549 cells was increased with Ad.CMVMnSOD and Ad.CMVGPx1, either alone or in combination. When tested separately, GPx1 alone did confer additional protection relative to controls, but the survival rate was not as high as observed with MnSOD (Fig. 4). The cotransduction of MnSOD and GPx1 increased survival significantly more than either gene alone (Fig. 4). However, in this case, MnSOD (alone) was able to overcome the growth inhibitory effect of hyperoxia in the first 48 h.



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Fig. 4. Transient expression of MnSOD and GPx1 alone and in combination improves survival. A549 cells were infected with Ad.CMVMnSOD and Ad.CMVGPx1 alone or in combination. Cells were harvested after 24 h and reseeded onto 100-mm plates at a fixed density of 2.5 x 105, allowed to adhere overnight, and then exposed to hyperoxia for the times indicated. P values represent differences compared with controls [Fisher's paired least significant difference (PLSD) post hoc test].

 
Dose response of MnSOD. The use of adenovirus also provided an opportunity to determine the optimal dose response of MnSOD as it relates to viability in hyperoxia. Duplicate cultures were transduced with increasing amounts of Ad.CMVMnSOD. Both virally transduced and mock-transduced cells were then reseeded and exposed to hyperoxia for up to 4 days. As shown in Fig. 5, increasing expression approximately twofold offered the most consistent protection with the highest number of viable cells at 4 days. Inducing expression up to three- to fourfold offered some additional improvement relative to LacZ or untransduced controls after 2 days of 95% O2 (data not shown) but minimal protection at 4 days. These results suggest that inability to derive epithelial cell lines with increased amounts of expression may be due to high levels of expression hampering cell replication (25).



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Fig. 5. Dose response of Ad.CMVMnSOD. A549 cells were transduced for 17 h with Ad.CMVMnSOD at 50–200 particles/cell. Cells were harvested, counted, and replated 48 h posttransduction and then allowed to adhere overnight. Cells were then exposed to 95% O2 for 4 days, and cell viability was assayed by dye exclusion. Enzymatic activity was assessed in duplicate cultures before exposure to hyperoxia. Cell numbers were normalized to original seeding density assessed in duplicate cultures before exposure. {lozenge}, Mock and Ad.CBLacZ controls (n = 12). All other values represent means of 3 samples from 2 independent experiments ± SD. P values represent differences compared with mock and Ad.CBLacZ controls (Fisher's PLSD post hoc test).

 
Effects of coexpression of CuZnSOD and MnSOD on cell survival in hyperoxia. Several studies suggest that the dismutation of superoxide to H2O2 is extremely important in preventing hyperoxic injury and by extension hyperoxic growth arrest. Based on these studies, one would predict that coexpression of MnSOD and CuZnSOD would provide additional protection. Contrary to this notion, increasing the amount of intracellular MnSOD beyond twofold over baseline values was clearly detrimental, but these experiments did not formally test whether increasing cytosolic SOD activity in the presence of MnSOD might be beneficial. For this reason, we tested the effect of both stable and transient-induced overexpression of MnSOD and CuZnSOD. We generated MLSOD1/SOD2–3, a cell line that expresses 1.98- and 1.8-fold increases of CuZnSOD and MnSOD activity, respectively. Coexpression of these enzymes reduced viability in hyperoxia (Fig. 6A) compared with cells with increased MnSOD alone (Fig. 1B). In fact, the survival curves were similar to that of MLE12 and MLV-0 (empty vector) cells (Fig. 1A). To determine whether this represented a cloning anomaly, viral transduction was again utilized. Transiently induced coexpression had an intermediate effect on cell viability. These cells fared better than controls but worse than MnSOD alone (Fig. 6B). Exogenous expression of CuZnSOD at levels corresponding to 1.5- to 3-fold over baseline were roughly equivalent, with a 2-fold increase offering the best protection during the 3-day exposure (Fig. 6C). The advantage of expressing SOD is most evident in the 2- to 3-day time frame, suggesting that the impact of these enzymes is largely on the growth inhibitory effects of hyperoxia.



