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Abstract
Introduction
Materials & Methods
Results
Discussion
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p21WAF/CIP1 is an important regulator of cell cycle progression (1). When induced, p21WAF/CIP1 protein inhibits cell cycle progression at the G1/S interface, resulting in growth arrest of the cell. To determine if p21WAF/CIP1 is involved in growth arrest and lung injury during hyperoxia, several cell lines were exposed to high levels of hyperoxia. p21WAF/CIP1 was found to be induced by 72 h in all three cell lines. Next, using an in vivo model, p21WAF/CIP1 was found to be induced at both the mRNA and protein level in neonatal murine lung born and maintained in hyperoxia. Localization of p21WAF/CIP1 was found in the peripheral airway cells. Hyperoxia-induced p21WAF/CIP1 expression was then shown to be mediated through the p53 pathway, using adult p53 mutant mice. These studies demonstrated that p21WAF/CIP1 is induced both in cells grown in culture and in neonatal and adult lung exposed to high levels of hyperoxia. Localization of p21WAF/CIP1 expression to the peripheral airway cells suggests that p21WAF/CIP1 may act to inhibit growth of alveoli in neonatal lung and delay repopulation of alveolar cells during hyperoxic administration.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
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p21WAF/CIP1 was first described as a potent and universal inhibitor of cyclin-dependent kinases (cdks) (1). p21 functions as a checkpoint in the cell cycle by inhibiting cdks at the G1/S interface. p21WAF/CIP1 has been shown to bind to cyclin-cdk complexes, preventing phosphorylation of the retinoblastoma protein. When this happens the E2F pathway is blocked and the cell cycle is arrested at the G1/S interface (5). Overexpression of p21WAF/CIP1 has been shown to result in growth arrest of cells in culture (1). Induction of p21WAF/CIP1 may occur through either a p53-dependent or independent pathway. p21WAF/CIP1 induction through a p53-mediated pathway has been demonstrated in cells damaged by ionizing irradiation or genotoxic agents (7, 8). p21WAF/CIP1 induction independent of the p53 pathway has been demonstrated in cells undergoing terminal differentiation (9, 10).
Frequently, chronic lung disease develops in infants exposed to a combination of hyperoxia, barotrauma, and
prematurity, although any one of these factors alone can
lead to impaired lung growth in the neonate (11). The
molecular mechanisms underlying the impaired lung growth
in these infants is not well understood. Factors involved in
the development of lung injury and impaired lung growth
may include decreased levels of antioxidant enzymes (14);
increased levels of cytokines (such as transforming growth factor 
[TGF-] leading to impaired growth, inflammation, and fibrosis) (15); and induction of genes such as
those encoding p21WAF/CIP1 or p27, which inhibit cellular
growth.
Antioxidant genes have been shown to have a protective role during hyperoxic exposure. The antioxidants CuZn superoxide dismutase (SOD) and MnSOD are known to be induced during hyperoxia and mRNA levels of CuZnSOD, glutathionine peroxidase, and catalase are increased in cultured endothelial cells exposed to hyperoxia (16, 17).
Growth arrest of cells exposed to hyperoxia may be
regulated by increased levels of TGF- and platelet-
derived growth factor protein 2. The genes encoding both
of these factors were shown to be induced in the serum of
type 2 immortalized pneumocytes undergoing growth arrest during hyperoxic exposure (18). p27, another G1 cyclin-cdk inhibitor involved in cell cycle arrest, has been
shown to be regulated by TGF-. Overexpression of p27-like p21WAF/CIP1 leads to cell cycle arrest at the G1/S interface (19, 20). The role of p27 during hyperoxia-induced
growth arrest is unclear at this time.
Heme oxygenase-1 is induced during hyperoxic exposure in adult rat lung and is believed to ameliorate oxidant-mediated lung injury (21). The genes encoding tumor necrosis factor (TNF), metallothionein, Na+,K+-ATPase, and heat shock protein are also induced during hyperoxic stress (22, 23).
In this study the expression pattern of p21WAF/CIP1 was investigated in murine lung exposed to high concentrations of oxygen for relatively short durations. High oxygen exposures, such as those used in this study, are frequently used in the treatment of acutely ill neonates or of people with adult respiratory distress syndrome. Because p21WAF/CIP1 overexpression can cause growth arrest in cells in culture and p21WAF/CIP1 is induced during certain types of cell injury, it appeared to be a likely candidate for being involved in growth arrest and injury during hyperoxic exposure.
