The Role of p53 in Bleomycin-Induced DNA Damage in the Lung
1999; Elsevier BV; Volume: 155; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)65236-4
ISSN1525-2191
AutoresKoji Okudela, Takaaki Ito, Hideaki Mitsui, Hiroyuki Hayashi, Naoko Udaka, Masayoshi Kanisawa, Hitoshi Kitamura,
Tópico(s)Cholangiocarcinoma and Gallbladder Cancer Studies
ResumoTo elucidate the role of p53 and apoptosis in the pathogenesis of lung injury, we examined histological changes, expressions of p53 and p21waf1/cip1 (p21), apoptosis, DNA double strand breaks, cell kinetics, and DNA synthesis in C57/BL6 mice (p53+/+) and mice deficient for p53 (p53−/−) at 2 hours to 7 days after a single intravenous administration of bleomycin. We also compared these parameters between the lung cells and small intestinal epithelial cells to explore potential differences in their response to DNA damage. Bleomycin induced p21 expression in a p53-dependent manner in p53+/+ mice but neither p53 nor p21 expression in p53−/− mice. In the lung of both groups of mice, focal inflammation followed by fibrosis was observed, but there was no evidence of apoptosis. Cells with DNA breaks and those undergoing DNA synthesis were unequivocally increased, but the cycling cell fraction remained unchanged, suggesting that the DNA synthesis detected in the lung reflected unscheduled DNA synthesis for repair of damaged DNA. DNA breaks and unscheduled DNA synthesis were prolonged in p53−/− mice compared to p53+/+ mice. By contrast, in the small intestine, marked cell cycle arrest and extensive apoptosis were evoked in the cycling crypt cells of both groups of mice, but these changes were milder and DNA breaks remained detectable for a longer time in p53−/− mice than in p53+/+ mice. Among the resting enterocytes in the villi, apoptosis was observed almost equally in both groups, but repair of DNA breaks was significantly delayed in the p53−/− mice. These observations imply that apoptosis is mediated largely by the p53-dependent pathway in the crypts but exclusively by the p53-independent pathway in the villi, that this pathway is particularly important in DNA repair in the villi, and that despite this difference in the significance of apoptosis, p53 plays an important role in DNA repair in both the crypts and villi. Our results suggest that the lung cells and small intestinal cells respond to the bleomycin treatment in different ways in terms of the induction of apoptosis and that p53 carries out an essential role in the early response to and repair of DNA damage by a non-apoptotic mechanism which appears to be crucial in the noncycling lung cells and enterocytes. Importantly, the p53-p21 pathway and apoptosis are unlikely to be essential for bleomycin-induced tissue injury in the lung. To elucidate the role of p53 and apoptosis in the pathogenesis of lung injury, we examined histological changes, expressions of p53 and p21waf1/cip1 (p21), apoptosis, DNA double strand breaks, cell kinetics, and DNA synthesis in C57/BL6 mice (p53+/+) and mice deficient for p53 (p53−/−) at 2 hours to 7 days after a single intravenous administration of bleomycin. We also compared these parameters between the lung cells and small intestinal epithelial cells to explore potential differences in their response to DNA damage. Bleomycin induced p21 expression in a p53-dependent manner in p53+/+ mice but neither p53 nor p21 expression in p53−/− mice. In the lung of both groups of mice, focal inflammation followed by fibrosis was observed, but there was no evidence of apoptosis. Cells with DNA breaks and those undergoing DNA synthesis were unequivocally increased, but the cycling cell fraction remained unchanged, suggesting that the DNA synthesis detected in the lung reflected unscheduled DNA synthesis