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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 890-897, (2002)
© 2002 American Thoracic Society

Original Articles

Bleomycin Sensitivity of Mice Expressing Dominant-Negative p53 in the Lung Epithelium

Sushmita Ghosh, Tamra Mendoza, Luis A. Ortiz, Gary W. Hoyle, Cesar D. Fermin, Arnold R. Brody, Mitchell Friedman and Gilbert F. Morris

Program in Lung Biology, Section of Pulmonary Diseases, Critical Care and Environmental Medicine; and Department of Pathology, Tulane/Xavier Center for Bioenvironmental Research and Tulane Cancer Center, New Orleans, Louisiana

Correspondence and requests for reprints should be addressed to Gilbert F. Morris, Department of Pathology, SL-79, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70118. E-mail: gmorris2{at}tulane.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chemotherapeutic drug bleomycin causes DNA damage and apoptosis in the lungs of mice within hours of endotracheal instillation followed by inflammation and fibrosis weeks later. The p53 tumor suppressor protein mediates cellular responses to DNA damage, including induction of apoptosis, but the effects of p53 activation in the various cell types of the lung during bleomycin-induced pulmonary fibrosis remain unclear. We show here that a transgene with a dominant-negative mutant form of human p53 expressed from the surfactant protein C promoter sensitizes mice to bleomycin-induced lung injury. The bleomycin-exposed transgenic animals display more severe lung pathology with associated collagen deposition and more pronounced lung eosinophilia than simultaneously exposed nontransgenic littermates. These observations suggest that compromising p53 function in the alveolar epithelium impairs recovery of the lung from bleomycin-induced injury.

Key Words: p53 • bleomycin • pulmonary fibrosis • transgenic mice


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pulmonary fibrosis is a devastating disease that resists current therapies (1). Injury of the alveolar and bronchiolar epithelium followed by inflammation and consequent mesenchymal cell proliferation and deposition of extracellular matrix characterizes pulmonary fibrosis (2). The efficiency of regeneration of the epithelium is an important factor in the severity of the fibrogenic response to lung injury (2, 3). Endotracheal instillation of the antitumor agent bleomycin into mice, which causes DNA damage in the lung epithelium (4), provides a useful experimental model to study the pathogenesis of pulmonary fibrosis (reviewed in 5–7), although the utility of the bleomycin model may be limited to a subgroup of the human interstitial lung diseases (8). DNA damage in, and apoptosis of, bronchiolar and alveolar epithelial cells have been observed in fibrotic lesions of idiopathic pulmonary fibrosis, but not in normal lung parenchyma from the same patient (9).

DNA damage elevates cellular levels of the p53 tumor suppressor protein and activates a latent form of the protein through post-translational modification (10, 11 and references therein). Activated p53 prevents cell cycle progression of cells with damaged DNA through induction of growth arrest and DNA repair or apoptosis. These biologic effects of p53 are largely the result of the protein's function as a transcription factor. Wild-type p53 forms a tetramer and binds DNA in a sequence-specific manner to activate transcription of numerous target genes. Among the targets activated by wild-type p53: p21/WAF1 promotes cell cycle arrest (12, 13); bax (14), p53 apoptosis-inducing protein 1 (15), and/or redox regulatory proteins (16); and induce apoptosis and ribonucleotide reductase (17), GADD45 (18, 19), and proliferating cell nuclear antigen (20) potentiate DNA repair. Many oncogenic mutant forms of p53 lack the ability to bind DNA and activate transcription of these target genes. In addition to this loss of activity, some mutant p53 proteins can interact with the wild-type protein (21) and thereby inhibit p53 function in a dominant-negative manner (22, 23).

Endotracheal instillation of bleomycin activates p53 expression in the lung by 1-hour postexposure (24–26). The observation that p53 appears in fibrotic areas but not in normal lung tissue of idiopathic pulmonary fibrosis (9) suggests that p53 may participate in the pathogenesis of fibrotic lung disease. After bleomycin-induced injury and loss of the alveolar epithelium, alveolar type II cells proliferate and differentiate to regenerate the alveolar surface (3). The consequences of p53 activation, that is, growth arrest and DNA repair or apoptosis, would affect the proliferative capacity of alveolar type II cells and, hence, regeneration of the alveolar epithelium. In support of this view, bleomycin exposure of sensitive mice induces expression of the p53-regulated genes, p21, bax, proliferating cell nuclear antigen, and GADD45 at early times postexposure before the development of pulmonary fibrosis (24–27). Expression of p21 and GADD45 is relatively reduced in bleomycin-resistant mice lacking the ß6 integrin subunit (27).

