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Abstract
Introduction
Materials and Methods
Results
Discussion
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Previously it was reported that hyperoxia induced death of the human lung adenocarcinoma cell line (A549 cells) by necrosis, not by apoptosis. This study examined proliferation and death of untransformed human small airway epithelial (SAE) cells in normoxia or hyperoxia in comparison with A549 cells. We tested the hypothesis that SAE cells respond differently to hyperoxic injury than do A549 cells. We measured total cell number and viability, thymidine incorporation (SAE cells only), lactate dehydrogenase (LDH) release, and apoptotic changes as markers for cell proliferation and death. Protective effects of antioxidant vitamins also were examined in SAE cells. In normoxia, subconfluent SAE cells had less apoptosis and fewer detached cells, but higher thymidine incorporation than did near-confluent cells. Hyperoxia suppressed thymidine incorporation and augmented apoptosis in both subconfluent and near-confluent SAE cells. Hyperoxia decreased the total cell number only in subconfluence, whereas SAE cell viability declined with hyperoxia in near confluence, but not in subconfluence. For SAE cells, necrosis assessed by LDH release was minimal in all conditions and was not augmented by hyperoxia in SAE cells. In contrast, normoxic A549 cells proliferated more rapidly than did SAE cells with a large number of cells detached during the culture. A549 cells underwent necrotic cell death under confluent or in hyperoxic conditions, but had much less apoptotic cell death. In SAE cells, vitamin E partially prevented the decline of thymidine incorporation with hyperoxia in subconfluence and protected against apoptotic changes with hyperoxia in both subconfluent and near-confluent conditions. Vitamin C prevented apoptosis with hyperoxia only in near-confluent SAE cells. Thus, SAE cells maintained balanced apoptosis and cell proliferation that were altered by cell density and hyperoxia and demonstrated very little necrosis with hyperoxia. Although A549 cells underwent cell death mainly by necrosis, they also were influenced by cell density and hyperoxia. Cell density also determined specific antioxidant vitamin protection in SAE cells.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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The small airway epithelium forms a selectively permeable layer that serves as a first-line barrier of defense against exogenous agents. The lung is exposed to higher oxygen partial pressures than most other tissues and may be more prone to oxidant injury caused by endogenous and exogenous reactive oxygen species (ROS). ROS can damage epithelial monolayers (1), and antioxidant defenses are probably vital to maintain epithelial cell integrity, and prevent pathogen invasion and allergen sensitization.
Hyperoxic injury in the lung has been studied as a model of the respiratory distress syndrome (RDS) (1). Oxidant injury probably is important in the pathogenesis of common lung diseases such as asthma, lung cancer, and chronic obstructive pulmonary disease (COPD). However, less research has examined the effects of hyperoxia on small airway epithelium. In this study, we examined hyperoxic injury and antioxidant vitamin protection in human small airway epithelial (SAE) cells, which demonstrate characteristic features of basal cells and differentiate into Clara cells or ciliated epithelial cells when they become confluent (Clonetics, San Diego, CA). For comparison, A549 human lung cancer cells derived from type II alveolar cells were used as control transformed cells that are known to undergo necrosis with prolonged hyperoxia (4).
Epithelial cell death and proliferation likely change depending on the conditions of an epithelium (5). We found previously that cell density and specific antioxidant protection determined the effects of hyperoxia on cell death and proliferation of Madin-Derby canine kidney (MDCK) epithelial cells that have been extensively used as a model epithelium for epithelial cell death and proliferation (8). Unlike MDCK cells, SAE cells depend on multiple growth factors, grow much slower than MDCK cells, and differentiate into mature cells (Clara cells or ciliated epithelial cells) during subcultivation; SAE cells can be maintained in culture for only five to six passages. In other words, SAE cells may more closely mimic in vivo growth and differentiation of normal epithelial cells than do transformed MDCK cells or A549 cells. In this study, we determined the effects of hyperoxia and antioxidant vitamins on subconfluent and near-confluent SAE cells by examining cell proliferation (measured by cell number and viability and thymidine incorporation) and death (measured by lactate dehydrogenase [LDH] release and apoptosis).