for repair of damaged DNA. DNA breaks and unscheduled DNA synthesis were prolonged in p53−/− mice compared to p53+/+ mice. By contrast, in the small intestine, marked cell cycle arrest and extensive apoptosis were evoked in the cycling crypt cells of both groups of mice, but these changes were milder and DNA breaks remained detectable for a longer time in p53−/− mice than in p53+/+ mice. Among the resting enterocytes in the villi, apoptosis was observed almost equally in both groups, but repair of DNA breaks was significantly delayed in the p53−/− mice. These observations imply that apoptosis is mediated largely by the p53-dependent pathway in the crypts but exclusively by the p53-independent pathway in the villi, that this pathway is particularly important in DNA repair in the villi, and that despite this difference in the significance of apoptosis, p53 plays an important role in DNA repair in both the crypts and villi. Our results suggest that the lung cells and small intestinal cells respond to the bleomycin treatment in different ways in terms of the induction of apoptosis and that p53 carries out an essential role in the early response to and repair of DNA damage by a non-apoptotic mechanism which appears to be crucial in the noncycling lung cells and enterocytes. Importantly, the p53-p21 pathway and apoptosis are unlikely to be essential for bleomycin-induced tissue injury in the lung. 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One line of evidence suggests that the p53-p21waf1/cip1 pathway, as well as apoptotic cell death, contributes to lung injuries induced by various agents.12Hagimoto N Kuwano K Nomoto Y Kunikake R Hara N Apoptosis and expression of Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice.Am J Respir Cell Mol Biol. 1997; 16: 91-101Crossref PubMed Scopus (246) Google Scholar, 13Kuwano K Hagimoto N Nomoto Y Kawasaki M Kunikake R Fujita M Miyazaki H Hara N p53 and p21 (Waf1/Cip1) mRNA expression associated with DNA damage and repair in acute immune complex alveolitis in mice.Lab Invest. 1997; 76: 161-169PubMed Google Scholar, 14Barazzone C Horowitz S Donati YR Rodriguez I Piguet PF Oxygen toxicity in mouse lung: pathways to cell death.Am J Respir Cell Mol Biol. 1998; 19: 573-581Crossref PubMed Scopus (235) Google Scholar, 15Hayashi H Miyamoto H Ito T Kameda Y Nakamura N Kubota Y Kitamura H Analysis of p21waf1/cip1 expression in normal, premalignant, and malignant cells during the development of human lung adenocarcinoma.Am J Pathol. 1997; 151: 461-470PubMed Google Scholar An anticancer drug, bleomycin, generates superoxide radicals to induce DNA double strand breaks in vitro16Harrison Jr, JH Hoyt DG Lazo JS Acute pulmonary toxicity of bleomycin: DNA scission and matrix protein mRNA level in bleomycin-sensitive and -resistant strains of mice.Mol Pharmacol. 1989; 36: 231-238PubMed Google Scholar and is well known to cause severe progressive pulmonary fibrosis.16Harrison Jr, JH Hoyt DG Lazo JS Acute pulmonary toxicity of bleomycin: DNA scission and matrix protein mRNA level in bleomycin-sensitive and -resistant strains of mice.Mol Pharmacol. 1989; 36: 231-238PubMed Google Scholar, 17Thrall RS McCormick JR Jack RM McReynold RA Ward PA Bleomycin-induced pulmonary fibrosis in the rats.Am J Pathol. 1979; 95: 117-130PubMed Google Scholar, 18Smith RE Strieter RM Phan SH Kunkel S C-C chemokines: novel mediator of the profibritic inflammatory response to bleomycin challenge.Am J Respir Cell Mol Biol. 1996; 15: 693-702Crossref PubMed Scopus (91) Google Scholar, 19Gharaee-Kermani M Phan SH Lung interleukin-5 expression in murine bleomycin-induced pulmonary fibrosis.Am J Respir Cell Mol Biol. 1997; 16: 438-447Crossref PubMed Scopus (68) Google Scholar, 20Hoyt DG Lazo JS Murine strain difference in acute lung injury and activation of poly (ADP-ribose) polymerase by in vitro lung slices to bleomycin.Am J Respir Cell Mol Biol. 1992; 7: 645-651Crossref PubMed Scopus (31) Google Scholar, 21Piguet PF Vesin C Pulmonary platelet trapping induced by bleomycin: correlation with fibrosis and involvement of the β integrin.Int J Exp Pathol. 