To assess the significance of p53 activation in bleomycin-induced pulmonary fibrosis, experiments have been performed with p53 null mice (24, 28, 29). Relative to wild-type mice, bleomycin exposure of p53 null mice produces more DNA damage (24) and/or apoptosis and inflammation (29). However, the results with bleomycin-exposed p53 null mice have been variable, and the phenotype of the bleomycin-exposed p53 null animals is difficult to interpret because the null mutation may produce profibrogenic or antifibrogenic effects in a manner dependent on the cell type. To evaluate the p53-mediated response to DNA damage in the lung epithelium, mice that express a dominant-negative mutant form of human p53 from the surfactant C promoter (SPC-DNp53 transgenic mice [30]) were exposed to bleomycin by endotracheal instillation. Similar to previous findings with p53 null mice (29), the SPC-DNp53 transgenic mice display enhanced lung inflammation and scarring after exposure to bleomycin relative to simultaneously exposed nontransgenic littermates.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic Mice
A DNA construct was prepared with the human SPC promoter fused upstream and SV40 sequences downstream of a cDNA sequence encoding a dominant-negative mutant form (arginine to histidine change at position 175) of human p53 as previously described (30). Transgenic mice were generated by microinjection of the linear SPC-DNp53 fragment into fertilized B6SJLF2 mouse eggs as described (31). After selection (30), two SPC-DNp53 transgenic lines, 70-2 and 70-3, were propagated by breeding transgenic mice with B6SJLF1 hybrid mice (Jackson Laboratory, Bar Harbor, ME). The mice were housed under pathogen-free conditions in a modified barrier facility. SPC-DNp53 transgenic mice and nontransgenic littermates greater than 6 weeks old from both transgenic lines were used for experiments.

Bleomycin Administration
We exposed mice to bleomycin using a well-established procedure described previously (32). Bleomycin sulfate was obtained from Bristol-Meyers (Princeton, NJ). Sterile solutions of 15 U of bleomycin per ml of normal saline were stored in aliquots at -70°C. Immediately before use, the bleomycin was thawed on ice and administered to mice at a dose proportional to the animal's body weight, usually 4 U/kg. The mice were anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol (Sigma-Aldrich, St. Louis, MO), and the trachea was exposed by a midline incision in the neck. Bleomycin was administered in 0.05- to 0.06-ml aliquots via a needle inserted into the lumen of the trachea. In control experiments, mice received sterile saline via the same procedure. After exposure, the incisions were closed with surgical clips, and the mice were allowed to recover on a warm plate at 37°C, after which they were put back in their cages. Food and water were provided ad libitum. The animals were weighed periodically after exposure to monitor their health.

Tissue Preparation and Histopathology
At various times after exposure (usually 3 weeks), mice were anesthetized by intraperitoneal injection of tribromoethanol and were killed by exsanguination after cutting the descending aorta. The chest cavity was opened. The left bronchus was closed with a suture, and the left lung was removed and frozen in liquid nitrogen. The lobes of the right lung were inflated and fixed by endotracheal perfusion of 10% neutral buffered formalin (Sigma) at a constant pressure of 30 cm of water. After 20 minutes of perfusion, the trachea was closed with a suture; the cardiopulmonary tree was removed and placed in fresh formalin overnight at 4°C. The fixed lung tissue was rinsed in phosphate-buffered saline. The lobes were separated and placed in a cassette for automated paraffin embedding. The paraffin blocks were sectioned at 4- to 5-µm thickness. Sections were stained with hematoxylin and eosin, and the stained sections were digitized into a computer via a Polaroid PrintScan 35 and Meyer's Histopath attachment at 2,700 dpi. This method permitted image acquisition of the whole section with identification of fibrotic areas. Affected and unaffected areas of each image were objectively demarcated by tracing with the mouse on the screen within Image Pro from Media Cybernetics (Silver Spring, MD). After transfer of the area measurements to Microsoft Excel, the percentage of affected areas for each section was calculated.