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Fig. 6. Effect of MnSOD and CuZnSOD coexpression on cell viability in hyperoxia. A: subconfluent cultures of MLSOD1/SOD2–3 cells were exposed to 95% O2 in the absence of antibiotics and harvested at the days indicated. Cell viability was assessed in triplicate cultures by dye exclusion. Values represent triplicate samples from at least 2 independent experiments. B: A549 cells were transduced with Ad.CMVCuZnSOD alone or in the presence of Ad.CMVMnSOD. Cells were harvested, counted, and replated 48 h posttransduction and then allowed to adhere overnight. Cells were then exposed to 95% O2 for the times indicated, and cell viability was assayed by dye exclusion. Values represent mean ± SD of triplicate samples. C: dose response of Ad.CMVCuZnSOD. A549 cells were transduced with Ad.CMVCuZnSOD at moi values ranging from 50 to 200. Cells were harvested, counted, and replated 48 h posttransduction and then allowed to adhere overnight. Cells were then exposed to 95% O2 for times indicated, and cell viability was assayed by dye exclusion. Enzymatic activity was assessed in duplicate cultures and values indicated in parentheses. P values represent differences compared with mock-transduced controls (Fisher's PLSD post hoc test).

 
AUC survival analysis. Because the pattern of protection differed between groups, area under the survival curves were calculated to further assess which of these combination(s) provided optimal protection. These analyses utilized the data from growth curves of the stable cell lines and transduced cells, with results shown in Fig. 7. Figure 7A demonstrates that, in terms of stable, constitutive expression, the relative order was MnSOD + GPX > MnSOD > CuZnSOD > MnSOD + CuZnSOD. A similar pattern was observed when gene expression was induced transiently, with the exception that this analysis suggested that MnSOD expression improved cell survival in the presence of CuZnSOD (Fig. 7B).



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Fig. 7. Viability in hyperoxia assessed by transgene. A: survival curve data of all clones from each combination of transgene(s) were pooled, and the area under the curve (AUC) was calculated and analyzed by ANOVA. Transgene and mean relative increases of expression are indicated. Values represent means ± SD of a minimum of 2 independent trials performed in triplicate (n = 6). B: data from transduction experiments, where induced expressions that were equivalent were pooled. Transgene and mean relative increases of expression are indicated. Note that the number of viable cells was normalized to controls (Ad.CBLacZ and/or mock-infected) to obtain the percent improved survival. This was performed because the initial seeding density was not the same between experiments. The AUC was calculated for at least 2 independent trials performed in triplicate for the single transgene experiments and triplicate samples for the dual transgene experiments. Permutation analysis was performed as described (MATERIALS AND METHODS). P values for both A and B were obtained using Bonferroni's post hoc test.

 
Effects of AOE expression on aconitase activity. CuZnSOD and MnSOD differ in their contribution to total intracellular SOD levels as well as their subcellular distribution. In this cell line, CuZnSOD is responsible for ~65% of the total SOD levels, with only 3–4% of this activity found in mitochondria (data not shown). This suggests that it is critical to protect the mitochondria from oxidation, primarily with MnSOD. Aconitase is a mitochondrial enzyme involved in respiration and is a target of oxidation in cells exposed to hyperoxia (9). Aconitase activity serves as a marker for mitochondrial function and oxidation status. To determine whether the protective effects of MnSOD was due to a moderation of mitochondrial oxidation, aconitase activity was determined before hyperoxic exposure and after 72 h of 95% O2. Aconitase activity in all groups was similar (mean range 7.80–9.05 mU/mg protein) before hyperoxic exposure. The best protection (i.e., highest aconitase activity) after 72 h was provided by overexpression of MnSOD (Fig. 8). Although MnSOD + GPx1 was significantly better than controls (P < 0.001), there was no statistical difference between this group and MnSOD alone. Indeed, expression of GPx1 alone decreased aconitase activity (Fig. 8). CuZnSOD did not provide protection from mitochondrial oxidation relative to the LacZ control, and coexpression of MnSOD and CuZnSOD did not confer any additional protection relative to MnSOD alone.