1994; 75: 321-328PubMed Google Scholar, 22Zhang K Flanders KC Phan SH Cellular localization of transforming growth factor-β expression in bleomycin-induced pulmonary fibrosis.Am J Pathol. 1995; 147: 352-361PubMed Google Scholar, 23Santana A Saxena B Noble NA Gold LI Marshall BC Increased expression of transforming growth factor β isoforms (β1, β2, β3) in bleomycin-induced pulmonary fibrosis.Am J Respir Cell Mol Biol. 1995; 13: 34-44Crossref PubMed Scopus (134) Google Scholar Recent studies demonstrated that excessive apoptotic cell death was responsible for acute lung injury leading to pulmonary fibrosis induced by bleomycin.12Hagimoto N Kuwano K Nomoto Y Kunikake R Hara N Apoptosis and expression of Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice.Am J Respir Cell Mol Biol. 1997; 16: 91-101Crossref PubMed Scopus (246) Google Scholar To elucidate the role of the p53-p21waf1/cip1 pathway in the pathogenesis of chemical-induced lung injury, we examined the effects of bleomycin on the lung cells in p53 knockout mice in comparison with wild-type mice. To the best of our knowledge, a study of bleomycin-induced lung injury using the p53 knockout mice has not previously been conducted. We evaluated alterations of cell kinetics, expression of p53 and p21waf1/cip1, apoptotic cell death, DNA double strand breaks, and DNA synthesis at various time points after the bleomycin treatment. These parameters in the bronchial (epithelial) and alveolar cells were also compared with those in the small intestinal epithelial cells, because these two tissues are different in terms of cell kinetics under both physiological and pathological conditions. The small intestinal epithelium belongs to the renewal system consisting of clearly separated cycling and resting cell compartments, and its response to DNA damage has been examined extensively.2Willson JW Prichard DM Hickman JA Potten CS Radiation induced p53 and p21WAF1/CIP1 expression in the murine intestinal epithelium. Apoptosis and cell cycle arrest.Am J Pathol. 1998; 153: 899-909Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 24Hall PA Mckee PH Menage HD Dover R Lane DP High level of p53 protein in UV-irradiated normal human skin.Oncogene. 1993; 8: 203-207PubMed Google Scholar, 25Clarke AR Howard LA Harrison D Winton DJ p53, mutation frequency and apoptosis in the murine small intestine.Oncogene. 1997; 14: 2015-2018Crossref PubMed Scopus (40) Google Scholar, 26Merritte AJ Allen TD Potten CS Hickman JA Apoptosis in small intestinal epithelia from p53-null mice: evidence for a delayed, p53-independent G1/M-associated cell death after a γ-irradiation.Oncogene. 1997; 14: 2759-2766Crossref PubMed Scopus (197) Google Scholar The respiratory epithelium belongs to the conditional renewal system, the study of which has been rather limited.12Hagimoto N Kuwano K Nomoto Y Kunikake R Hara N Apoptosis and expression of Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice.Am J Respir Cell Mol Biol. 1997; 16: 91-101Crossref PubMed Scopus (246) Google Scholar, 13Kuwano K Hagimoto N Nomoto Y Kawasaki M Kunikake R Fujita M Miyazaki H Hara N p53 and p21 (Waf1/Cip1) mRNA expression associated with DNA damage and repair in acute immune complex alveolitis in mice.Lab Invest. 