Immunohistochemistry
Immunohistochemical detection of p53 expression was as described previously (33). Briefly, after xylene washes and rehydration, the slides were heated at greater than 90°C two times for 10 minutes each in 10 mM of sodium citrate, pH 6.0, to unmask the antigen. After blocking, the slides were incubated with a sheep polyclonal primary antibody to p53 (Oncogene Research Products, Boston, MA) diluted 1:500 in 0.1% gelatin and 1% bovine serum albumin in phosphate-buffered saline. A control section on each slide was processed similarly with normal sheep serum. The secondary antibody was a biotin-conjugated rabbit anti-sheep antibody (Oncogene Research) diluted 1:10,000. Visualization was achieved by streptavidin-conjugated horseradish peroxidase (1:2,000) with diaminobenzidine as the chromagen and hematoxylin as the counterstain.

Lung Hydroxyproline
The lung hydroxyproline content was measured by a modification of the colorimetric assay described previously (34, 35). Briefly, the left lobe of each mouse was hydrolyzed overnight at 110°C in 6 N HCl. The hydrolysate was neutralized with 6 N NaOH and was diluted. Color was developed using chloramine T, perchloric acid, and dimethylaminobenzaldehyde. The amount of hydroxyproline was determined by comparing the absorbance of the sample at 562 nm to a standard curve that was generated using known amounts of hydroxyproline.

Eosinophilia
Eosinophilia was assessed by counting the number of cells per section that were positive by immunostaining with an antibody specific to murine eosinophil major basic protein as previously described (36). Briefly, paraffin-embedded lung tissue sections were deparaffinized and hydrated. After inactivation of endogenous peroxidase by incubation in H2O2/methanol for 20 minutes at room temperature, the slides were immersed in 0.01% trypsin (containing 0.1% CaCl2 in Tris-HCl, pH 7.6) for 20 minutes at 37°C to retrieve the antigen. Nonspecific antibody binding was blocked by incubation of the slides in 5% normal goat serum before incubation at room temperature for 1 hour with the primary rabbit polyclonal antibody to murine eosinophil major basic protein (kindly provided by James Lee, Scottsdale, AZ) diluted at 1:2,000. Subsequently, bound primary antibody was detected with a biotinylated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:500 at room temperature for 30 minutes followed by a 30-minute incubation in streptavidin-horseradish peroxidase complex (Jackson ImmunoResearch Laboratories). Visualization was achieved with diaminobenzidine (Sigma) as the chromagen and hematoxylin as the counterstain. For negative control purposes, a second section on each slide was processed simultaneously with control serum at the same dilution. Eosinophils were identified by the positive brown stain and were quantified at high power (x400) with the observer unaware of the section identity.

RNA Analyses
Total lung RNA was prepared by the guanidinium thiocyanate–phenol method as previously described from samples frozen in liquid nitrogen (37). RNase protection assays were performed as described previously (38). The radiolabeled RNA probe for mouse p21/WAF1 corresponded to a PstI–StuI fragment of the mouse p21 cDNA (kindly provided by Bert Vogelstein, Baltimore, MD), which was subcloned into Bluescript II KS+ (Stratagene, La Jolla, CA). The antisense probe for mouse cyclophilin mRNA was prepared by T7 transcription of a DNA fragment purchased from Ambion (Austin, TX). The radiolabeled RNA probes were gel purified before use.

Statistics
Data are presented as the mean for the number of animals in the group, indicated in parentheses. The statistical significance of differences between various groups of mice was determined by unpaired t test or Mann-Whitney test with two-tailed p values using InStat version 1.14.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To compromise p53-mediated effects in the lung epithelium during bleomycin-induced fibrogenesis, mice were prepared that harbor a transgene with the human SPC directing expression of a dominant negative mutant form of human p53 (arginine to histidine change at codon 175). Progeny of two transgenic founders, 70-2 and 70-3, displayed higher levels of mutant p53 on immunoblots of total lung extracts than three other transgenic lines analyzed (30). In both the 70-2 and 70-3 SPC-DNp53 transgenic lines, the pattern of mutant p53 expression in the lung appeared to be restricted to alveolar type II cells and epithelial cells of the small airways (Figure 1) .



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  		Figure 1. Detection of p53 expression in SPC-DNp53 transgenic mice by immunohistochemistry. Histologic sections from formalin fixed, paraffin-embedded lung tissue were immunostained with an antibody specific for p53. A brown nuclear stain marked p53-positive cells. The nuclei of negative cells appeared blue with the hematoxylin counterstain. (A) p53-Specific immunostaining in the parenchyma of a mouse from line 70-2 (x100). Note that the interspersed pattern of immunostaining in the alveoli is consistent histologically with the pattern expected for type II pneumocytes. (B) Parenchyma of a nontransgenic animal processed for p53 immunostaining simultaneously with that shown in A.