This study has found that p21WAF/CIP1 is induced both in cells in culture and in neonatal and adult murine lung exposed to high levels of hyperoxia. The hyperoxic conditions that were used were sufficient to induce growth arrest of cells in culture and decrease cellular proliferation in neonatal lung. The distribution of p21WAF/CIP1 protein in neonatal lung exposed to hyperoxia was predominantly peripheral, suggesting that p21WAF/CIP1 induction may inhibit alveolar growth and impair healing in neonatal lung. Finally, p21WAF/CIP1 induction was found to be mediated through a p53-dependent pathway.
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Materials and Methods |
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Materials & Methods
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Cell Lines
nMuLi and MRC5 cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and grown to 50% confluence. Cells were placed in a modular incubator chamber (Billups-Rothenberg, del Mar, CA) and exposed to either 95% O2 and 5% CO2 or room air and 5% CO2. Cells were then harvested at specific time periods and RNA was obtained by the RNAzol B technique. Fifteen micrograms of total RNA was electrophoresed on a 1% formaldehyde gel, transferred, and probed with a full-length p21 cDNA probe.
Primary type 2 epithelial cells from 19-d fetal rat lung were isolated, resuspended in trypsin, counted, and grown in modified Eagle's medium (MEM) plus 10% fetal bovine serum (FBS), with penicillin and streptomycin (24). After 72 h in culture, cells were placed either in 95% oxygen and 5% CO2 or room air and 5% CO2. Cells were then harvested for RNA extraction.
[3H]Thymidine Uptake in nMuLi Cells
nMuLi cells were plated in 24-well plates at a cellular density of 25,000 cells/well. Cells were then placed either in 5% CO2 and room air or 95% O2 and 5% CO2. Cells were labeled with 5 µCi of [3H]thymidine per well 24 h before harvesting the cells. Cells were washed and lysed (0.5% sodium dodecyl sulfate [SDS], 100 mM NaCl, 10 mM Tris, 20 mM EDTA), 50% trichloroacetic acid (TCA) was added, and the cells were placed on ice overnight. Cells were then filtered using a vacuum manifold on 24-mm Whatman glass microfiber filters and counted at 24, 44, and 68 h. The viability of cells was determined. Cells were trypsinized, then stained with 0.4% trypan blue. Cells were counted on a hemocytometer slide. The percentage viability was determined by calculating the percentage of unstained cells.
RNA Extraction and Northern Blot Analysis
Timed pregnant CD-1 mice (Charles River, Wilmington, MA) were placed in a hyperoxic chamber at 18.5 d of gestation. Mice were born under hyperoxic conditions (92- 93% FIO2) the following morning. Mothers were rotated every 12 to 24 h to prevent death from acute oxygen toxicity. These experiments were done according to the animal protocol approved by the Animal Care Use Committee of the Johns Hopkins University School of Medicine (Baltimore, MD). Lung tissue was harvested at specific time points from both hyperoxia-exposed and age-matched control neonatal mice raised in room air. RNA was isolated by either the RNAzol method or extracted by guanidium thiocyanate followed by centrifugation on a cesium chloride cushion (25). For the original 84-h time point total RNA was poly(A) selected twice through an oligo(DT) column as described (25), otherwise total RNA was used for Northern blot analysis.
Five micrograms of poly(A)-selected RNA (tissue) or 15 µg of total RNA (cell extracts) was run on a 1% agarose-formaldehyde gel, transferred to a filter (Genescreen Plus; Du Pont, Wilmington, DE) and baked for 2 h at 80°C in a vacuum oven. p21WAF/CIP1, p27, and p53 cDNA clones (generous gifts of B. Vogelstein, The Johns Hopkins Oncology Center, Baltimore, MD) were labeled with 32P and randomly primed as previously described (26). Northern analysis was performed using hybridization solution (5× SSPE [0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA, pH 7.7], 10% dextran sulfate, 50% formamide, 1% SDS, 200 µg/ml salmon DNA, and 0.1% each of bovine serum albumin, ficoll and polyvinylpyrrolidone) at 42°C overnight, and the blots were then washed in 0.1% SDS, 0.2% SSC 5× [0.15 M NaCl plus 0.015 M sodium citrate] for 20 min at 55°C.
Western Blot Analysis
Lung tissue homogenates from neonatal murine lung, exposed for different periods of time to hyperoxia, and control lung were equally loaded as determined by Coomassie staining and run on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose. Western blot analysis was performed using a p21WAF/CIP1 polyclonal antibody (13436E; Pharmingen, San Diego, CA) 1 µg/ml as recommended in 5% BLOTTO, Tris-buffered saline (TBS), and 0.05% Tween for 1 h; the blots were then washed three times in TBS-Tween, incubated with a biotinylated goat anti-rabbit second antibody (RPN 2108; Amersham, Arlington Heights, IL) at a 1:10,000 dilution for 30 min, washed again, then developed using enhanced chemiluminescence (ECL) (RPN 2106; Amersham). Control and hyperoxia-exposed murine tissues from brain and liver were also obtained from 3.5-d-old mice and used for Western blot analysis.