1997; 76: 161-169PubMed Google Scholar, 14Barazzone C Horowitz S Donati YR Rodriguez I Piguet PF Oxygen toxicity in mouse lung: pathways to cell death.Am J Respir Cell Mol Biol. 1998; 19: 573-581Crossref PubMed Scopus (235) Google Scholar In this paper, we discuss the significance of the p53-p21waf1/cip1 pathway and refer to the difference between these two epithelial systems in response to bleomycin-induced DNA damage. Specific-pathogen-free male C57/BL6 mice (wild-type mice), 10 weeks of age, were obtained from Japan SLC (Sizuoka, Japan). Age-and sex-matched C57/BL6 mice homozygously deficient for p53 (p53 knockout mice)27Donehower LA Harvey M Slagle BA McArthur MJ Montogomery Jr, CA Butel JS Bradley A Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumor.Nature. 1992; 356: 215-221Crossref PubMed Scopus (4191) Google Scholar were obtained from Immuno-Biological Laboratories (Gunma, Japan). Experimental animals were treated with 100 mg/kg body weight of bleomycin hydrogen chloride (Nippon Kayaku Co. Ltd., Tokyo) by a single intravenous injection through the tail vein as previously described.20Hoyt DG Lazo JS Murine strain difference in acute lung injury and activation of poly (ADP-ribose) polymerase by in vitro lung slices to bleomycin.Am J Respir Cell Mol Biol. 1992; 7: 645-651Crossref PubMed Scopus (31) Google Scholar, 21Piguet PF Vesin C Pulmonary platelet trapping induced by bleomycin: correlation with fibrosis and involvement of the β integrin.Int J Exp Pathol. 1994; 75: 321-328PubMed Google Scholar This dose corresponded to one-third of the Lethal dose 50% (LD50) described in the instructions (315 mg/kg body weight). In our preliminary study, 2 of 10 mice died within 7 days after the treatment and this dose was sufficient for the treated animals to develop pulmonary fibrosis. Control animals were treated with sterile saline alone. Wild-type and p53 knockout mice were divided into eight groups (controls and 2, 4, 6, and 12 hours and 1, 3, and 7 days after bleomycin injection) with 3 animals in each group. Mice were sacrificed under a pentobarbital anesthesia, and the lungs and small intestines were obtained. One hour before the sacrifice, mice were given 2 mg/kg body weight of bromodeoxyuridine (BrdU, Sigma Chemical, St. Louis, MO) by intraperitoneal injection. Tissues from the lower lobe of the right lung and from the small intestine were fixed in a buffered 4% paraformaldehyde solution for 3 days and embedded in paraffin. Other pieces of the right lung and small intestine were embedded in O.C.T. compound (Sakura, Tokyo) and snap-frozen in liquid nitrogen. Tissue sections were prepared from paraffin-embedded or frozen tissues for routine hematoxylin-eosin staining, immunohistochemistry, and in situ nick end labeling. For p21waf1/cip1, Ki-67, and BrdU staining, 5-μm sections made from paraffin-embedded tissues and placed on silane-coated slides were dewaxed and rehydrated. The sections were immersed in 0.01 mol/L citrate buffer, pH 6.0, for p21waf1/cip1 or 0.05 mol/L Tris-HCl buffer containing 5% urea, pH 9.5, for Ki-6728Ito T Udaka N Hayashi H Okudela K Kanisawa M Kitamura H Ki-67 (MIB5) immunostaining of mouse lung tumors induced by 4-nitroquinoline 1-oxide.Histochem Cell Biol. 1998; 110: 589-593Crossref PubMed Scopus (28) Google Scholar and were heated in a microwave oven at 97°C for 30 minutes to retrieve the antigenic activities. For BrdU staining, the sections were immersed in a 4-N HCl solution for 20 minutes to denature double-strand DNA. The sections were incubated with 3% hydrogen peroxide/methanol at room temperature for 10 minutes to inactivate the endogenous peroxidase, and nonimmunospecific protein bindings were blocked with 5% normal goat serum and avidin-biotin blocking reagent (Vector Laboratories Inc., Burlingame, CA). The sections were incubated with one of the following primary antibodies at room temperature for 90 minutes, each diluted at 1:100: anti-p21waf1/cip1 rabbit