 
To evaluate the response of the SPC-DNp53 mice to bleomycin, five transgenic mice (two from line 70-2 and three from line 70-3) and five nontransgenic littermates were instilled endotracheally with bleomycin at 5 U/kg body weight. None of the five transgenic animals survived more than 2 weeks after administration of the drug. With one exception, all of the bleomycin-treated nontransgenic littermates survived to the 3-week time point.

In a separate experiment, bleomycin was administered endotracheally to three groups of mice from line 70-2 (10 animals: five transgenic and five nontransgenic in each group) at decreasing incremental doses of 0.5 log (4 U, 1.26 U, and 0.4 U/kg), and the mice were sacrificed 3 weeks later. On average, SPC-DNp53 mice and nontransgenic littermates in the high-dose group (n = 8) lost significantly (p < 0.0001) more body weight (an indicator of morbidity) than mice in the low-dose group (n = 10), 27% versus 0%, respectively (Table 1) . Moreover, a comparison of weight loss in all three exposure groups revealed that transgenic mice (n = 12) exposed to bleomycin lost significantly (p = 0.006) more of their initial body weight than nontransgenic littermates (n = 13), 16% versus 3%, respectively (Table 1). Assessment of lung sections prepared from the bleomycin-treated mice indicated that increasing doses of bleomycin led to increasing histopathologic changes in both SPC-DNp53 transgenic mice and nontransgenic littermates (Table 1). The enhanced lung pathology and weight loss in SPC-DNp53 transgenic mice administered bleomycin agree with the results in the first experiment, indicating that SPC-DNp53 mice display shortened survival after bleomycin treatment. Fibrogenesis and weight loss appeared more obvious in two transgenic female mice in the high-dose group (4 U/kg) relative to the two nontransgenic female littermates (data not shown). Therefore, additional experiments to assess bleomycin-induced fibrogenesis in SPC-DNp53 mice focused on transgenic and nontransgenic females.


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  		TABLE 1. Response of surfactant protein c–dominant-negative mutant form of human P53 transgenic mice and nontransgenic littermates to decreasing doses of bleomycin

 
To establish better the bleomycin sensitivity of the female SPC-DNp53 transgenic mice, the drug was endotracheally administered at 4 U/kg to female mice in the 70-2 line. For control purposes, transgenic and nontransgenic females in the 70-2 line were administered saline by endotracheal instillation. On Day 21 after treatment, the lung morphology of the mice was assessed. Both transgenic mice and nontransgenic littermates exposed to saline showed normal lung morphology (data not shown). Lung sections of nontransgenic animals administered bleomycin exhibited inflammation and subpleural fibrosis but little intra-alveolar fibrosis (Figure 2A) . In transgenic animals, bleomycin-induced lesions were scattered about the lung parenchyma with subpleural fibrosis, but the airways were generally unaffected (Figure 2B). Developing lesions displayed organization of extracellular matrix into scars that filled many alveolar spaces and became contiguous in more advanced regions of the injured lung (Figure 2B, arrows). There were numerous mesenchymal cells in the organizing matrix and great variability in the numbers of inflammatory cells associated with the scarring process. Alveolar walls adjacent to developing lesions exhibited varying degrees of interstitial thickening. Some of the transgenic mice had advanced disease with contracted scars and enlarged anomalous spaces characteristic of honeycombing (Figure 2B, arrowheads). Morphometric analyses by light microscopy at low power demonstrated more lung involvement in the bleomycin-treated SPC-DNp53 transgenic animals than in the simultaneously exposed nontransgenic littermates (Figure 3) . These observations suggested that SPC-DNp53 transgenic mice were more sensitive to bleomycin-induced injury and consequent fibrogenesis than nontransgenic littermates.



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  		Figure 2. Lung histology of bleomycin-exposed SPC-DNp53 transgenic mice and nontransgenic littermates. Female SPC-DNp53 transgenic mice and nontransgenic female littermates in the 70-2 line were instilled intratracheally with saline or 4 U/kg bleomycin. Twenty-one days later, the mice were killed, and the right lung was fixed by intratracheal perfusion of 10% neutral buffered formalin. Histologic sections were prepared from the paraffin-embedded lung tissue and the sections were stained with hematoxylin and eosin to reveal the anatomic details. The panels show areas of more severe pathology from hematoxylin and eosin–stained lung sections of a bleomcyin-exposed nontransgenic mouse (A) and a SPC-DNp53 transgenic littermate (B). The arrows mark areas of fibrosis with enlarged spaces indicated by arrowheads.

