Abstract
Bleomycin damages DNA and causes lung injury and fibrosis. To determine whether bleomycin is associated with the appearance of DNA damage–inducible proteins, C3H mice received either 0.4 mg bleomycin or normal saline intratracheally and were killed 1 to 14 d later. The lungs were examined for expression of p53, p21WAF1/PiCl, and proliferating cell nuclear antigen (PCNA) using immunohistochemistry and Western blotting. p53-positive cells first appeared at 5 d after treatment and peaked at 7 d; PCNA-positive cells appeared at 1 d after treatment and peaked at 7 d; and p21-positive cells appeared at 5 d and peaked at 9 d. Western blot analysis confirmed that bleomycin upregulated the DNA damage–inducible proteins in a similar fashion. This is the first evidence that bleomycin causes a p53-dependent response associated with acute injury in the lung.
Bleomycin, a potent cancer chemotherapeutic agent, causes fibrogenic lung disease in rats and mice similar to that observed in human subjects (1, 2). Bleomycin is typically administered to animals by either intraperitoneal (3, 4) or intratracheal (5) instillation as a model of lung fibrosis. Bleomycin causes acute lung injury before the onset of fibrosis, and this injury is associated with a proliferative response by lung cells as the injury-repair process evolves (6-8).
Bleomycin affects many cellular pathways, but the cytotoxic effect of bleomycin is generally believed to be related to its ability to bind and cleave DNA (9, 10). A number of reports indicate that bleomycin generates reactive oxygen species in vitro (11-13), and one report confirmed that activated oxygen is generated in the lungs of rodents after bleomycin treatment in vivo (14). Consistent with the production of oxygen-free radicals, bleomycin also induces antioxidant enzymes in the lung (15). Release of reactive oxygen radicals activates expression of cytokines (16) and may induce chromosomal aberrations, DNA damage, and DNA strand breaks (17-19). Bleomycin-mediated injury may prevent progression of the cell cycle with initiation of DNA repair. The DNA damage in the lung appears necessary for the development of pulmonary fibrosis (20).
The p53 tumor suppressor protein is a DNA damage– inducible protein that may mutate in cancer cells (21, 22). Signals from damaged DNA can activate a p53-dependent transduction pathway that includes G1/S and G2/M arrest and repair of DNA damage (23). An important function of p53 is to protect the integrity of the genome (21, 22). If bleomycin damages DNA in the lung, it is possible that expression of a cascade of genes, including the p53 gene, will occur.
Cells with elevated levels of p53 will typically arrest in the G1 phase of the cell cycle and will either institute DNA repair or undergo apoptosis (21-24). One mechanism whereby p53 inhibits cell cycle progression is by transcriptionally activating expression of the gene for p21WAF1/PiC1, a family of small proteins that negatively regulate the cell cycle. The gene products of p21WAF1/PiC1 act as inhibitors of both proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinases (25-27). PCNA is an essential DNA replication and repair protein that increases significantly in proliferating cells (28-32). Consistent with its role in DNA repair, PCNA is induced concomitantly with the increase in p53 levels (33). An understanding of bleomycin-induced p53-mediated upregulation of p21 and PCNA may help explain the mechanism of programmed cell death in bleomycin pulmonary toxicity.
Using the intratracheal administration of bleomycin as a model of pulmonary fibrosis (8), we demonstrated that a single intratracheal administration of bleomycin induced expression of DNA repair or growth arrest proteins within 3 to 5 d. The expression of DNA damage–inducible proteins in bleomycin-induced pulmonary injury suggests that DNA damage and its sequelae may be important harbingers of lung-cell injury and lung fibrosis in this disorder.
Pathogen-free, C3H mice (Harlan Sprague Dawley, Inc., Indianapolis, IN), 5 to 6 wk old and weighing 18 to 22 g, were given 0.4 mg bleomycin (Bristol-Myers Squibb Co., Princeton, NJ) intratracheally in 50 μl saline; control mice received equal amounts of saline alone. At different time intervals (1 to 14 d), five mice per group were anesthetized by intraperitoneal injection using 0.1 ml of 100 mg ketamine hydrochloride/ml and killed by exsanguination. Neutral 4% paraformaldehyde in phosphate buffer, pH 7.4, was instilled in the lungs at a pressure of 22 cm H2O2 for 60 min (34). The lungs were surgically removed, the trachea was clamped, and the lungs were placed in fresh fixative for 24 h at 4°C. After fixation, the lung tissue was embedded in paraffin, and 5 μm sections were cut and affixed to positively charged slides for routine hematoxylin and eosin staining.