Our results indicate that SAE cells maintain an epithelial monolayer with balanced cell proliferation and apoptosis depending on cell density. In subconfluence, hyperoxia suppressed SAE cell proliferation and augmented apoptosis, whereas near-confluent cells were less vulnerable to the injurious effects of hyperoxia. In contrast, as previously reported (4), A549 cells underwent necrosis without much apoptosis when they became confluent or with hyperoxia, as reported previously (4). Protection by vitamins E and C against these changes in SAE cells was partly determined by cell density.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Experimental Design
Effects of hyperoxia. The first set of experiments determined the in vitro effects of hyperoxia on SAE cells (Clonetics) and control A549 cells (American Type Culture Collection, Rockville, MD) following 2, 4, and 7 d of hyperoxia. SAE and A549 cells were plated at a density of 104 cells/ml in 35 × 35-mm petri dishes and studied at two different degrees of cell density: subconfluent cells covered less than 25% of the tissue culture plate at the start of normoxia or hyperoxia (95% O2/5% CO2), whereas near-confluent cells covered > 85% of the plate at the start of exposure. The medium was changed the day after plating cells and every 2 d afterward for both cells; SAE cells were maintained in the best condition if the medium was changed every other day. This also measured LDH release from SAE and A549 cells in the similar culture conditions. In hyperoxic experiments, cells were gassed every day. The subconfluent A549 and SAE cells typically were exposed to hyperoxia or normoxia 1 to 2 or 3 to 4 d after plating, respectively. For the near-confluent cell experiments, A549 and SAE cells were exposed 5 to 6 or 7 d after plating, respectively. In the time-course study, we measured the number of dead and live cells, apoptotic changes by acridine orange (AO) fluorescent staining (9), and LDH levels in the culture supernatant. In selected samples, terminal transferase dUTP nick end-labeling (TUNEL) staining and flow cytometry also were performed to confirm the findings with AO staining (10). In separate experiments, A549 cells were also cultured, and the medium was changed every day.
Because the preliminary study revealed significant changes in the parameters described previously when SAE cells were cultured for 2 d in hyperoxia, we also examined changes in thymidine incorporation and apoptotic changes following 2 d of normoxia and 1 d of hyperoxia (95% O2/ 5% CO2) plus 1 d of normoxia.Vitamin effects.
The second set of experiments was designed to test the protective effects of antioxidant vitamins
against hyperoxic injury in SAE cells. During exposure to
hyperoxia or normoxia, subconfluent and near-confluent
SAE cells prepared as described were cultured in media
supplemented with lipid-soluble or water-soluble antioxidant vitamins: vitamins E and C, respectively. Vitamins
were added to the culture at the start of oxygen exposure.
The cells were exposed to normoxia or hyperoxia for 2 d.
After 48 h, the cells were harvested and assayed as in the
first set of experiments. In preliminary experiments, vitamins C and E were tested at concentrations of 10
8 to 106
mol/liter. For both vitamins C and E, the concentration of
107 mol/liter was mainly used in later experiments because it provided the best cell viability with improvement
of other parameters tested for hyperoxic injury. The effects of organic solvents used for preparing vitamin E were
tested in four experiments to validate the results. At vitamin E concentrations lower than 5 × 107 mol/liter, no
toxicity of organic solvent above was detected with regard
to the parameters tested.
Analytical Methods
SAE and A549 cell culture and hyperoxic exposure. SAE cells were maintained in serum-free SAE basal medium (CCMD 160; Clonetics) supplemented with growth factors (bovine pituitary extract [30 mg/liter], hydrocortisone [0.5 mg/liter], human recombinant epidermal growth factor [0.5 µg/liter], epinephrine [0.5 mg/liter], transferrin [10 mg/liter], insulin [5 mg/liter], retinoic acid [0.1 µg/liter], triiodothyronine [6.5 µg/liter]; Clonetics) and gentamicin (50 g/liter), amphotericin-B (50 µg/liter), and fatty acid-free bovine serum albumin (BSA) (50 g/liter). Cells were fed every 2 d, subcultivated when confluent (once per week), and studied at less than five passages. Adherent SAE cells were passaged by detachment with trypsin (0.25 mg/ml) and ethylenediaminetetraacetic acid (EDTA; 0.1 mg/ml) in Hanks' balanced salt solution (HBSS) (Clonetics) for 5 min at room temperature without scraping. Trypsin neutralization solution (Clonetics) then was added to the cell suspension and the cells were briefly centrifuged at 1,000 cpm for 2 to 3 min and resuspended in the culture medium. A549 cells were cultured in F12K medium (Life Technologies, Gaithersburg, MD), supplemented with fetal calf serum (100 ml/liter), L-glutamine (2 mmol/liter), penicillin (105 U/liter), and streptomycin (100 mg/liter). Cells were passaged twice per week by detaching cells with trypsin solution (GIBCO BRL, Gaithersburg, MD). Representative plates were examined with a phase-contrast microscope (Nikon, Melville, NY) after the trypsin treatment and contained virtually no residual adherent cells. For culturing SAE and A549 cells in hyperoxia, cells were cultured in a humidified incubator chamber (MIC-101; Billups-Rothenberg, Del Mar, CA) in a condition of normobaric hyperoxia (95% O2/5% CO2). All experiments included simultaneous assessment of normoxic and hyperoxic cells from the same preparation.