Immunohistochemistry
Ten-micron cryostat sections were made of 84-h hyperoxia-exposed and control neonatal lung. Sections were fixed in cold acetone for 10 min. Slides were then washed in phosphate-buffered saline (PBS), placed in 0.2% H2O2 for 10 min, and washed again in PBS. They were next placed in 1.5% normal goat serum, incubated with a 1:200 dilution of primary polyclonal antibody (sc-397; Biotechnology Inc., Santa Cruz, CA) alone or antibody plus a 10-fold excess of peptide (sc-397 P; Santa Cruz Biotechnology Inc.) overnight at 4°C. Slides were then incubated with a secondary antibody (Santa Cruz rabbit ABC Immunostain System SC-2018) for 30 min at room temperature, washed and processed as per the ABC kit, and developed with diaminobenzidene (DAB), dehydrated, and mounted with Permount.
BrdU Staining in Neonatal Lung
Neonatal mice (84 h) were born into either 92-93% oxygen or room air. They were injected intraperitoneally with 1 mg of 5-bromo-2'-deoxyuridine (BrdU), 2 h before the animals were killed. Five-micron cryostat sections were made of 84 h hyperoxia-exposed and control neonatal lung. Sections were fixed in 80% ethanol-10% chloroform-10% acetic acid. The antibody staining was performed as per the Amersham cell proliferation kit (RPN 20). Nuclei that were stained with BrdU antibody were counted from random fields taken from three mice born into hyperoxia and three mice born into room air. The magnification of the lung fields was ×200.
p53 Mutant Mice
p53 mutant mice were obtained from Jackson Laboratory (Bar Harbor, ME). Adult p53 homozygote mutant mice (C57BL/6J-Trp53<tm1Tyj>) (Jackson Cat. No. JR2101.2) and age-matched control wild-type mice (Jackson Cat. No. 0000664) were used for hyperoxic experiments. Genotypes were verified by polymerase chain reaction (PCR) using a three-primer mix (Jackson Laboratory) to distinguish p53 homozygote mutant mice from wild-type mice. Mice were kept either in room air or placed in a 92-93% oxygen chamber for 50 h. Lung tissue was then harvested and RNA was extracted. Northern blot analysis was performed as described previously.
Statistical Analysis
Statistical calculations were performed using the Stata statistical package (Stata Corporation, College Station, TX). Differences in measured variables between experimental and control groups were assessed using an unpaired t test. Statistical difference was accepted at P < 0.05.
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Materials & Methods
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Discussion
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Expression of p21WAF/CIP1 in Primary Type and Immortalized Cell Lines
Respiratory and nonrespiratory cells were grown in culture and exposed to either 95% O2 and 5% CO2 or room air and 5% CO2. nMuLi cells (mouse liver epithelial cells, ATCC CRL 1638) were found to have significant induction of p21WAF/CIP1 at 72 h of hyperoxia. Cells exposed to room air expressed significantly less p21WAF/CIP1 at 72 h by Northern blot analysis. A time course was then undertaken to determine the onset of p21WAF/CIP1 mRNA expression. At 6 h, p21WAF/CIP1 mRNA levels were minimal in both room air- and hyperoxia-exposed cells. At 12 h, p21WAF/CIP1 levels were moderately increased in hyperoxia-exposed cells only. By 20 h, p21WAF/CIP1 mRNA levels in the hyperoxia-exposed cells were significantly increased relative to cells exposed to room air (Figure 1). At 28 h of hyperoxia p21WAF/CIP1 mRNA levels were also significantly increased relative to cells in room air. The room air cells, however, had increased p21WAF/CIP1 mRNA levels relative to the other control lanes. This could possibly represent a change in basal level of p21WAF/CIP1 expression in cell culture or an effect on cell density at this time period. Two other cell lines, MRC-5 cells (derived from human embryonal lung, ATCC CCL-171) and 19-d fetal rat primary type 2 alveolar cells (Figure 2), were found to have p21WAF/CIP1 induction at 72 h of hyperoxia.
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[3H]Thymidine Uptake in nMuLi Cells
To determine the degree of cellular proliferation taking place both in cells exposed to hyperoxia and cells exposed to room air, nMuLi cells were labeled with [3H]thymidine. Cell growth was markedly inhibited in the cells exposed to hyperoxia compared to the cells grown in room air (Figure 3). Cell viability determined by trypan blue staining revealed less then 5% cell death in both room air- and hyperoxia-exposed cells.