 


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  		Figure 3. Enhanced histopathologic changes in bleomycin-exposed SPC-DNp53 transgenic mice relative to nontransgenic littermates. Hematoxilyn and eosin–stained sections from bleomycin-exposed mice (see Figure 2) were assessed for bleomycin-induced alterations with the observer unaware of the sample identity. Morphometric analyses to assess the degree of lung inflammation and scarring were performed on low-power images of each section by computer-assisted area measurements. The graph shows the mean percentage abnormal area in the lung section with SD in transgenic mice and nontransgenic littermates administered 4 U/kg bleomycin. The number in parentheses indicates the number of animals in each group. There were significantly more histopathologic alterations in bleomycin-exposed SPC-DNp53 transgenic mice than in simultaneously exposed nontransgenic littermates.

 
Homogenates of the left lung of both transgenic and nontransgenic mice in line 70-2 were evaluated for hydroxyproline content to relate the increased fibrogenesis in the bleomycin-treated SPC-DNp53 transgenic mice with a biochemical measurement of lung scarring. Bleomycin exposure elevated relative lung hydroxyproline levels in both SPC-DNp53 transgenic mice and nontransgenic littermates as compared with saline-treated control animals (Figure 4) . However, the amount of lung hydroxyproline was significantly higher in bleomycin-exposed SPC-DNp53 transgenic mice relative to nontransgenic littermates. These results indicated that SPC-DNp53 transgenic mice in line 70-2 developed more injury after endotracheal administration of bleomycin than nontransgenic littermates. To confirm that this bleomycin sensitivity was due to pulmonary expression of mutant p53 and not some other genetic trait of this inbred line, the bleomycin sensitivity of the 70-3 line of SPC-DNp53 transgenic mice was also assessed in separate studies. Both SPC-DNp53 transgenic mice and nontransgenic littermates in the 70-3 line treated with bleomycin manifested increases in lung hydroxyproline relative to saline-treated control animals (Figure 4). Again, the bleomycin-exposed transgenic mice displayed more lung hydroxyproline than the simultaneously treated nontransgenic littermates. These observations substantiate that mutant p53 expression in the lung epithelium enhances bleomycin sensitivity. Thus, bleomycin sensitivity, as determined by survival, weight loss, lung histopathologic changes, and lung hydroxyproline content, appeared greater in bleomycin-treated SPC-DNp53 transgenic mice than simultaneously exposed nontransgenic littermates.



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  		Figure 4. Elevated lung hydroxyproline levels in SPC-DNp53 transgenic mice relative to nontransgenic littermates after bleomycin exposure. Upon death of saline or bleomycin-exposed SPC-DNp53 mice (both line 70-2 and line 70-3) on Day 21 after exposure, the left lung was removed and quickly frozen. After acid hydrolysis of the frozen lung tissue, levels of hydroxproline were determined by a colorimetric assay. The graph depicts the average hydroxyproline content (± SEM) of the left lung (µg) per gram body weight for the indicated number (parentheses) transgenic and nontransgenic mice treated with saline (white) or bleomycin (line 70-2, black bars; line 70-3, hatched bars) from several different exposures. The statistical significance of differences between bleomycin-exposed transgenic mice and nontransgenic littermates is indicated.

 
Prior studies showed that elevated numbers of lung eosinophils correlated with a poor prognosis in patients with pulmonary fibrosis (39–41). Similarly, in bleomycin-exposed mice, increased eosinophilic inflammation correlated with the severity of fibrosis (42, 43), although eosinophils are not required for fibrosis development (44). Lung sections from bleomycin- or saline-exposed SPC-DNp53 transgenic mice and nontransgenic littermates were assessed with an immunostain specific for eosinophils (Figure 5) . Few or no eosinophils were detected in lung sections from SPC-DNp53 transgenic mice or nontransgenic littermates exposed to saline (Figures 5A and 5B, respectively). Lung eosinophils were most obvious in SPC-DNp53 mice exposed to bleomycin (Figure 5C) relative to simultaneously exposed nontransgenic littermates (Figure 5D). The extent of lung eosinophilia correlated with the severity of histopathologic alterations displayed in Figure 2. Thus, the SPC-DNp53 transgenic mice with more pronounced bleomycin-induced histopathologic changes also displayed elevated numbers of lung eosinophils relative to simultaneously exposed nontransgenic littermates (Figure 6) .