Cell number. The numbers of dead and live cells were determined by trypan blue dye exclusion of SAE or A549 cells resuspended in phosphate buffered saline (PBS), pH 7.4. The number of nonadherent cells floating in the medium and those attached to the tissue culture plate were counted separately in a hemacytometer. The percentage of live adherent SAE cells was also assessed by staining cells with erythrosin-B without detaching cells in some occasions (9); the percentage of live adherent cells declined 10% to 15% following the treatment of trypsin/EDTA solution in SAE cells.
Assays for Cell Proliferation
Thymidine incorporation.
Proliferation of SAE cells
was assessed by thymidine incorporation as an indicator of
DNA synthesis. Subconfluent or near-confluent SAE cells
were cultured in 35 × 10-mm tissue culture plates (Costar, Cambridge, MA) under hyperoxia or normoxia for 2 d, as
described in the experimental design. Then the cells were
cultured for an additional 12 to 16 h in normoxia in the
presence of [3H]thymidine (1 µCi/well) (25 Ci/mmol, 3.77 GBq/mg; Amersham, Buckinghamshire, UK). Then the
supernatants were removed, and the plates were washed once with ice-cold PBS, fixed with 100% methanol for 10 min
on ice, and washed with 1 ml of distilled water. After 1 ml
of ice-cold trichloroacetic acid (TCA; 100 mg/ml) was added,
the plates were placed on ice for 10 min, washed once,
treated with TCA again, and washed two more times. Following solubilization with 0.3 mol/liter NaOH (300 µl/well),
the cell lysates in NaOH solution were added to scintillation solution (5 ml/vial Ecoscint; National Diagnostics, Atlanta, GA) and counted in a 
-scintillation counter (LS3100; Beckman, Fullerton, CA). The amount of [3H]thymidine
incorporated into SAE cells in each well was calculated. Triplicates were tested for each sample.
Assays for necrosis and apoptotic cell death. Necrotic and apoptotic cell death was assessed by measuring (1) LDH release into the culture medium and (2) morphologic changes detected by AO staining (9). The results of AO staining were confirmed by TUNEL stain and flow cytometry in selected samples (10).
- LDH levels: Culture supernatants were harvested and
frozen at
20°C until the time of measurement. LDH
levels in the culture supernatant were measured using a
commercial LDH kit (EC1.1.1.27 UV test; Sigma Chemical Co., St. Louis, MO). LDH levels varied less than 5%
when the same aliquoted samples were tested on three different occasions.
- Nuclear and cytoplasmic morphology by acridine orange staining: Cells were cultured in 35 × 10-mm tissue culture petri dishes, washed once with PBS, and fixed in 70% ethanol for 10 min on ice. Then the plate was air-dried for 10 to 15 min and stained with AO (6 µg/ ml; Sigma) in a 2:1 ratio mixture of distilled water and PBS for 3 to 4 h at room temperature. The staining solution was decanted and the plate was rinsed twice in a 2:1 mixture of distilled water and PBS and examined using a phase-contrast fluorescence microscope (450 to 490 nm; Nikon). Apoptotic cells were identified by their yellow fragmented and condensed nuclei as well as by their condensed red cytoplasm (9). The percentage of cells with apoptotic nuclei was calculated on the basis of 500 to 600 cells per dish in duplicate dishes.