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mRNA Expression of p21WAF/CIP1 in Murine Lung
Mice were born into either hyperoxia or room air and murine lung was examined for p21WAF/CIP1 expression by Northern blot analysis. Neonatal lung was initially examined at 84 h of life. This time point was selected for study because a previous study by Northway and coworkers (40) found that little [3H]thymidine uptake occurred in neonatal murine lung exposed to hyperoxia prior to 96-120 h of life. This is in contrast to neonatal murine lung exposed to room air, which showed significant [3H]thymidine uptake by 48 h of life (8). Marked induction of p21WAF/CIP1 as determined by Northern blot analysis was found in lung exposed to hyperoxia compared to control lung. No differences, however, were found in either p27 or p53 mRNA levels between hyperoxia- or room air-exposed murine lung (Figure 4). Several earlier and later time points were then obtained. Increased p21 mRNA levels as determined by Northern blot analysis at 36 h of life were found in murine lung exposed to hyperoxia. Increased p21 induction was found through 6.5 d of life (Figure 5), which was the last time point obtained. By 6.5 d of hyperoxia the mortality rate was approximately 50% in the neonatal mice.
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Adult CD-1 mice were then placed in hyperoxia. p21WAF/CIP1 was found to be induced in hyperoxia-exposed adult murine lung at 28 and 50 h of 92-93% O2 exposure compared to mice kept in room air (data not shown).
These results demonstrate that induction of p21WAF/CIP1 mRNA takes place in both neonatal and adult murine lung during hyperoxic exposure and that induction of p21WAF/CIP1 during hyperoxia remains elevated throughout the exposure period.
Protein Expression of p21WAF/CIP1 in Murine Lung
Protein expression of p21WAF/CIP1 was determined by Western blot analysis. p21WAF/CIP1 expression was first detected in neonatal murine lung exposed to oxygen for 48 h (Figure 6). Peak expression of p21WAF/CIP1 was evident by 84 h of oxygen exposure and remained elevated through 6.5 d of oxygen exposure. Both neonatal murine brain and liver from control and hyperoxia-exposed neonatal mice at 84 h of life were examined for increased levels of p21WAF/CIP1 protein. No p21WAF/CIP1 protein was detected in 84-h murine brain or liver in either the control or hyperoxia-exposed animals (data not shown). These experiments demonstrate that p21WAF/CIP1 induction during hyperoxia is specific to the lung irrespective of the other effects that hyperoxia may be having on the total animal.
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Localization of p21WAF/CIP1 in Hyperoxia-exposed Neonatal Lung
Immunohistochemistry revealed pronounced nuclear staining in peripheral pneumocytes and small bronchiolar epithelial cells in neonatal lung exposed to 92-93% hyperoxia for 84 h. Minimal nuclear staining in a similar distribution was found in neonatal lung from mice raised in room air. Peptide competition was used as a control (Figure 7). These findings suggest that exposure to hyperoxia induces p21WAF/CIP1 expression predominantly in the peripheral airway cells of the neonatal lung.
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To determine the degree of cellular proliferation in neonatal lung under conditions of hyperoxia versus room air, BrdU was injected into three 84-h neonatal mice born into hyperoxia and three neonatal mice born into room air. BrdU antibody staining was markedly increased in the lungs of mice raised in room air compared to mice raised under hyperoxic conditions (Figure 8). Random fields of lung were counted from different animals. The mean number of nuclei labeled with BdrU in lungs from mice raised in room air was 36, whereas the mean number of labeled nuclei in lungs from mice raised in hyperoxia was 11 (P < 0.001).
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mRNA Expression of p21WAF/CIP1 in p53 Mutant Mice
p53 homozygote mutant mice were obtained and exposed
to hyperoxia to determine if p21WAF/CIP1 induction was mediated through a p53 pathway. Adult male C57/B6 p53 homozygote mutant mice and age-matched control wild-type
mice were placed in a hyperoxic chamber for 50 h. All
adult mice survived and no mice appeared in distress.
mRNA was obtained from the lungs of each individual
mouse and p21WAF/CIP1 induction was measured by Northern blot analysis. Induction of p21WAF/CIP1 was quantitated
using a phosphor screen using the program ImageQuant (Molecular Dynamics, Sunnyvale, CA). Blots were normalized using murine -tubulin. Wild-type mice exposed
to hyperoxia demonstrated marked induction of p21WAF/CIP1
compared to wild-type mice in room air. p53 mutant mice
exposed to hyperoxia showed no induction of p21WAF/CIP1
compared to p53 mutant mice exposed to room air (Figure
9). The message level of p21WAF/CIP1 was similar between
wild-type mice exposed to room air and p53 mutant mice
exposed to room air and hyperoxia. These findings indicate that hyperoxia-induced p21 expression is mediated
predominantly through a p53-dependent pathway.