Biochemical evidence of injury and fibrosis was confirmed by a lactate dehydrogenase (LDH) of bronchoalveolar lavage (BAL) and by hydroxyproline determination of lung tissue, respectively (35, 36). LDH, a commonly used marker of cytotoxicity, is determined using a kinetic reaction in microtiter well plates. To cell-free BAL fluid (40 μl) in each well, 250 μl of Tris buffer/nicotinamide adenine dinucleotide, reduced/pyruvate reagent was added. The change in optical density at 340 nm over time was monitored in a TMAX microplate reader (Molecular Devices Corp., Menlo Park, CA) using SOFTmax software.
Hydroxyproline measurements were performed as an index of lung collagen content (37). Briefly, the excised lungs were homogenized, lyophilized, and assayed using 1.4% chloramine T, 10% n-propanol, and 0.05 M sodium acetate (pH 6.0) for 20 min, followed by 1 ml Erlich's solution for a 15-min incubation at 65°C. Absorbances were measured at 550 nm. Hydroxyproline in each sample was determined from a standard curve and expressed as micrograms of hydroxyproline per total lung.
Immunohistochemical staining was performed as previously described (38). In brief, deparaffinized tissue sections were preincubated in 0.3% hydrogen peroxide in methanol for 20 min to inhibit endogenous peroxidase activity. After washing with phosphate-buffered saline (PBS), nonspecific antibody binding was blocked by a 30-min incubation in 2.5% fish gelatin–5% normal goat serum in 1% bovine serum albumin (BSA)–PBS (pH 7.4). The slides were incubated at room temperature with three different antibodies for the immunohistochemical recognition of p53, p21 (Oncogene Sciences, Cambridge, MA), and PCNA proteins (DAKO Corp., Carpinteria, CA). The sections were incubated with biotinylated goat antimouse immunoglobulin (Ig) G (Jackson Immunoresearch, West Grove, PA), diluted 1:4,000 in 0.1% gelatin–1% BSA in PBS for 1 h, washed three times for 5 min, and incubated for 1 h in streptavidin–horseradish peroxidase (HRP) with a dilution of 1:2,000 (Jackson Immunoresearch). Peroxidase activity was visualized after a 10-min incubation in 50 ml of 0.05 M Tris-HCl buffer (pH 7.6), containing 10 mg diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and 100 μl 3% hydrogen peroxidase. The slides were counterstained by hematoxylin. One specimen on each slide was incubated with an equal titer of mouse IgG as a negative control for p53, p21, and PCNA.
Expression of DNA damage–inducible proteins was identified by standard avidin-biotin complex immunostaining technique (39). Deparaffinized lung sections were boiled with 6 M urea twice for 30 min at 100°C before blocking the nonspecific binding sites with normal goat serum. p53 immunostaining was performed using sheep polyclonal anti-p53 (Ab-7) antibody (Oncogene Sciences). p21 and PCNA immunostaining were performed using mouse monoclonal Ab-4–anti-p21 antibody (Oncogene Sciences) and mouse monoclonal PC-10 anti-PCNA antibody (DAKO Corp.), respectively. To enhance the antigen retrieval, lung sections with 100 mM citrate buffer (pH 7.0) were microwaved at 100°C twice for 10 min, followed by the same immunostaining procedure described earlier. Positive immunostaining with p53, p21, and PCNA was determined counting a minimum of 200 cells/ high-power field. The percentage of positively stained cells was determined for each section of lung tissue at each time point, and the values were expressed as mean ± standard deviation (SD).
Double immunohistochemical staining was performed on paraffin-embedded lung tissues by avidin-biotin complex and alkaline phosphatase-Histomark red report reagent (KPL, Inc., Gaithersburg, MD). The lung sections were first immunostained for DNA damage–inducible proteins (p53, p21, and PCNA) by a HRP method previously described (39) and then immunostained for the detection of type II alveolar epithelial cells using anti–pro-surfactant protein (SP) C antibody (R68514; gift of Dr. Jeffrey Whitsett, Children's Hospital Medical Center, Cincinnati, OH).