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  		Figure 5. Detection of eosinophils by immunostaining. Eosinophils were detected in paraffin-embedded lung sections by immunostaining with a rabbit polyclonal antibody to murine eosinophil major basic protein. Brown cytoplasmic staining and irregular nuclei identified eosinophils, whereas other cell types stained only with the blue nuclear counterstain (see inset of C). Areas with some inflammatory cells are shown for the indicated mouse/treatment group. (A) SPC-DNp53 transgenic mouse exposed to saline. (B) Nontransgenic littermate exposed to saline. (C) SPC-DNp53 transgenic mouse exposed to bleomycin (arrowheads denote eosinophils, see inset). (D) Nontransgenic littermates exposed to bleomycin.

 


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  		Figure 6. More prominent eosinophilia in bleomycin-exposed SPC-DNp53 transgenic mice relative to nontransgenic littermates. The immunostained lung sections (see Figure 5) from transgenic and nontransgenic mice were assessed for eosinophils with the observer unaware of the sample identity. The graph displays the mean number (± SEM) of lung eosinophils per section for bleomycin-exposed (black bars) or saline-exposed (white bars) mice of the indicated genotype. The number of lung eosinophils in bleomycin-exposed SPC-DNp53 transgenic mice was significantly different (p < 0.016, Mann–Whitney test) from that of simultaneously treated nontransgenic littermates.

 
Previous experiments suggested that p53 attenuated bleomycin-induced lung injury by inhibiting apoptosis in the lung (29). It was proposed that p53-mediated activation of p21/WAF1, a cellular target of activated p53, may play an important role in promoting lung cell survival in the bleomycin model (29). Consistent with this postulate, expression of p21/WAF1 prevents lung injury induced by hyperoxia (45). To determine the effects of bleomycin instillation on levels of p21/WAF1 in the lungs of SPC-DNp53 transgenic mice and nontransgenic littermates, total lung RNA was isolated 18 hours after administration of the drug or saline. Equal amounts of RNA from each mouse were probed for p21/WAF1 and cyclophilin mRNAs (Figure 7A) , the latter to normalize for RNA recovery and integrity in each assay. The results of two separate experiments are summarized in Figure 7B. Relative to saline-exposed nontransgenic mice, the basal level of p21/WAF1 mRNA was significantly lower, approximately 30%, in saline-exposed SPC-DNp53 transgenic mice. This observation agrees with previous findings that basal expression of p21/WAF1 mRNA in the lung is p53 dependent (46). Bleomycin exposure significantly increased expression of p21/WAF1 mRNA in the lungs of both SPC-DNp53 mice and nontransgenic littermates, but the levels in the transgenic animals remained approximately 70% of that observed in simultaneously exposed nontransgenic littermates (Figure 7B). These data are consistent with compromised p53 function in the lung and correlate with the observation that activation of p21 expression in the lungs of bleomycin-exposed mice is p53 dependent (24). However, the difference in p21/WAF1 levels in bleomycin-exposed SPC-DNp53 mice and nontransgenic littermates is only marginal, and more direct testing of the function of p21/WAF1 in the bleomycin model will be necessary to determine any possible protective effect.




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  		Figure 7. Reduced p21/WAF1 mRNA levels in the lungs of SPC-DNp53 mice. (A) SPC-DNp53 mice and nontransgenic littermates were exposed to saline or 4 U/kg bleomycin by endotracheal instillation. At 18 hours after exposure, the animals were killed, the lungs were removed and quickly frozen in liquid nitrogen. Total lung RNA prepared from the frozen lung samples and equal amounts of RNA were assayed by RNase protection assay with antisense RNA probes specific for p21/WAF1 mRNA and cyclophilin mRNA. The protection products were separated in a denaturing polyacrylamide gel. The dried gel was exposed overnight to X-ray film, and a scan of the autoradiograph is shown. (B) The protection products from A and a similar assay of animals from a separate bleomycin exposure were quantified by phosphoimage analyses. The signal for p21/WAF1 in each sample was normalized to the signal for cyclophilin. The graph shows the relative p21 mRNA levels in total lung RNA from saline or bleomycin-exposed SPC-DNp53 transgenic mice (black bars) and nontransgenic littermates (white bars) with the value in saline-exposed nontransgenic mice arbitrarily set to one. The results are the average (± SEM) of determinations from the indicated number of animals in parentheses. The significance of differences is indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our findings indicate that mutant p53 expression in the lung epithelium sensitizes B6SJL hybrid mice to bleomycin-induced lung injury. Endotracheal administration of bleomycin to SPC-DNp53 transgenic mice shortens survival and produces more lung pathology, greater weight loss, and increased lung hydroxyproline and lung eosinophilia relative to that observed in nontransgenic littermates. Similar findings in two independent lines of SPC-DNp53 transgenic mice demonstrate that the enhanced bleomycin sensitivity is attributed to mutant p53 expression in the lung epithelium and not some other characteristic of these mice.