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Materials & Methods
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Discussion
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Exposure to high oxygen tensions is known to be a contributory factor in the development of chronic lung disease or bronchopulmonary dysplasia (BPD) in infants (12, 13). Infants with BPD have impaired lung growth manifested by decreased numbers of alveoli (11). In this study p21WAF/CIP1, a known G1 cdk inhibitor that is induced in response to cell injury and leads to growth arrest, was found to be induced in cells in culture and in neonatal and adult murine lung during hyperoxic exposure. The induction of p21WAF/CIP1 was found to be mediated through a p53-dependent pathway. Neither p53 nor p21WAF/CIP1 has been shown previously to be induced during hyperoxia. The induction of p21WAF/CIP1 in the lung, particularly in the distribution of the peripheral respiratory cells, suggests that it may have an inhibitory role in the growth of alveolar cells during hyperoxia. This may be important because the potential for lung growth is limited. Alveolar growth ceases by 8 years of age in the human, with the majority of alveolar growth occurring much earlier (27). p21WAF/CIP1 induction during hyperoxic conditions may also inhibit repair of epithelial cells after lung injury by maintaining cells in a state of growth arrest.
p53 is an important regulator of the cell cycle and has been shown to be inactivated in many types of cancers (7, 28). Induction of p53 results in either growth arrest of the cell, to allow repair of the genome, or programmed cell death or apoptosis. p53 has been shown to act independently of p21WAF/CIP1 during apoptosis; however, during cell injury, the p21WAF/CIP1 pathway is induced. In this study p21WAF/CIP1 induction is dependent on an intact p53 pathway during hyperoxic exposure. In the absence of functional p53, p21WAF/CIP1 induction did not occur during hyperoxia. Basal levels of p21WAF/CIP1, however, were equally present in both p53 mutant and intact mice, suggesting that p53 is not involved in the regulation of p21WAF/CIP1 at the basal level.
An increase in p53 mRNA level was not demonstrated in this study. Although p53 protein levels were not determined, others have shown that equal amounts of p53 mRNA may result in different amounts of p53 protein synthesis (29). Alternatively, p53 mRNA increases may be transient and the induction of p53 mRNA may occur earlier than the time point that was evaluated. Although it is not clear which mechanism is taking place, this study finds that in the absence of p53, induction of p21WAF/CIP1 does not take place during hyperoxia, indicating that in this model p21WAF/CIP1 induction is dependent on p53 regulation.
p53 has been shown to regulate cell cycle progression by detecting specific signals in the environment of the cell and responding through either a p21WAF/CIP1-dependent or -independent pathway. p21WAF/CIP1 functions as an inhibitor of the G1 cdks (2, 6) and affinity of p21WAF/CIP1 for the cdks is enhanced when they are bound to cyclins (30). Loss of p21WAF/CIP1 function has been shown to result in defective G1 checkpoint control (31) and cells lacking p21WAF/CIP1 develop uncoupling of the S phase and polyploid nuclei, and undergo apoptosis when treated with Adriamycin (32). Previous studies have demonstrated that p53 is induced during cell injury, hypoxia, oncogene activation, and certain viral infections (28). Hyperoxia appears to be another condition that induces p53. Whether p53 is sensing the effect of high oxygen tensions on the cell or the effect of cell damage induced by hyperoxia is not clear. In nMuLi cells the induction of p21WAF/CIP1 occurs early, by 12 h of hyperoxia, and is associated with growth arrest but not cell death. This suggests that hyperoxia is the primary signal leading to p53-mediated p21WAF/CIP1 expression and growth arrest during hyperoxic exposure. Cell injury, however, caused by hyperoxia may also be an important signal and cannot be ruled out. It has been shown that both oxygen- and radiation-induced cell injury result from free radical toxicity (14). Previous studies have found that irradiation of cells can cause induction of p21WAF/CIP1 through a p53 pathway (7, 8). The induction of p21WAF/CIP1 during hyperoxia may be secondary to the toxic effects of oxygen and excess release of free oxygen radicals. The development of free oxygen radicals has been shown to occur and to be a source of toxicity in cells exposed to hyperoxia (33, 34). Cell injury may also induce apoptosis through a p53 pathway independent of p21. Hypoxia has been shown to induce apoptosis in tumor cells through a p53 pathway (35). The presence of the p53- induced apoptosis pathway in hyperoxia-induced lung injury was not investigated in this study.