- TUNEL stain: To confirm the presence of apoptosis in selected experiments, TUNEL staining was done using a commercially available kit (In Situ Cell Death Detection Kit, fluorescein, and TUNEL AP; Boehringer Mannheim, Indianapolis, IN) and following its protocol (10, 11). Adherent cells in a 35 × 10-mm petri dish were washed twice with PBS supplemented with BSA (10 g/liter; Sigma) and fixed in a fixing solution (40 g/liter paraformaldehyde in PBS, pH 7.4) for 30 min at room temperature. The plate was washed twice with PBS, placed in a permeabilization solution (1 g/liter Na citrate with Triton X-100 [1 g/liter]) for 2 min on ice, washed with PBS again, and stained with TUNEL reaction mixture for 60 min at 37°C. The plate was then washed with PBS, treated with antifluorescein Ab (Fab fragment) conjugated with alkaline phosphatase (AP) for 30 min at 37°C, washed with PBS, and treated with substrate for AP for 10 min at room temperature. The plates were examined under a light microscope.
- Cell cycle analysis: At the end of the culture, cells were harvested, washed once with HBSS, and fixed in ice-cold ethanol (75%) for more than 30 min at 4°C. Then cells were centrifuged and resuspended into 1 ml of staining solution (50 mg/liter propidium iodide in PBS, pH 7.4, with Triton X-100 [0.1%], EDTA [0.1 mmol/ liter], and RNase [2.5 U/ml]) (12). Then cells were analyzed by FACScan (Becton Dickinson, San Jose, CA), quantitating the percentage of cells with < 2c, 2c, intermediate, and 4c DNA content.
Antioxidant Vitamins
Vitamin E (
-tocopherol; Sigma) was dissolved in a mixture of ethanol, methanol, and isopropanol (19:1:1 ratio)
at a concentration of 103 mol/liter. The stock solutions
then were further diluted with the culture media to their
final concentrations. In control experiments, the effects of
the organic solvents alone were assessed for each of the
parameters tested. At the solvent concentrations used, the
bioassay results were unchanged from those of controls
with media and without solvents.
Vitamin C (Sigma) was dissolved in PBS on the day of
experiment to yield a stock concentration around 104 M
and further diluted with the culture medium for SAE cells.
Statistics
For comparison of test values with control values, Student's t test or Mann-Whitney U test was used depending on the results of Levene's test for equality of variances (13). For comparison of effects of antioxidant vitamins, a one-way analysis of variance (ANOVA) was employed, and statistical significance was assessed by Duncan's test or the Kruskal-Wallis test (13). P < 0.05 was considered to be significant. At least five replicate experiments were performed with duplicate or triplicate samples for each parameter.
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Introduction
Materials and Methods
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Effects of Hyperoxic Injury on A549 and SAE Cells
Subconfluent A549 cells. In normoxia, the cell number reached a plateau at Day 2 of incubation and remained at a similar level for up to Day 7 of culture (Figure 1A). In hyperoxia, a large number of nonviable cells detached at Day 2 of culture. The number of detached cells declined steadily afterward. The number of adherent A549 cells in hyperoxia was lower than in normoxia (Figure 1A), as was the number of live hyperoxic cells (Figure 2B). By the end of 7 d of culture, most hyperoxic A549 cells were dead (Figure 2). Apoptotic changes were minimal in normoxic A549 cells during the entire culture period, but LDH levels in the culture supernatant gradually increased in normoxia. Hyperoxia did not increase the apoptotic changes (Figure 3A), but increased LDH levels at Day 2 of the culture (Figure 4A). LDH levels remained at a similar level afterward in hyperoxic conditions, possibly because of rapid decline of A549 cell numbers with hyperoxia. When the medium was changed every day, we also observed minimal apoptotic changes but an increase of LDH levels in hyperoxia in two experiments.
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Subconfluent SAE cells. Under normoxia, the number of adherent and nonadherent SAE cells increased steadily during 7 d of culture, but the number of total and adherent cells was much lower than A549 cells (Figure 1B). Hyperoxia decreased the number of adherent SAE cells (Figure 1B), but surprisingly the number of live SAE cells was not significantly altered with hyperoxia (Figure 2B). This was also confirmed by erythrosin B staining. More apoptotic cells were observed in normoxic SAE cells than in A549 cells by AO staining during the entire culture period and hyperoxia further augmented the apoptotic changes in a dose-dependent manner (Figure 3A). We confirmed these results with TUNEL stain and flow cytometry in SAE cells following 2 d of culture in normoxia or hyperoxia (Table 1). Although about 10% of cells were lost secondary to trypsin treatment prior to fluorescent-activated cell sorter analysis, the results of flow cytometry were equivalent to those obtained with AO and TUNEL stain, in which adherent cells were directly stained without trypsin treatment. The amount of LDH released to the culture medium remained low in SAE cells (Figure 4A). The decrease of cell number was paralleled by a decline in thymidine incorporation (Figure 5). These results suggest that in subconfluence, hyperoxia suppressed cell proliferation and induced significantly more apoptotic cell death in SAE cells.