polyclonal (sc-397, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Ki-67 mouse monoclonal (MIB5, Immunotech, Marseille, France), and anti-BrdU mouse monoclonal antibody (Br3, Caltag, San Francisco, CA). Subsequently, they were incubated with biotinylated animal-matched secondary antibodies at room temperature for 60 minutes. The protein expressions were visualized by the avidin-biotin complex immunoperoxidase method using the SLAB kit (DAKO Japan Co., Ltd., Kyoto, Japan) with diaminobenzidine as substrate. Nuclear counterstaining was performed lightly with hematoxylin. For p53 staining, 6-μm frozen sections were made and fixed with a buffered 4% paraformaldehyde solution for 5 minutes, then washed with distilled water. After treatment with 5% normal goat serum, the sections were incubated with 1:500 diluted rabbit polyclonal antibody against p53 (CM5, Novocastra, Newcastle, UK) at room temperature for 90 minutes. Then, tissue sections were incubated with the fluorescein isothiocyanate-conjugated goat antibody against rabbit immunoglobulin (DAKO) at room temperature for 60 minutes. The protein expression was observed with a fluorescence microscope (HB10101, Nikon, Tokyo). DNA double strand breaks and/or apoptotic nucleosomal degradation were detected on paraffin tissue sections by the ISNEL method using the Trevigen apoptotic cell system (Trevigen, Gaithersburg, MD). Six-micron sections were dewaxed, rehydrated, and incubated first with a proteinase K solution at room temperature for 15 minutes, then with a 2% hydrogen peroxide solution for 5 minutes. After being immersed in a labeling buffer for 3 minutes, the sections were incubated with a reaction mixture containing Klenow fragment and dNTP in labeling buffer at 37°C for 60 minutes. After a brief wash with PBS, the streptavidin-biotin peroxidase method was applied with diaminobenzidine as a substrate. Light nuclear counterstaining was performed with hematoxylin. Fresh tissues from the left lung were homogenized in 1 ml of a homogenizing buffer solution containing 5 mmol/L Tris-HCl, pH 7.5, 0.25 mol/L sucrose, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.5 mmol/L DDT, 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 5 μg leupeptin with a politron homogenizer at 1000 rpm for 1 minute, and then filtrated through a nylon mesh (100 μm in pore size). The filtrates were centrifuged at 600 × g for 10 minutes. The pellets were resuspended in 100 μl of buffer A (50 mmol/L Tris-HCl, pH 7.5, 0.25 mol/L sucrose, 25 mmol/L KCl, 25 mmol/L MgCl2, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.5 mmol/L DDT, 0.5 mmol/L PMSF, 5 μg leupeptin) and were incubated for 10 minutes on ice. Then, 200 μl of buffer B (50 mmol/L Tris, 2.3 mol/L sucrose, 25 mmol/L KCl, 25 mmol/L MgCl2, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.5 mmol/L DDT, 0.5 mmol/L PMSF, 5 μg leupeptin) were added and mixed well. Each suspension was layered over 200 μl of buffer B and centrifuged at 12,000 × g for 30 minutes. The pellets were resuspended in hypotonic lysis buffer containing 25 mmol/L HEPES, pH 7.5, 0.4 mmol/L KCl, 5 mmol/L EDTA, 10 mmol/L NaF, 5 mmol/L DDT, 5 mmol/L EDTA, and 1% NP-40 and incubated for 60 minutes on ice. After centrifugation at 12,000 × g for 15 minutes, the supernatants were recovered as protein lysates. Equal volumes of protein lysate and 2× sodium dodecyl sulfate (SDS) sample buffer (0.1 mol/L Tris-HCl, pH 6.8, 4% SDS, 12% 2-mercapteethanol, 10% glycerol, and 0.0025% bromophenol blue) were mixed and boiled for 5 minutes. The protein concentration of the samples was determined by Bradford's method. Nuclear protein (30 μg) was subjected to SDS-polyacrylamide gel electrophoresis on 12% acrylamide gel and then transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Nonspecific