Organs other then lung were evaluated for p21WAF/CIP1 protein induction during hyperoxia. Increased levels of p21WAF/CIP1 protein were not found in either liver or brain in 84-h neonatal mice born into hyperoxia. Although blood gases were not obtained from these animals, this observation suggests that the direct effect of hyperoxia and not the arterial pO2 of the blood perfusing the organ is the signal for p21WAF/CIP1 p53-dependent induction. The induction of p21WAF/CIP1, however, does not appear to be specific only to lung because several different cell lines in this study, including mouse liver epithelial cells, showed induction of p21WAF/CIP1 in response to hyperoxia.
The minimal concentration of oxygen needed to induce
p21WAF/CIP1 expression was not determined in this study
and will need to be addressed in a later study. The level of
oxygen needed to induce p21WAF/CIP1 may also be dependent on the age and degree of prematurity of the lung.
High concentrations of oxygen were used in this study because previous studies have demonstrated impaired lung
growth using these levels (36). High oxygen tensions
have been shown to cause growth arrest in cells in culture
and in neonatal lung (18, 40). The levels of hyperoxia used
in this study resulted in profound growth arrest of nMuLi
cells in culture. Neonatal lung exposed to a 92-93% hyperoxic environment also demonstrated markedly decreased
DNA synthesis as determined by BrdU uptake. This response to hyperoxia occurred during a time of marked induction of p21WAF/CIP1 mRNA and protein, suggesting that
it may be a major factor involved in growth impairment
secondary to hyperoxia. The pronounced expression pattern of p21WAF/CIP1 in peripheral airway cells and small
bronchial epithelial cells of neonatal murine lung exposed
to hyperoxia also suggests that p21WAF/CIP1 is affecting the
growth of respiratory units in the lung specifically. Other
genes have previously been shown to be induced during
hyperoxia, such as those encoding TGF- and components
of the insulin growth factor system (18). These gene products may interact with p21WAF/CIP1; however, this will need
to be studied further.
p21WAF/CIP1 expression has previously been found in lung tissue. In areas of normal lung from human lung tumors, p21WAF/CIP1 expression was found in low abundance in type 2 alveolar and bronchial epithelial cells (41). Nuclear localization of p21 has also been described in cells undergoing G1 arrest in cell culture (8). In this study p21WAF/CIP1 was expressed in lungs from mice raised in room air but to a much lesser extent than in lungs of mice born into hyperoxia or adult mice exposed to hyperoxia. The role of p21WAF/CIP1 may be minimal during normal homeostasis as demonstrated by the p21WAF/CIP1 mutant mice, which survive to adulthood with no obvious phenotype (31). From these studies, a significant role for p21WAF/CIP1 may be during hyperoxic stress. Its induction may induce growth arrest in the lung, allowing time for repair of the genome before undergoing further DNA synthesis; the problem may arise if growth impairment is maintained during the normal period of alveolar growth that is necessary for normal lung development.
In conclusion, this study showed marked induction of p21WAF/CIP1 in vivo and in vitro during hyperoxia. p21WAF/CIP1 induction during hyperoxic exposure was found to be mediated through a p53-dependent pathway and the distribution of p21WAF/CIP1 in hyperoxia-exposed neonatal lung suggests that p21WAF/CIP1 is involved in growth arrest of peripheral respiratory cells during hyperoxic exposure. Future studies will be directed at determining how the absence of p21WAF/CIP1 affects cell growth during hyperoxic exposure, the level of oxygen required to induce p21WAF/CIP1 expression, and the role of p53 in apoptosis in hyperoxia-induced lung injury.
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Footnotes |
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Address correspondence to: Dr. Sharon McGrath, Department of Pediatric Pulmonary, Johns Hopkins Hospital, Park 316 N. Wolfe St., Baltimore, MD 21287-2533.
(Received in original form March 14, 1997 and in revised form June 10, 1997).
Acknowledgments: The author thanks Dr. Se-Jin Lee, for these studies would not have been performed without his guidance and support. The author also thanks Dr. Michael Kaston and Teresa Zimmers for helpful discussions, Dr. Mary Greene and Dr. Stephen Goodman for assistance with the statistical analysis, and Dr. Carol Murray for kindly providing the primary type 2 fetal epithelial cells.
This work was supported by NIH-K08 award HL03624, a Solo Cup Foundation Award Grant, a Children's Health Research Center Grant, and a Maryland American Lung Association Research Grant.
Abbreviations
cdk, cyclin-dependent kinase;
CIP, cdk-interacting protein;
PBS, phosphate-buffered saline;
SDS, sodium dodecyl sulfate;
SOD, superoxide
dismutase;
TGF-, transforming growth factor ;
WAF, wild-type p-53 activated fragment 1.