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Near-confluent A549 cells. The number of normoxic A549 cells remained at the similar levels during the entire culture period (Figure 1C). A large number of cells detached from the plate during the culture, most of which were not viable. The cell number temporarily increased at Day 4 of culture in hyperoxia, possibly reflecting an increase of nonadherent, dead cells, but hyperoxia decreased the number of live adherent cells (Figures 1C and 2D), as observed in subconfluence. Apoptotic changes remained minimal as in subconfluent A549 cells and hyperoxia did not increase apoptosis (Figure 3B). In normoxia, LDH levels were much higher than in SAE cells, highest at Day 4 of culture, and decreased slightly by the end of 7 d of culture (Figure 4). LDH tended to be higher in near-confluent A549 cells at Days 2 and 4 than in subconfluent cells. Hyperoxia did not alter LDH levels in near confluence. This was also true when the culture medium was changed every day in two experiments.
Near-confluent SAE cells. Under the normoxic conditions, the number of nonadherent and adherent SAE cells remained stable, and much less thymidine was incorporated in near confluence than in subconfluence (Figures 1D and 5). However, more apoptotic changes were observed in near-confluent adherent SAE cells than in subconfluent cells (Figure 3B), whereas LDH release remained low (Figure 4B). This suggested that the cell number was held constant by balanced apoptosis and proliferation. Hyperoxia did not alter the total cell number (Figure 1D) or LDH release, but the number of live adherent cells assessed by trypan blue and erythrosin B staining declined in hyperoxia in near confluence accompanied by the increase of apoptotic changes (Table 1 and Figure 6). Hyperoxia suppressed thymidine incorporation (Figure 5), but the decline of thymidine incorporation was not as striking as in subconfluence. At near confluence, hyperoxia altered the balance of SAE cell apoptosis and proliferation with less suppression of proliferation but more potent induction of apoptosis than in subconfluent conditions.
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Antioxidant vitamin protection on hyperoxic injury in SAE cells. After we observed significant changes in proliferation and apoptotic changes of SAE cells with 2 d of hyperoxia, we then determined protective effects of antioxidant vitamins in SAE cells following 2 d of hyperoxia. The total and live SAE cell numbers were not altered by supplemental vitamins E or C, irrespective of cell density and oxygen partial pressure (data not shown). These vitamins had no effect on thymidine incorporation in normoxia, but vitamin E partially prevented the hyperoxia-induced decline of thymidine incorporation in subconfluence (Figure 7A). Vitamin E slightly decreased thymidine incorporation by normoxic near-confluent cells (Figure 7B). Neither vitamin E nor C supplementation prevented the decrease of thymidine incorporation with hyperoxia in near confluence (Figure 7B). Under normoxia, supplemental vitamins had no effect on basal levels of apoptosis (Figure 8) or LDH release (data not shown), irrespective of cell density. However, vitamin E partially protected against the increase of apoptosis with hyperoxia in both subconfluence and near confluence (Figure 8). The protective effect of vitamin C on apoptotic changes was observed only in near confluence (Figure 8). These results indicate that specific antioxidant protection differed in subconfluence and near confluence.
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Understanding the regulation of cell death and proliferation of human airway epithelium in the face of environmental insults has been difficult, partly because of a lack of an appropriate in vitro model system employing normal airway epithelial cells. Transformed lung cancer cell lines have been used as convenient model systems (4, 9, 14). However, the mode and rate of cell death and proliferation may have changed in transformed cell lines and the results obtained may not apply to in vivo cellular proliferation and death of airway epithelium. This study examined proliferation and death of human untransformed SAE cells in normoxic and hyperoxic culture conditions. Our results suggest that SAE cells maintained epithelial homeostasis with balanced cell proliferation and apoptosis depending on cell density under normoxic conditions. In contrast, A549 cells (human lung cancer cells derived from type II alveolar cells) underwent necrosis when confluent in normoxia or with hyperoxia, as reported previously (4). We have demonstrated that hyperoxia augmented apoptosis in SAE cells, but not in A549 cells. Cell density also determined the protective effects of vitamin E and C against the injurious effects of hyperoxia in SAE cells. Thus the pathways through which hyperoxia induces cell death differ depending on the target cell type.