protein binding was blocked with 5% nonfat milk in 0.01 mol/L PBS containing 0.1% Tween 20 (Tween-PBS) at room temperature for 60 minutes. The membranes were incubated with 1:500 diluted rabbit polyclonal antibody against p53 (CM5) or p21waf1/cip1 (sc-397) at room temperature for 90 minutes. After a brief washing with Tween-PBS, the membranes were incubated with a horseradish peroxidase-conjugated goat antibody against rabbit immunoglobulin (Amersham Life Science, Buckinghamshire, UK) at room temperature for 60 minutes. The membrane was briefly washed again with Tween-PBS. Immunoblotted proteins were visualized with the enhanced chemiluminescence system (Amersham). Fresh tissues from the middle lobe of the right lung and those from the small intestine were minced with a razor blade and were suspended in a 500 μl DNA extraction buffer containing 50 mmol/L Tris-HCl, pH 7.7, 100 mmol/L NaCl, 100 mmol/L EDTA, 1% SDS, and 0.1 mg/ml proteinase K (Sigma). The samples were incubated at 55°C overnight and then subjected to phenol/chloroform extraction. After isopropanol precipitation, pellets of DNA were washed 3× with 70% ethanol. DNA samples (10 μg) were electrophoresed on a 1% agarose gel containing 1 μg/ml of ethidium bromide and visualized on an ultraviolet transilluminator. More than 1000 cell nuclei of the bronchial epithelia, lung alveoli including epithelial cells, fibroblasts, endothelial cells, and macrophages, small intestinal crypts, and villi epithelia were counted. Percentages of cells positive for Ki-67, BrdU, and ISNEL were determined and used as the labeling index. The percentage of cells showing the morphological characteristics of apoptotic cell death, such as cell shrinkage and chromatin condensation and fragmentation, was determined on hematoxylin-eosin sections and used as the apoptotic index. The difference in mean values was analyzed by Student's t-test. A P value <0.05 was considered significant. From 2 hours to 1 day after bleomycin treatment, no significant pathological changes were observed in the lungs of either the wild-type or p53 knockout mice. At 3 days, infiltration by small numbers of inflammatory cells, such as neutrophils, lymphocytes, and monocytes, was observed focally around the bronchioles and adjacent small blood vessels, as well as within the alveolar spaces (Figure 1, A and B). The inflammatory infiltrates increased with time, and these changes were almost equivalent in the wild-type and p53 knockout mice. The small intestinal mucosa showed severe epithelial cell desquamation in the crypts of wild-type mice from 12 hours to 1 day after the treatment (data not shown). In the p53 knockout mice, epithelial desquamation also occurred in the crypts, but the changes were milder than in the wild-type mice. From day 1 to day 3, regeneration of epithelia with numerous mitotic figures was observed. These changes had almost completely disappeared by day 7 in both groups (data not shown). In neither the wild-type nor the p53 knockout mice did bronchial and alveolar cells show signs of apoptotic cell death throughout the experimental period histologically (Figure 1, A and B, and Figure 2B, top, second panel). Agarose gel electrophoresis did not reveal evidence of DNA ladders at any time point examined (Figure 2A). In contrast to the lung, apoptotic cell death was observed in the small intestine in both the crypts and villi, not only in the wild-type but also in the p53 knockout mice after treatment. In the crypts, the frequency of apoptotic cells increased rapidly after treatment in both groups (Figure 2B, third panel). The peak of apoptotic index was seen at 4 hours in the wild-type mice and at 12 hours in the p53 knockout mice, with the maximal value in the former