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References |
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Abstract
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Materials & Methods
Results
Discussion
References
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1. Harper, J. W., G. R. Adami, N. Wei, K. Keyomarsi, and S. J. Elledge. 1993. The p21 cdk-interacting protein CIP1 is a potent inhibitor of G1 cyclin- dependent kinases. Cell 75: 805-816 [Medline].
2. Xiong, Y., G. J. Hannon, H. Zhang, D. Casso, R. Kobayashi, and D. Beach. 1993. p21 is a universal inhibitor of cyclin kinases. Nature (London) 366: 701-704 [Medline].
3. El-Diery, W. S., T. Tokino, V. Velculescu, D. B. Levy, R. Parsons, J. M. Trent, D. Lin, W. E. Mercer, K. Kinzler, and B. Vogelstein. 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75: 817-825 [Medline].
4. Harper, J. W., S. J. Elledge, K. Keyomarsi, B. Dynlacht, L. Tsai, P. Zhang, S. Dobrowolski, C. Bai, L. Connell-Crowley, E. Swindell, M. P. Fox, and N. Wei. 1995. Inhibition of cyclin-dependent kinases by p21. Mol. Biol. Cell 6: 387-400 [Abstract].
5.
Sherr, C. J..
1996.
Cancer cell cycles.
Science
274:
1672-1677
6.
Elledge, S. J..
1996.
Cell cycle checkpoints: preventing an identity crisis.
Science
274:
1664-1672
7.
Hartwell, L. H., and
M. B. Kastan.
1994.
Cell cycle control and cancer.
Science
266:
1821-1828
8.
El-Diery, W. S.,
J. W. Harper,
P. M. O'Connor,
V. E. Velculescu,
C. E. Canman,
J. Jackman,
J. A. Pietenpol,
M. Burrell,
D. E. Hill,
Y. Wang,
K. G. Winman,
W. E. Mercer,
M. B. Kastan,
K. W. Kohn,
S. J. Elledge,
K. W. Kinzler, and
B. Vogelstein.
1994.
WAF/CIP1 is induced in p53-mediated
G1 arrest and apoptosis.
Cancer Res.
54:
1169-1174
9. Steinman, R. A., B. Hoffman, A. Iro, C. Guillouf, D. A. Liebermann, and M. E. El-Houseini. 1994. Induction of p21 (WAF-1/CIP) during differentiation. Oncogene 9: 3389-3396 [Medline].
10.
Halevy, O.,
B. G. Novitch,
D. B. Spicer,
S. X. Skapek,
J. Rhee,
G. J. Hannon,
D. Beach, and
A. B. Lassar.
1995.
Correlation of terminal cell cycle
arrest of skeletal muscle with induction of p21 by myoD.
Science
267:
1018-1021
11. O'Brodovich, H. M., and R. B. Mellins. 1985. Unresolved neonatal acute lung injury. Am. Rev. Respir. Dis. 132: 694-709 [Medline].
12. Erickson, A. M., S. M. de la Monte, G. W. Moore, and G. Hutchins. 1987. The progression of morphologic changes in bronchopulmonary dysplasia. Am. J. Pathol. 127: 474-484 [Abstract].
13. Rush, M. G., and T. A. Hazinski. 1992. Current therapy of bronchopulmonary dysplasia. Clin. Perinatol. 19: 563-590 [Medline].
14. Frank, L.. 1991. Developmental aspects of experimental pulmonary oxygen toxicity. Free Rad. Biol. Med. 2: 463-494 .
15. Kotecha, S., A. Wangoo, M. Silverman, and R. J. Shaw. 1996. Increase in the concentration of transforming growth factor beta-1 in bronchoalveolar lavage fluid before development of prematurity. J. Pediatr. 128: 464-469 [Medline].
16. Quinlan, T., S. Spivack, and B. T. Mossman. 1994. Regulation of antioxidant enzymes in lung after oxidant injury. Environ. Health Perspect. 102: 79-87 .
17. Yoo, J.-H., S. C. Erzurum, J. G. Hay, P. Lemarchand, and R. G. Crystal. 1994. Vulnerability of the human airway epithelium to hyperoxia. J. Clin. Invest 93: 297-302 .
18.
Cazals, V.,
B. Mouhieddine,
B. Maitre,
Y. Le Bouc,
K. Chadelat,
J. S. Brody, and
A. Clements.
1994.
Insulin-like growth factors, their binding
proteins, and transforming growth factor- 1 in oxidant-arrested lung alveolar epithelial cells.
J. Biol. Chem.
209:
14111-14117
.
19.
Polyak, K.,
J. Kato,
M. J. Solomon,
C. J. Sherr, and
J. Massague.
1994.
p27Kip, a cyclin-Cdk inhibitor, links transforming growth factor- and contact inhibition to cell cycle arrest.