Lung-tissue slices were suspended in 500 μl of ice-cold PBS (pH 7.2) containing 1 μM pepstatin A, bestatin, leupeptin, and phenylmethylsulfonyl fluoride. The protein content of the homogenate was analyzed using a BCA protein assay (Pierce Chemical Co., Rockford, IL), and 100 μg of each lung homogenate sample was mixed with sample buffer (2% sodium dodecyl sulfate [SDS], 10% glycerol, and 10 mM dithiothreitol in 62 mM Tris-HCl [pH 6.8]) and heated to 95°C for 5 min. Equivalent amounts of protein in each lane were run on a SDS-polyacrylamide gel (10% for p53 and 12% for p21) at 50 mV for approximately 6 h and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham, Arlington Heights, IL) by using a wet electroblotter (Bio-Rad Laboratories, Hercules, CA). Membranes were probed for p53, p21, and PCNA by immunoblot analysis using a specific monoclonal primary and [125I]-labeled mouse IgG secondary antibody. Specific antibodies, as described earlier for immunohistochemistry staining, were used for the immunoblot detection of each protein. p53 immunoblotting was performed using a 6-h incubation with Ab-4–anti-p53 mouse monoclonal primary antibody, followed by 30-min washes and incubation with [125I]-labeled mouse IgG secondary antibody (ICN, Costa Mesa, CA) for 2 h. The membrane was washed six times with Tris-buffered saline containing Tween-20 (TBST) for 30 min, dried at room temperature, and developed using X-ray film. p21 immunoblotting was performed with mouse monoclonal Ab-4–anti-p21 monoclonal primary antibody using the same procedure as described earlier for p53. After protein transfer to nitrocellulose membrane, PCNA immunoblotting was performed by incubating the membrane for 3 h with mouse monoclonal PC-10 anti-PCNA primary antibody, followed by 2 h of six 20-min washes with TBST. The nitrocellulose membrane was further incubated with [125I]-labeled mouse IgG secondary antibody for 2 h, followed by several 20- to 30-min washes with TBST. The membrane was dried at room temperature and then developed with X-ray film.
Statistical significance comparing bleomycin treatments with control was determined by two-way analysis of variance (40).
Our histopathologic findings of bleomycin injury are consistent with the findings of other investigators (3-8). In brief, on Day 1 after bleomycin treatment, alveolar epithelial cells were hyperplastic and pleomorphic with condensed nuclear material. At 3 d, the alveolar walls revealed evidence of thickening with mild infiltration of lymphocytes, which continued to increase 7 d after treatment. The proliferation of fibroblasts and collapse of alveolar spaces at 7 d after treatment were accompanied by a significant elevation of LDH in the lung lavage and hydroxyproline content of the lung tissue (data not shown). No evidence of cell proliferation and fibrosis was observed in any of the saline-treated control mice.
The lung sections of each group at the various time points were processed for immunohistochemical evaluation of p53, p21, and PCNA expression. The lung tissue of saline-treated animals at each time point exhibited a normal histology with few alveolar macrophages and interstitial cells. The saline-treated lungs exhibited no evidence of p53- or p21-positive cells (Figures 1A and 2A), whereas a typical background of PCNA staining (< 1%) in a variety of epithelial and mesenchymal cells was found (as discussed later with Figure 3A).
Fig. 1. Time-dependent expression of wild-type p53 in the lung sections of mice treated with intratracheal bleomycin (0.4 mg). Paraffin lung sections (5 μm) were immunostained with anti-p53 antibody by standard avidin-biotin complex technique. Overexpression of p53 was identified by reddish-brown nuclei (arrows indicate positively stained cells). Saline control mice exhibited no evidence of p53 expression (A). The number of positive cells in bleomycin-treated mice increased by Day 5 (B), peaked at Day 7 (C), and returned to normal at Day 14 (D) after treatment.