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Fig. 8. Effect of transient expression of MnSOD, CuZnSOD, and GPx1 on aconitase activity in hyperoxia. Transient expression of MnSOD, CuZnSOD, and GPx1 was induced by a 17-h transduction of A549 cells with Ad.CMVMnSOD, Ad.CuZnSOD, and Ad.CMVGPx1, respectively. Controls include Ad.CBLacZ and mock-transduced cells. Cells were harvested, counted, and replated 48 h posttransduction and then allowed to adhere overnight. Cells were then exposed to 95% O2 for 72 h, and aconitase activity was assayed and normalized to protein content. Values represent means of triplicate samples ± SD. *P ≤ 0.0001; **P ≤ 0.005 relative to LacZ controls (Bonferroni's post hoc test).

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In this report, we have demonstrated that overexpression of both MnSOD and GPx allows lung epithelial cells to survive and divide in 95% O2 for a prolonged period of time. This combination was effective whether overexpression was increased constitutively in a stable manner or induced transiently. The distribution studies of SOD provided some insight as to why this occurs. Transgene expression of MnSOD with its natural mitochondrial localization signal resulted in increases in both cytosolic and mitochondrial SOD levels. Several lines of evidence suggest that increasing expression in the cytosol is not as beneficial compared with increases primarily in mitochondria. First, cells with increased CuZnSOD activity (primarily in the cytosol) did not survive as well as cells with comparable increases in MnSOD activity (localized to the mitochondrial). Second, MnSOD represents only ~35% of the total SOD activity in these cells. Doubling the expression of MnSOD increases the intracellular SOD level only by 35%, whereas doubling CuZnSOD activity would increase the total intracellular SOD level by 65%.

Both stable and transient expression of SOD revealed that the most effective dose was a twofold increase. Using the drug-resistance selection strategy allowed us to select for cells expressing multiple transgenes. Although multiple colonies were selected, maximum expression of either SOD gene was 2.5-fold higher than baseline, suggesting that higher expression effects cell division. The ability of MnSOD to impair cell division has also been documented in human melanomas where transfection with a cDNA encoding MnSOD reverted the malignant phenotype (4). Conversely, a mild oxidative stress induces normal healthy cells to divide (18), whereas more severe stress inevitably leads to growth arrest and cell death. Future studies to delineate the pathways and mechanism of SOD to impair cell division in room air and partially overcome the growth inhibitory effects of hyperoxia would be of interest.

A model of ischemia-reperfusion in isolated rabbit heart demonstrated a bell-shaped dose-response curve for both CuZnSOD and MnSOD (21, 22). Nelson et al. (16) demonstrated that overscavenging of superoxide radicals results in a net increase of iron catalyzed lipid peroxidation that can be prevented with selective inhibitors. This may also account for the observation that coexpression of MnSOD and CuZnSOD reduced cell survival relative to cells with increased MnSOD alone. Although CuZnSOD is predominantly nonmitochondrial, electron microscopy (3) and fractionation experiments (20) demonstrate that a small fraction is present in the mitochondria and may result in excessive scavenging of ROS.

Although intratracheal administration of CuZnSOD has been shown to reduce hyperoxia-induced inflammation and lung damage in neonatal piglets and more recently shown to significantly improve pulmonary outcome in premature infants, overexpression of AOEs in transgenic mice has not consistently improved outcome in response to oxidative stress. Overexpression of CuZnSOD does not protect mice from hyperoxia; however, overexpression of MnSOD in airway epithelium does confer protection from 95% O2. Overexpression of other AOE gene products (e.g., catalase) can protect cells from other oxidant-induced injuries (e.g., peroxide, ozone, etc.), but they are not effective against hyperoxia. Our results suggest two possible explanations. First, only a single gene is targeted in these animals, and a combination of AOE may be necessary to provide optimal long-term protection (see Fig. 7). Second, overexpression of excessive AOE can impact cell growth. The role of ROS in cell proliferation has already been demonstrated in endothelial cells and primary alveolar type II cells (15, 26). Thus preventing ROS-induced injury will likely require conditional rather than constitutive expression, especially to not interfere with normal cell replication.