Genes Dev.
8:
9-22
20. Toyoshima, H., and T. Hunter. 1994. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78: 67-74 [Medline].
21. Lee, P. J., J. Alam, S. L. Sylvester, N. Inamdar, L. Otterbein, and A. M. Choi. 1996. Regulation of heme oxygenase-1 expression in vivo and in vitro in hyperoxic lung injury. Am. J. Respir. Cell Mol. Biol. 14: 556-568 [Abstract].
22.
Horowitz, S.,
N. Dafni,
D. L. Shapiro,
B. A. Holm,
R. H. Notter, and
D. J. Quible.
1989.
Hyperoxic exposure alters gene expression in the lung.
J. Biol. Chem.
264:
7092-7095
23. Nici, L., R. Dowin, M. Gilmore-Hebert, D. Jamieson, and D. H. Ingbar. 1991. Upregulation of rat lung Na-K-ATPase during hyperoxic injury. Am. J. Physiol. 262: L307-L314 .
24.
O'Brodovich, H.,
C. Canessa,
J. Ueda,
B. Rafii,
B. C. Rossier, and
J. Edelson.
1993.
Expression of the epithelial Na+ channel in the developing lung.
Am. J. Physiol.
265:
C491-C496
25. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Extraction, purification and analysis of messenger RNA from eukaryotic cell. In Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
26. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13 [Medline].
27. Murray, J. F. 1986. Postnatal growth and development of the lung. In The Normal Lung: The Basis for Diagnosis and Treatment of Pulmonary Disease, 2nd ed. W.B. Saunders Co. Philadelphia, PA.
28. Jacks, T., and R. A. Weinberg. 1996. Cell-cycle control and its watchman. Nature (London) 381: 643-644 [Medline].
29. Oren, M., and C. Prives. 1996. p53: upstream, downstream and off stream. Review of the 8th p53 workshop. Biochim. Biophys. Acta 1288: R13-R19 [Medline].
30. Gartel, A. L., M. S. Serfas, and A. L. Tyner. 1996. p21-negative regulator of the cell cycle. Proc. Soc. Exp. Biol. Med. 213: 138-149 [Medline].
31. Deng, C., P. Zhang, J. W. Harper, S. J. Elledge, and P. Leder. 1995. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82: 675-684 [Medline].
32. Waldman, T., C. Lengauer, K. W. Kinzler, and B. Vogelstein. 1996. Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature (London) 381: 713-716 [Medline].
33.
Imlay, J. A., and
S. Linn.
1988.
DNA damage and oxygen radical toxicity.
Science
240:
1302-1309
34. Crapo, J. D.. 1986. Morphologic changes in pulmonary oxygen toxicity. Annu. Rev. Physiol 48: 721-731 [Medline].
35. Graeber, T. G., C. Osmanian, T. Jacks, D. E. Housman, C. J. Koch, S. W. Lowe, and A. J. Giaccia. 1996. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors. Nature (London) 379: 88-91 [Medline].
36. Northway, W. H., R. C. Rosan, and D. Y. Porter. 1967. Pulmonary disease following respiratory therapy of hyaline membrane disease. N. Engl. J. Med 276: 357-368 .
37. Wilson, W. L., M. Mullen, P. M. Olley, and M. Rabinovitch. 1985. Hyperoxia-induced pulmonary vascular and lung abnormalities in young rats and potential for recovery. Pediatr. Res. 19: 1059-1067 [Medline].
38. Randell, S. H., R. R. Mercer, and S. L. Young. 1990. Neonatal hyperoxia alters the pulmonary alveolar and capillary structure of 40-day-old rats. Amer. J. Pathol. 136: 1259-1266 [Abstract].
39. Shaffer, S. G., D. O'Neil, S. K. Bradt, and D. W. Thibeault. 1987. Chronic vascular pulmonary dysplasia associated with neonatal hyperoxia exposure in the rat. Ped. Res. 21: 14-20 [Medline].
40. Northway, W. H., R. Petriceks, and L. Shahinian. 1972. Quantitative aspects of oxygen toxicity in the newborn: inhibition of lung DNA synthesis in the mouse. Pediatrics 49: 67-72 .
41. Marchetti, A., C. Doglioni, M. Barbareschi, F. Buttitta, S. Pellegrini, G. Bertacca, A. Chella, G. Merlo, A. Angeletti, P. Dalla, Palma, and G. Bevilacqua. 1996. p21 RNA and protein expression in non-small cell lung carcinomas: evidence of p53-independent expression and association with tumoral differentiation. Oncogene 12: 1319-1324 [Medline].
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