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Fig. 3. Time-dependent expression of PCNA in lung sections of bleomycin-treated mice. Paraffin lung sections (5 μm) were immunostained with anti-PCNA antibody by standard avidin-biotin complex technique. Overexpression of PCNA was identified by brown nuclei (arrows indicate positively stained cells). Saline control mice exhibited minimal or no PCNA background expression (< 1%) (A). The number of positive cells in bleomycin-treated mice increased at 24 h (B), peaked at Day 7 (C), and decreased at Day 14 (D).
[More] [Minimize]Agents that damage cellular DNA show an increase in cellular p53 levels, and these elevated p53 levels correlate with cell-cycle arrest or with apoptosis (22, 24). Mice given bleomycin revealed an increase in p53 immunostaining in the lung cells (Figures 1B–1D). Positive cells were found in all cell types, including macrophages and epithelial and interstitial cells, with a large number of positive cells present at fibrotic sites. In contrast, very few positive cells were present at nonfibrotic sites. p53-positive cells appeared at 5 d (Figure 1B), and their number was increased further at 7 d after treatment (Figure 1C). The expression was decreased at 9 d, and no evidence of p53 expression was found at 14 d after exposure (Figure 1D). Clara cells in the bronchioles, as well as an occasional airway cell, also showed positive p53 staining. No expression of p53 occurred in control lungs (Figure 1A). Specificity of staining was demonstrated by substituting the primary antibody with mouse IgG at an equivalent titer, and no nuclear staining was found at any time.
p21WAF1/PiC1 is important for p53 function and negatively regulates the cell cycle (26). Bleomycin increased p21 expression in the lung, and the immunoreactivity for p21 revealed a nuclear pattern with variable intensities (Figures 2B–2D). p21 overexpression in the lung parenchyma was detected at 5 to 7 d after exposure (Figure 2B). By 9 d after treatment, the most intense nuclear staining was apparent in epithelial cells, alveolar macrophages, and interstitial cells (Figure 2C). The immunoreactivity in the lung cells was decreased at 14 d (Figure 2D). Cuboidal epithelial cells at alveolar bifurcations and interstitial cells of the bronchioles and airways expressed p21 immunostaining. The specificity of p21 staining was again demonstrated by substituting the primary antibody with mouse IgG at an equivalent titer. No nuclear staining was observed in any lung sections.
Fig. 2. Time-dependent expression of p21 is shown in lung sections of bleomycin-treated mice. Paraffin-embedded lung sections (5 μm) were immunostained with anti-p21 antibody by standard avidin-biotin complex technique. Overexpression of p21 was identified by brown nuclei (arrows indicate positively stained cells). Saline control mice exhibited no evidence of p21 expression (A). The number of positive cells in bleomycin-treated mice increased by Day 5 (B), peaked at Day 9 (C), and decreased at Day 14 (D) after treatment.
[More] [Minimize]PCNA, an accessory protein for DNA polymerase-δ, is present in proliferating cells and is required for DNA replication and repair (28, 41). Bleomycin induced significant overexpression of PCNA in the lungs (Figures 3B–3D). The lungs from bleomycin-treated mice at 3 d exhibited dense nuclear staining in alveolar macrophages and epithelial and interstitial cells in the lung parenchyma (Figure 3B). The pattern of PCNA immunostaining in bleomycin-treated animals increased at 3 to 5 d after exposure, increased maximally at 7 d (Figure 3C), and waned by 14 d (Figure 3D). Very few positive lung cells were found at 14 d after treatment as compared with 3 or 7 d after treatment. Staining specificity was demonstrated by substituting the primary antibody with mouse IgG at an equivalent titer. This IgG fraction was associated with a very low level of nonspecific diffuse background staining.
Lung tissue from control mice revealed minimal staining with the monoclonal PCNA antibody and was almost negligible at all time intervals (Figure 3A). A low level of diffuse background staining was found in the bronchoalveolar epithelial cells. The cells lining the ducts or airways showed no evidence of positive nuclear staining for PCNA (Figure 3A). Visible alveolar macrophages occasionally showed some cytoplasmic staining, but there was no evidence of nuclear staining.