Mitochondria play a central role in the regulation of cell growth and apoptosis. As the site of respiration, mitochondria generate ATP necessary for cell division and hyperoxia inhibits several key mitochondrial metabolic enzymes. Our results, and those reported by O'Donovan et al. (19) targeting glutathione reductase to the mitochondria, demonstrate that, although O2 is pleiotropic, the best strategy to attenuate growth inhibition is to prevent oxidative damage to the mitochondria. This hypothesis is supported by the aconitase activity assay, which demonstrated that overexpression of MnSOD offered optimal protection. GPx1 (alone or in combination with MnSOD) did not confer any additional protection from oxidative injury to mitochondrial enzymes, suggesting that O2 is the predominant factor causing oxidative damage. Recently, the role of ROS in cell signaling has received increased attention. Our data do not discount the possibility that the AOEs are acting at the level of oxidant signaling. In fact, it is possible that any protective effects of GPx1 (seen in survival experiments) are likely due to the impact on these signaling pathways. The role of mitochondrial-generated ROS in hyperoxia-induced cell death remains controversial. Several studies have demonstrated that the ROS responsible for inducing cell death in hyperoxia are cytosolic (i.e., nonmitochondrial). These studies utilized diphenylene iodonium, an ROS inhibitor specific for the NADH/NADPH oxidase system present in the membrane of various cells (24, 32). In support of this hypothesis, Budinger et al. (2) generated cells lacking mitochondrial DNA ({rho}°). These cells do not generate mitochondrial ROS during exposure to hyperoxia and are not protected against cell death from hyperoxia (95% O2). In contrast, when HeLa cells were similarly depleted, the {rho}°-HeLa cells were protected against hyperoxia (80% O2)-induced cell death and growth inhibition (13). Furthermore, introduction of normal mitochondria into the {rho}°-HeLa cells by creating "cybrids" with platelets restored the susceptibility. Given that the studies utilized different cell lines, methodologies, and O2 concentrations, it is difficult to reconcile these results. Note that only the study by Li and colleagues (13) assessed the effect on growth inhibition, and our data confirm the importance of moderating mitochondrial ROS generation for the attenuation of growth inhibition but not necessarily cell death in 95% O2. Whether this strategy has a greater impact at lower concentrations of O2 remains to be determined. Furthermore, whether the ROS pathways involved in hyperoxia-induced growth inhibition and cell death are divergent, convergent, or overlapping remains to be determined.

In summary, we demonstrate that expression of MnSOD in combination with GPx1 prevents growth inhibition within the first 48 h of exposure to 95% O2. In addition, our data suggest that there is a relatively narrow therapeutic range of AOE activity that is beneficial for protection against hyperoxia. Although 1.5- to 3-fold increases in AOE activities prevented hyperoxic injury over the 3-day study period, cells with higher AOE activities (≥4-fold) did not grow or survive as well. This has important implications in the development of strategies to prevent oxidative damage from O2 therapy.


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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This study was supported National Heart, Lung, and Blood Institute Grant HL-64158-03 (J. M. Davis and J. A. Kazzaz) and a grant from the Cystic Fibrosis Foundation (J. A. Kazzaz).


    ACKNOWLEDGMENTS
 
We thank Dr. Lin Mantell for suggestions, criticisms, and insights.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Kazzaz, CardioPulmonary Research Institute, Winthrop Univ. Hospital, 222 Station Plaza North, Suite 604, Mineola, NY 11501 (E-mail: jkazzaz{at}winthrop.org)

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. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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