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From the Department of Pathology, Yokohama City University School of Medicine, Yokohama, Japan
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and UV irradiation, anticancer drugs,
and so on, they undergo cell cycle arrest to allow repair of damaged
DNA and/or apoptotic cell death to remove unrepairable
DNA.1-3
In these processes, p53 has been demonstrated to
play key roles through its transcriptional activity and other
unclarified mechanisms.1,3,4
A cyclin-dependent kinase
inhibitor, p21waf1/cip1, is a crucial downstream factor of p53, which
mediates cell cycle arrest by binding to and inhibiting the functions
of cyclin-dependent kinases and proliferating cell nuclear
antigen.5,6
Bax, another downstream factor of p53, is
shown to mediate apoptotic cell death in
vitro,7
though its in vivo role in
p53-mediated apoptotic cell death has been disputed.8-10
Whether cells bearing DNA damage undergo cell cycle arrest or apoptotic
cell death appears to depend on the type of cell (organ specificity)
and/or conditions surrounding the cells, whereas the determinant of
these two disparate cellular outcomes has not yet been
clarified.11 The lung is subject to damage caused by a variety of exogenous and endogenous insults. 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.12-15 An anticancer drug, bleomycin, generates superoxide radicals to induce DNA double strand breaks in vitro16 and is well known to cause severe progressive pulmonary fibrosis.16-23 Recent studies demonstrated that excessive apoptotic cell death was responsible for acute lung injury leading to pulmonary fibrosis induced by bleomycin.12
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.2,24-26 The respiratory epithelium belongs to the conditional renewal system, the study of which has been rather limited.12-14 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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)27 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.20-21 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.
Tissue Preparation
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.
Immunohistochemistry
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-6728 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).
In Situ Nick End Labeling (ISNEL)
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.
Western Blotting
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 x 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 x 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 x g for 15 minutes, the supernatants were recovered as protein lysates. Equal volumes of protein lysate and 2x 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).
Electrophoresis of DNA
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 3x 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.
Labeling Index and Apoptotic Index
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.
Statistical Analysis
The difference in mean values was analyzed by Student's t-test. A P value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Apoptotic Cell Death
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)
.
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, 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)
. ISNEL
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).
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, 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). DNA Synthesis Determined by BrdU Labeling
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 knockout mice. There was no significant difference in the basal
level of BrdU labeling between the two groups. After the treatment, the
uptake of BrdU was observed in the bronchial and alveolar cells from
both groups of mice (Figure 4, A and B)
.
The BrdU labeling in the wild-type mice rapidly increased, peaking at
12 hours, whereas in the p53 knockout mice it continued to increase for
up to 3 days and showed significantly higher maximal values than in the
wild-type mice (P < 0.05) (Figure 4C
, top,
second panel, and Table 2
). Within 7
days, the BrdU labeling returned to the basal level in both the
wild-type and p53 knockout mice.
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, third panel, and
Table 2
). In the wild-type mice, BrdU labeling in the crypts rapidly
decreased after treatment, with the lowest level reached at 6 hours.
Then it increased, peaking at 12 hours before returning to the basal
level 7 days later. By contrast, in the p53 knockout mice BrdU labeling
in the crypts transiently increased from 2 to 6 hours, then decreased
to a low at 24 hours. Thus, in the crypts the initial decrease in BrdU
labeling was delayed in the p53 knockout mice by 6 hours compared to
the wild-type mice. It had also returned to the basal level by day 7 in
the p53 knockout mice. In the villi, no BrdU uptake was observed in the
untreated control, and its labeling was unchanged 6 hours after the
treatment in both groups (Figure 4C
, bottom, and Table 2
). From 6 to 12
hours, it started to increase rapidly, peaking at 12 hours in the
wild-type mice and at 24 hours in the p53 knockout mice. Thereafter, it
decreased and had returned to the basal level by day 3 in both groups.
Thus, the time at which the BrdU labeling peaked was delayed in the p53
knockout mice by 12 hours compared to the wild-type mice, but the
maximal values were not significantly different between the two. Expression of p53 and p21waf1/cip1
Western blot analysis showed that the expression of p53 in the
lung of wild-type mice was first detected at 2 hours after bleomycin
treatment and continued for 7 days (Figure 5A)
. The expression of p21waf1/cip1
coincided with that of p53 (Figure 5A)
. The expression of these
proteins was also detected immunohistochemically in the bronchial
epithelial and alveolar cell nuclei from 2 hours to 7 days after the
treatment (Figure 5, B and C)
. By contrast, in the p53 knockout mice
the expression of p53 and p21waf1/cip1 was not detected by either
immunohistochemistry (data not shown) or Western blotting at any time
point examined (Figure 5A)
. In the small intestine, the concomitant
expression of these proteins was immunohistochemically detected
exclusively in the crypts, but not in the villi, only in the treated
wild-type mice (data not shown). In both groups of mice, only a weak
expression of p21waf1/cip1 was seen in a few enterocytes of the villi,
where p53 expression was not detected.
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The labeling index of Ki-67 in the bronchial and alveolar cells
showed no significant change at any time point examined in either the
wild-type or the p53 knockout mice, although some fluctuation was
observed soon after the treatment (Figure 6
, top and second panels).