Bleomycin promotes cell killing by binding to DNA and causing single- and double-stranded breaks with oxygen and metal ions as cofactors in the cleavage reaction (47, 48). Because the half-life of bleomycin in the lungs of mice is short (less than 32 minutes [49]), the maximal DNA damage from the drug is achieved at early times after exposure and targets primarily the lung epithelium. An enhanced rate of DNA repair in the lung may correlate with resistance to bleomycin-induced lung disease (4). The bleomycin sensitivity of the SPC-DNp53 mice compares favorably with cell culture results, indicating that p53 promotes DNA repair after bleomycin-induced DNA damage (50). Therefore, the ability of dominant-negative p53 to compromise the wild-type p53-mediated response to DNA damage and thereby delay DNA repair could account for the enhanced fibrogenesis in the bleomycin-exposed SPC-DNp53 mice. It may be essential to the survival of a lung epithelial cell with DNA damage to resist proliferative signals. The reduced levels of p21 mRNA in bleomycin-exposed SPC-DNp53 transgenic mice suggest that growth arrest may be impaired in the injured epithelium of these animals. This scenario would be consistent with previous observations indicating that p53 expression protects the lung from bleomycin injury by inhibiting apoptosis (29). Coincidentally, pharmacologic inhibition of epithelial apoptosis abrogates fibrosis in both rat (51) and mouse (52) bleomycin models.

The data presented here indicate that compromising p53 function in the lung epithelium increases inflammation and fibrosis in mice exposed to bleomycin. Similarly, bleomycin-exposed p53 null mice develop extensive and persistent DNA damage/apoptosis (24, 29) with more severe inflammation (29). In contrast to the findings presented here, deposition of collagen and the histopathologic changes associated with fibrosis appear to be similar in bleomycin-exposed wild-type and p53 null mice administered bleomycin by subcutaneous (28) or tail vein (24) injection. However, bleomycin administration by endotracheal instillation promotes enhanced fibrogenesis in p53 null mice relative to similarly treated wild-type mice (29). Thus, the route of bleomycin administration may affect the relative fibrotic response in wild-type and p53 null mice. The enhanced fibrosis in bleomycin-exposed SPC-DNp53 mice results from the absence of wild-type p53 function in only epithelial cells and despite the presence of wild-type p53 in other cell types. The dependence of murine fibroblast proliferation on p53 status in response to transforming growth factor-ß (53) is consistent with cell type-specific consequences of the p53 null mutation. Moreover, p53-mediated stimulation of thrombospondin (54) may be essential for clearance of apoptotic debris by phagocytic cells. The SPC-DNp53 transgenic mice provide the means of evaluating p53 function in the lung epithelium while maintaining these and additional p53-dependent activities in other cells of the lung. An alternate scenario to explain the enhanced sensitivity of SPC-DNp53 mice is that the mutant p53 protein possesses "gain-of-function" activities (55), such as forming heterotetramers with and compromising the activity of other p53 family members, p63 and p73 (56, 57). These other members of the p53 family of proteins may provide compensating activities in the pulmonary epithelium. In apparent contradiction of this hypothesis, activation of a number of p53-regulated genes in the lungs of mice by ionizing radiation or by bleomycin is abrogated in p53 null animals (24, 46).

The increased eosinophilic inflammation that accompanies the more pronounced lung pathology of the bleomycin-exposed SPC-DNp53 transgenic mice (Figure 6) may account, at least in part, for the increased disease severity. Eosinophils secrete fibrogenic mediators, and lung eosinophilia correlates with a poor prognosis for patients with pulmonary fibrosis (42, 58, 59 and references therein). In agreement with these findings, inhibition of the activity of tumor necrosis factor-{alpha} or interleukin-5 in a mouse model of bleomycin-induced fibrosis diminishes the influx of eosinophils into the lung (42, 43) and decreases the extent of pulmonary fibrosis (28, 42, 43, 60, 61). A possible role for p53 in suppression of bleomycin-induced inflammation is consistent with the recent observation that p53 may be involved in suppression of chronic inflammation in a mouse model of rheumatoid arthritis (62).