being 1.7-fold higher than in the latter (P < 0.05). On the other hand, in the villi, the frequency of apoptotic cells gradually increased and peaked at 3 days in both groups (Figure 2B, bottom). The maximal value was slightly higher in the wild-type mice than in the p53 knockout mice, but the difference was not significant. In both groups, the apoptotic index in the crypts and villi had returned to the basal level 7 days after the treatment. Figure 2, C and D, shows typical features of apoptotic cell death observed in the crypts (left) and villi (right) of the wild-type and p53 knockout mice after the treatment, respectively. At this time point, a DNA ladder was observed on agarose gel electrophoresis (Figure 2A). In both the wild-type and p53 knockout mice, ISNEL-positive cells were observed among the bronchial and alveolar cells after the bleomycin treatment (Figure 3, A and B). The ISNEL index in the bronchial cells of the wild-type mice was elevated soon after the treatment and peaked at 6 hours, whereas in the p53 knockout mice the peak was observed at 3 days (Figure 3C, top, and Table 1). The ISNEL index then gradually decreased in both groups, but in the p53 knockout mice it remained at higher levels than in the wild-type mice (P < 0.05). The ISNEL index in the alveolar cells of the wild-type mice was rapidly elevated soon after the treatment and peaked at 6 hours (Figure 3C, second panel, and Table 1). In the p53 knockout mice, the peak was somewhat delayed and seen at 12 hours. Similarly, sustained higher levels in the ISNEL index were observed in the p53 knockout mice compared to the wild-type mice (P < 0.05).Table 1ISNEL IndexTime after bleomycin treatment (hours)Tissuep53 status0246122472168BronchiKnockout0.9 ± 0.58.9 ± 3.38.7 ± 1.313.2 ± 1.116.4 ± 5.722.4 ± 10.146.9 ± 20.037.2 ± 20.6Wild-type0.1 ± 0.14.2 ± 2.84.7 ± 3.211.0 ± 1.28.5 ± 1.86.2 ± 1.95.0 ± 3.47.2 ± 6.7AlveoliKnockout1.3 ± 1.115.6 ± 14.914.5 ± 2.649.9 ± 3.251.3 ± 3.248.6 ± 1.952.1 ± 3.930.2 ± 3.8Wild-type0.1 ± 0.216.4 ± 7.614.6 ± 10.920.2 ± 5.711.0 ± 2.513.6 ± 3.67.9 ± 1.95.4 ± 4.7CryptsKnockout1.9 ± 1.67.0 ± 4.615.5 ± 0.717.5 ± 0.916.5 ± 1.417.8 ± 2.012.5 ± 3.719.8 ± 6.73Wild-type0.0 ± 0.026.3 ± 25.412.5 ± 10.817.6 ± 5.012.0 ± 3.88.7 ± 7.60.1 ± 0.10.1 ± 0.1VilliKnockout0.4 ± 0.225.0 ± 11.68.8 ± 1.88.6 ± 5.717.1 ± 8.023.9 ± 7.128.7 ± 8.710.7 ± 9.4Wild-type0.1 ± 0.218.9 ± 7.24.2 ± 4.716. ± 1.62.6 ± 3.23.4 ± 2.110.0 ± 5.70.8 ± 1.2Values are presented as the mean ± SD (%). Open table in a new tab Values are presented as the mean ± SD (%). In the small intestine, the ISNEL index in the crypts of the wild-type mice was rapidly elevated, peaking at 2 hours, then returning to the basal level 3 days after treatment (Figure 3C, third panel, and Table 1). By contrast, in the p53 knockout mice the ISNEL index of the crypts showed a gradual elevation and peaked at 12 hours, with a delay of 10 hours compared to the wild-type mice. It did not return to the basal level throughout the experimental period. On the other hand, in the villi, the ISNEL index in both groups rapidly increased, peaking at 2 hours, then decreasing. It gradually increased from 6 hours and peaked again at 3 days, but had returned to the basal level by 7 days in the wild-type mice (Figure 3C, bottom, and Table 1). As was seen in the crypts, the ISNEL index in the villi of the p53 knockout mice also did not return to the basal level. Virtually all of the apoptotic cell nuclei were ISNEL-positive, but many non-apoptotic cell nuclei were also stained positively with ISNEL (data not shown). The pattern of change in BrdU labeling after the treatment was similar between the bronchial and alveolar cells in both the wild-type and p53 knock
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