In a blinded fashion, the number of stained cells were quantified using light microscopy. Five to six anatomic units were analyzed at each time point to establish the percentages of the various positive cells. The percentage of p53-positive cells was significantly increased at 5 d after bleomycin treatment, peaked at 7 d, and was undetectable at 14 d. The total numbers of positive cells were 12.6 ± 2.7, 25.4 ± 6.8, and 19.8 ± 9.7% at Days 5, 7, and 9, respectively (Figure 4a). p21-positive cells in bleomycin-treated lungs were 21.6 ± 4.2% at 5 d, peaked at 43.2 ± 7.3% at 9 d, and returned to 17.8 ± 3.4% by 14 d (Figure 4b). Expression of PCNA occurred at 3 d with 20.8 ± 5.4% of cells, peaked at 61.0 ± 9.8% at 7 d, and was reduced to 14.4 ± 4.3% of cells at 14 d (Figure 4c).
Fig. 4. Quantification of p53, p21, and PCNA immunostaining. More than 200 cells/high-power anatomic field of each lung section of five different mice were counted at various time intervals after bleomycin instillation, and data are expressed as means ± SD. Figure represents p53-specific immunostaining (a), p21-specific immunostaining (b), and PCNA-specific immunostaining (c).
Double immunostaining of lung sections detected upregulation of DNA damage and repair proteins in type II alveolar epithelial cells at 7 d after bleomycin treatment. The colocalization of SP-C with DNA damage–inducible proteins in cells morphologically consistent with type II alveolar epithelial cells identified the type II cells as an important cellular site for overexpressing the proteins. p53-positive staining that colocalized with SP-C staining was enumerated in the immunostained lung sections and found positive in 53.4 ± 11.3% of type II alveolar cells at Day 7. The p21 and SP-C colocalized positive type II alveolar epithelial cells were 43.6 ± 13.5%, whereas PCNA colocalized with SP-C in 83.0 ± 6.6% of type II alveolar cells (Figure 5).
Fig. 5. Identification of type II alveolar epithelial cells expressing cell-cycle growth arrest proteins. Lung sections were coimmunostained for pro– SP-C (red cytoplasmic stain) and the following DNA damage–inducible proteins (reddish-brown nuclear stain): SP-C alone (A), PCNA (B), p53 (C), and p21 (D) (arrows indicate positively stained cells).
[More] [Minimize]The upregulation of DNA damage–inducible proteins p53, p21, and PCNA in lungs was evaluated by Western blot at various times after bleomycin treatment. Equal amounts of protein in each sample on the gel were confirmed by PansoS staining of the nitrocellulose paper after transfer. Increased upregulation of p53 protein was evident within 3 d and was followed by a further increase at 7 d after bleomycin treatment (Figure 6). Elevation of PCNA occurred at 3 d and increased further at 7 and 14 d, whereas the upregulation of p21 was present at Day 7 and was reduced at Day 14 after treatment (Figure 6). The upregulation of these proteins was generally consistent with the pattern of immunohistochemical expression in the lung cells.
Fig. 6. Western blot analysis of bleomycin-induced time-dependent upregulation of p53 (top panel), PCNA (middle panel), and p21 (lower panel) in murine lung homogenate. Immunoblots for p53, PCNA, and p21 are shown from lung homogenates obtained from mice treated with saline or exposed to bleomycin for 1, 3, 7, and 14 d. BSA was used as a negative control.
[More] [Minimize]We have demonstrated that DNA damage to alveolar epithelial cells occurs in response to bleomycin and that p53, p21, and PCNA proteins are overexpressed within these cells. Bleomycin-treated lungs showed a patchy interstitial inflammation, fibrosis, and altered alveolar architecture at the same time that bleomycin induced a peak level of p53 followed by the peak expression of p21. PCNA was detectable very early in the bleomycin treatment but also revealed peak expression simultaneously with p53. The relationship of the dose and duration of bleomycin exposure to the upregulation of cell-cycle growth-arrest proteins adds to our understanding of the mechanisms of cell death and DNA repair in injured lung cells before the development of pulmonary fibrosis.