The mechanism whereby mutant p53 expression in the lung epithelium elevates the numbers of pulmonary eosinophils 3 weeks after exposure to bleomycin remains unclear. Unexposed SPC-DNp53 mice possess a normal lung morphology and bronchalveolar lavage fluid from these untreated animals does not contain elevated numbers of eosinophils (A. Nelson, personal communication). Therefore, the increased eosinophilia in bleomycin-exposed SPC-DNp53 mice originates from an altered injury response of the lung epithelium rendered by mutant p53 expression in these cells. As mentioned previously here, both tumor necrosis factor-{alpha} and interleukin-5 are associated with eosinophilia and fibrogenesis in the bleomycin model (42, 43). Although a link between interleukin-5 and p53 is lacking in the literature, interactions between the functions of p53 and tumor necrosis factor-{alpha} are described (63–65), but the significance of these interactions in bleomycin-induced lung fibrosis remains unclear. Immunostaining of lung sections from bleomycin-exposed SPC-DNp53 mice for tumor necrosis factor-{alpha} did not reveal significant differences with that in nontransgenic littermates (data not shown). Expression of granulocyte-macrophage colony-stimulating factor from a recombinant adenovirus promotes pulmonary eosinophilia and fibrosis (66). In accord with this observation, exposure of a lung epithelial cell line to bleomycin stimulates the release of eosinophil chemotactic activity, which can be partially neutralized by an antibody to granulocyte-macrophage colony-stimulating factor (67). This information taken together with the observation that wild-type p53 represses transcriptional activation of the granulocyte-macrophage colony-stimulating factor gene in some instances (68) suggests a granulocyte-macrophage colony-stimulating factor-dependent mechanism through which mutant p53 promotes eosinophilia and fibrogenesis. However, granulocyte-macrophage colony-stimulating factor appears to ameliorate bleomycin-induced fibrosis in both rats and mice, despite dramatically different regulation of the cytokine's expression after bleomycin exposure in these two species (69, 70). Additional experiments will be required to determine whether factors that promote chemotaxis or survival account for the pulmonary eosinophilia in the SPC-DNp53 mice.

A potential complication in the use of genetically altered mice is that compensatory changes occur during development that prevents functional analyses of the genetic alteration. p53 is expressed at high levels during mouse early embryonic development (71), and a number of reports indicate a role for p53 during embryogenesis (72). In particular, a subset of female p53 null mice dies in utero because of the development of exencephaly (73). Thus, in p53 null mice, there is evidence of selective pressure and the possibility that surviving female mice develop compensating alterations. However, expression of the mutant p53 transgene in SPC-DNp53 mice is limited to the lung, and the SPC promoter does not become active until embryonic Day 10 (74), by which time wild-type p53 levels (71) and activity (75, 76) have largely declined. Because there is no evidence of abnormal lung development in the SPC-DNp53 mice, there does not appear to be selective pressure for compensatory changes. The genetic alteration in SPC-DNp53 mice impinges primarily on the stress response in the lung. In the absence of stress, strong selective pressure for compensatory changes would not occur during lung development. Moreover, the SPC-DNp53 transgenic mice differ from wild-type mice in the response to bleomycin. If compensatory changes occur in these animals, they are not sufficient to provide the wild-type response to bleomycin.

In summary, our results show that SPC-DNp53 mice in the B6SJL hybrid background are more sensitive to bleomycin injury than nontransgenic littermates. In addition to p53, other factors that respond to genotoxic stress are likely to be important mediators of the response to bleomycin-induced acute alveolar injury. The interplay between the functions of p53 and other mediators of the cellular response to genotoxic stress will be an important consideration in elucidation of the fibrogenic response to lung injury.


   Acknowledgments
 
The authors thank Alona Zhuravel for technical assistance with morphometric analyses, which were performed at the Centralized Tulane Imaging Center, New Orleans, LA.


   FOOTNOTES
 
Supported by grant NIEHS R29 ES07856 (G.F.M.), Louisiana Cancer and Lung Trust Fund Board, LCLTFB-98–01–01 (G.F.M.), Department of Defense–Tulane/Xavier Center for Bioenvironmental Research, DSWA 01–97–0028 (G.F.M.); and the Wetmore Foundation (M.F.); and matching funds from the Tulane Cancer Center (S.G.)

Received in original form September 24, 2001; accepted in final form May 30, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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