The human distal airway epithelium predominantly consists of basal and Clara cells with few ciliated cells and less numerous goblet cells compared with proximal airway epithelium (17). Basal cells can be found as early as 10 wk of gestation and likely serve as a precursor to more differentiated cells in normal development or repair after injury (18). It has been difficult to maintain untransformed airway epithelial cells in the culture for prolonged periods because ciliated and Clara cells often do not survive. Recently it became possible to maintain human SAE cells for five to six passages in the serum-free medium supplemented with multiple growth factors. This enabled us to investigate factors determining cell death and proliferation of normal untransformed human epithelial cells in vitro.
In small airway diseases such as asthma and COPD, the airway epithelium may be disrupted by chronic inflammation. Repair occurs in response to these injuries, resulting in patchy areas of denuded, repaired, and hyperplastic or potentially dysplastic airway epithelium (17). In these areas, epithelial cell functions likely are altered and this may determine the effects of further or subsequent lung insults. Our previous studies of MDCK renal tubular epithelial cells demonstrated that cell density partly determined the effects of hyperoxia on cell death and proliferation and the protective effects of specific antioxidant vitamins (8). These results indicated the importance of evaluating oxidant injury depending on cell density. Therefore, the first set of experiments determined the effects of hyperoxia in subconfluent and confluent SAE cells that may partly mimic the repairing and intact in vivo airway epithelia, respectively.
We observed a striking difference in cell death and proliferation between A549 lung cancer cells and untransformed SAE cells. Normoxic subconfluent SAE cells proliferated actively with little cell death as evidenced by the increase of live cell numbers, active DNA synthesis (elevated thymidine incorporation), low percentage of apoptosis, and minimal LDH release. Normoxic near-confluent SAE cells appeared to maintain their epithelial monolayer with balanced apoptosis and low-grade proliferation. This was indicated by low but persistent thymidine incorporation in parallel with increases in the number of nonadherent dead cells and frequency of apoptotic cells. LDH levels in the SAE cell medium remained low, indicating that cell death occurred mainly through apoptosis. These findings with SAE cells contrast with our previous findings with near-confluent MDCK cells, in which cell death occurred through a combination of apoptosis and necrosis, but predominantly by necrosis (8). Similarly, near-confluent A549 cells underwent necrotic cell death with minimal apoptosis, consistent with a prior report (4). There are several possible explanations for these differences. First, in transformed cell lines like A549 cells, the mechanisms regulating cell death and proliferation may have been altered so that necrosis was a more dominant cause of cell death. Second, these differences may reflect other cell type-specific differences, such as the levels of apoptosis checkpoint molecules including bcl2 or interleukin-1 converting enzyme (19). Third, A549 cells presumably derived from alveolar type II cells; these cells may respond differently to hyperoxic insults. In summary, our results indicate that, as opposed to A549 cells, SAE cells likely maintain an intact epithelial monolayer by adjusting the rate of apoptosis, rather than necrosis.
This study examined the effects of hyperoxia on SAE cell death and proliferation as a model of an environmental oxidant insult. In subconfluent SAE cells, hyperoxia induced a short decline of thymidine incorporation in parallel to the decrease in the adherent cell number, indicating suppression of cell proliferation in hyperoxia. This is consistent with the cell cycle arrest seen in an alveolar type II cell line exposed to hyperoxia (14), but may also be associated with inactivation of growth factors such as fibroblast growth factor (23) or with toxic metabolites potentially generated during hyperoxia. However, we found no toxic effects when SAE cell medium without cells was kept in hyperoxia for 2 d and then added to cultures of SAE cells (H. Jyonouchi et al., unpublished observations). Hyperoxia also induced significant apoptosis, but not necrosis, in the adherent SAE cells. ROS are a putative trigger for apoptosis in many cell types. In the serum-free SAE cell culture conditions, ROS are most likely produced by lipid peroxidization of cell/plasma membranes that may have triggered increase of apoptosis in SAE cells by altering the apoptosis checkpoint (19). While present in A549 cells, hyperoxia increased necrotic cell death with suppression of cell proliferation as reported previously (4).