In response to DNA damage, G1–G2 cell-cycle arrest is an important cellular response that gives cells time for DNA repair before going into the S and M phases of the cell cycle (42). p53 is required for the G1 check point in mammalian cells (43, 44). p53 arrests cell growth by a p21-dependent mechanism. p21 either can inhibit the activity of the G1 cyclin Cdk complexes that are required for the cell to progress through the G1/S transition or can directly bind and inhibit PCNA (45). In our present study, the upregulation of p21 and PCNA in response to bleomycin-induced DNA damage may be a result of p53 induction in lung cells. p53 functions as a transcription factor for cell cycle proteins (21). Because of its regulatory function, one can predict that bleomycin-induced activation of p53 expression in the lungs may influence the fibroproliferative response. Presently, two pathways for the induction of p21 or PCNA exist: a DNA damage–induced p53-dependent activation pathway and a p53-independent activation by mitogens (46).
In this study we suggest that p53 mediates upregulation of PCNA and p21 in bleomycin-induced injury in the lung; however, this may not be the only mechanism. Inflammatory cells produce cytokines such as transforming growth factor–α and –β and platelet-derived growth factor that regulate fibroblast proliferation and upregulate PCNA and p21 expression in a p53-independent pathway (41, 43– 45). Therefore, bleomycin-induced lung inflammation may also be the cause of the upregulation of p21 or PCNA protein in the lungs.
p53 can transcriptionally activate PCNA as a DNA repair protein in injured lung cells (38). However, p53 and PCNA are also expressed in response to mitogenic agents for the purpose of DNA repair and replication. Normally, PCNA is expressed in the lung at low levels. However, as evidenced by our study, bleomycin increases lung PCNA expression, and the protein remains detectable for a prolonged period of time. Recently, a study has shown that p53 protein has an inverse relationship with mitogenic growth factors and in vivo wound repair (47). This suggests that during the healing process the expression of mitogenic growth factors is suppressed and p53 levels are increased by a control mechanism at the time of complete epithelization of the injured tissue (47).
p21 controls Cdk activity by inactivating the retinoblastoma gene product Rb (25), thereby affecting the cell cycle, whereas PCNA functions in both DNA replication and repair. p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin Cdk (27). In normal human cells, p21 exists in quaternary complex with cyclin, Cdk, and PCNA (27, 48). Further, p21 blocks the ability of PCNA to activate DNA polymerase-δ, the principal replicative DNA polymerase.
The overexpression of p21 in our bleomycin study suggests that p21 may be a key regulator of DNA replication, DNA repair, and the cell-cycle machinery during bleomycin-induced injury and repair. It is unclear whether p21/ WAF1-mediated growth inhibition in the epithelial cells results from the induction of apoptosis or from G1 arrest. We hypothesize that during bleomycin-induced lung injury, the upregulation of p53 transcriptionally activates p21, arrests the epithelial cells in G1 phase, and inhibits the function of PCNA during DNA replication (25-27). Induction of apoptosis in alveolar epithelial cells during bleomycin-induced lung injury may result from a p53- dependent mechanism, as occurs with transcriptional activation of Bax, a cellular protein that promotes apoptosis (49); or alternatively, apoptosis may result from a Fas–Fas ligand–mediated pathway associated with infiltration of lymphocytes bearing the Fas ligand during bleomycin-induced injury and repair (50, 51). During bleomycin pulmonary toxicity, widespread apoptosis of alveolar epithelial cells may interfere with re-epithelization of the alveolar spaces and may further contribute to lung impairment (50, 51). Further studies using p53 and p21 gene–deficient mice are needed to understand the complex mechanisms by which DNA damage–inducible proteins and inflammatory or immune cells influence cell injury and repair during bleomycin pulmonary toxicity.
On the basis of the present study, DNA damage and repair protein expression are clearly present during bleomycin-induced lung injury and repair, suggesting that these proteins play an important role in the regulation of cell death and proliferation in bleomycin toxicity. Inasmuch as bleomycin toxicity is a commonly used model of lung injury and fibrosis, it is possible that DNA damage and repair proteins are involved in many types of lung injury and repair. For example, asbestos and ionizing radiation, two agents known to injure the lung, have been shown to induce overexpression of p53 and PCNA in lungs (38) and in cultured cells (52), respectively. An improved understanding of how DNA-damaging agents injure the lung may permit development of strategies to control important regulatory mechanisms, to minimize lung cell injury, and to facilitate ordered repair of the lung.
This study was supported by NIH P01 CA75426.
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