In near-confluent SAE cells, hyperoxia augmented apoptosis and slightly suppressed DNA synthesis. The total cell number was not altered by hyperoxia, although the number of live cells decreased. The near-confluent SAE cells appeared less vulnerable to hyperoxic injury than did subconfluent SAE cells. That is, cell proliferation and death differed in subconfluent and near-confluent SAE cells in both normoxic and hyperoxic conditions. For near-confluent A549 cells, hyperoxia decreased the cell number as observed in subconfluent A549 cells. Thus, the injurious effects of hyperoxia were less dependent on cell density in A549 cells. Cell death induced by hyperoxia almost exclusively occurred through apoptosis in SAE cells. Taken together, our results indicate that degree of proliferation and death of SAE cells was greatly affected with hyperoxia depending on cell density.
In evaluating antioxidant vitamin protection, vitamins E and C were chosen as representative dietary lipid-soluble and water-soluble antioxidant vitamins (24). Lipid-soluble antioxidant vitamins prevent lipid peroxidation and help maintain the integrity of cellular organelles (25). Water-soluble antioxidant vitamins provide defense against aqueous free radicals and ROS. Vitamin E also has other biological functions including antiproliferative action on cells of various lineages (28).
Vitamin E protected subconfluent SAE cells against both the decline of thymidine incorporation and the increased apoptosis in hyperoxia. At near confluence, vitamin E had no effect on thymidine incorporation but prevented the increase of apoptosis in hyperoxia. In normoxia, vitamin E slightly suppressed thymidine incorporation at near confluence but not at subconfluence. Thus vitamin E can exert different actions depending on cell density and partial oxygen pressure (normoxia versus hyperoxia), possibly because of its multiple biological functions (28). The protective effect of vitamin E against hyperoxia-induced apoptosis indicates that ROS generated through lipid peroxidation in hyperoxia may be partly responsible for increase of apoptosis in SAE cells.
Vitamin C protected SAE cells against apoptotic changes with hyperoxia at near confluence, but not at subconfluence. This may be partly due to the greater amount of apoptosis at near confluence with hyperoxia; hence, protective effects of vitamin C may be more evident. Vitamin C, a water-soluble antioxidant, did not alter other parameters and appeared less effective than vitamin E. This may be partly attributed to the serum-free cultures of SAE cells; in this condition, ROS likely were generated by lipid peroxidation of cell and plasma membranes in hyperoxia.
In summary, we have shown that (1) SAE cells underwent oxidant-induced death primarily by apoptosis, in contrast to necrosis in A549 cells and mixed death in MDCK cells; (2) hyperoxia suppressed DNA synthesis by SAE cells; and (3) SAE cells were more susceptible to hyperoxic injury when subconfluent, probably because of more active proliferation. The cell density also determines the effects of hyperoxia and antioxidant vitamin protection in SAE cells. These descriptive studies showing differences between responses to hyperoxia of different cell types or with differing cell density provide a vehicle to analyze the mechanisms of these different responses that may be dictated by growth factors, specific ROS, matrix detachment, or the levels of apoptosis checkpoint molecules (19, 32).
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Address correspondence to: Harumi Jyonouchi, M.D., Department of Pediatrics, University of Minnesota, Box 610, UMHC, 420 Delaware St. S.E., Minneapolis, MN 55455. E-mail: jyono001{at}maroon.tc.umn.edu
(Received in original form December 2, 1996 and in revised form December 15, 1997).
Acknowledgments: This study was supported in part by grants from the Minnesota Medical Foundation (H.J.) and Viking Children's Foundation (H.J.), American Lung Association Career Investigator Award (D.H.I.), American Heart Association Research Grant In Aid (D.H.I.) and a National Institutes of Health SCOR in Acute Lung Injury HL50152 (D.H.I.).
Abbreviations AO, acridine orange; AP, alkaline phosphatase; ANOVA, analysis of variance; BSA, bovine serum albumin; COPD, chronic obstructive pulmonary disease; EDTA, ethylenediaminetetraacetic acid; HBSS, Hanks' balanced salt solution; LDH, lactate dehydrogenase; MDCK cells, Madin-Derby canine kidney cells; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SAE cells, small airway epithelial cells; TUNEL stain, terminal transferase dUTP nick end-labeling stain.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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