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, third panel). In the wild-type mice, the
Ki-67 labeling rapidly decreased after the treatment, bottoming out at
12 hours. It rapidly recovered and peaked at 24 hours, then decreased
again before returning to the basal level on day 7. In the p53 knockout
mice, the Ki-67 labeling transiently increased from 2 to 6 hours, then
decreased to a low at 24 hours. Thereafter, it increased again, peaking
at 3 days, and returned to the basal level by day 7. Thus, the lowest
level of Ki-67 labeling in the p53 knockout mice was obtained some 12
hours later and the value was significantly higher than in the
wild-type mice (P < 0.05). In the villi, Ki-67
labeling was very low, ranging from 0 to 0.2%, and was unchanged
throughout the experimental period (Figure 6
, bottom). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several recent studies emphasized the importance of the
p53-p21waf1/cip1 pathway and apoptotic cell death in acute alveolar
damage induced by bleomycin and other agents.12-14
Our
results were inconsistent with these and suggested it is unlikely that
the p53-p21waf1/cip1 pathway is obligatory for bleomycin-induced acute
alveolar damage and pulmonary fibrosis, as apoptotic cell death is.
There appear to be other essential factors initiating the early
inflammatory reactions.18-23
For example, a
transcriptional factor, NF-
ß, which is ubiquitously expressed and
activated in response to a variety of stimuli including DNA damage and
transactivates proinflammatory cytokines, is presumed to be involved in
the pathway mediating bleomycin-induced acute alveolar
damage.29-31
It would be of interest to examine the role
of p53 in transactivation of proinflammatory cytokines using this
experimental system with the aid of quantitative assessment of
bleomycin-induced pulmonary lesions.
In the small intestine, in contrast to the lung, a number of cells undergoing apoptotic cell death were observed in both the treated wild-type and p53 knockout mice. This result appears to support the conclusion of previous studies that this type of cell death was mediated not only in a p53-dependent, but also in a p53-independent manner.26 Consistent with previous studies,2,26 we confirmed here that p53-dependent apoptotic cell death occurred earlier and predominantly in the crypts, where p53 was expressed immediately after the bleomycin treatment, whereas p53-independent apoptotic cell death occurred a little later in the crypts and much later in the villi, where no p53 expression was detected. The differences in response to bleomycin treatment observed between the lung and small intestine, and between the crypts and villi, may be related either to the organ specificity of bleomycin toxicity16,32 or to the difference in cell types and particularly in cell kinetics, as discussed below.
As described above, no apoptotic cell death was seen in the bronchial and alveolar cells, but the ISNEL index was elevated soon after the bleomycin treatment. In the small intestine, the ISNEL index was also elevated in the crypts and villi; its change coincided with that in the apoptotic index in the wild-type mice, but it continued to be much higher in the p53 knockout mice. The ISNEL method can detect not only those DNA double strand breaks induced by endogenous apoptotic mechanisms, but also those directly caused by exogenous DNA-damaging agents.33,34 Thus, the ISNEL index in the small intestine is thought to reflect preferentially the apoptotic cell death in the wild-type mice, whereas in the p53 knockout mice it probably reflected both the apoptotic cell death and the DNA damage directly caused by bleomycin. However, it most likely reflected only the latter in the bronchial and alveolar cells of both groups of mice. The extremely high value of and delayed reduction in the ISNEL index observed in the lung of p53 knockout mice could be attributed exclusively to a delay of the repair of damaged DNA, implying that p53 is an important factor in the early response to and repair of DNA damage in vivo. The sustained high ISNEL index observed in the villi of the p53 knockout mice appears strongly supportive of the importance of this p53-dependent DNA repair in noncycling cells.
To evaluate the effect of the p53-p21waf1/cip1 pathway on cell kinetics, we determined the fraction of cycling cells as measured by Ki-67 antigen expression and the frequency of cells undergoing DNA synthesis as measured by BrdU uptake,28,35 and compared the values between the wild-type and p53 knockout mice. The incidence of cycling cells among the bronchial and alveolar cells as well as among the enterocytes of small intestinal villi was not changed significantly by the bleomycin treatment; it remained quite low in both the wild-type and p53 knockout mice. The BrdU uptake, in contrast, was rapidly elevated in these cell compartments in both groups of mice. Previous studies by others showed that the BrdU uptake in noncycling cells correlates well with unscheduled DNA synthesis in the repair of damaged DNA.36,37 Thus, the elevated BrdU uptakes observed here in the bronchial and alveolar cells and in the enterocytes of the villi most likely reflect the repair of damaged DNA. The delayed reduction of BrdU uptake as well as of the ISNEL index in the p53 knockout mice reconfirmed the significance of p53 in the early response to and repair of DNA damage in the lung. In the crypts of the small intestine, where Ki-67 expression and BrdU uptake showed almost coincidental changes, the BrdU uptake represents mostly DNA replication in the S phase and, to a small extent, unscheduled DNA synthesis. It is suggested that the p53-p21waf1/cip1 pathway is essential for induction of cell cycle arrest in response to bleomycin-induced DNA damage in the small intestinal crypt cells, because both the Ki-67 expression and BrdU uptake rapidly decreased in the wild-type mice, whereas these parameters increased, transiently but significantly, in the p53 knockout mice. This transient increase of cycling cells may be associated with the effects of early responsible genes, such as jun and fos, which are reported to be immediately expressed after DNA damage and to promote various cellular mechanisms including cell cycle progression.38,39
Taken together, our results confirmed that p53 plays important roles in the early response to and repair of the bleomycin-induced DNA damage in both the lung and small intestine in vivo. However, as observed here and in other studies,16,32 cellular responses to bleomycin treatment are different between these two tissue systems, and even in the small intestine they are distinctly different between the crypts and villi. This difference may be attributable mostly to cell type, because DNA damage was unequivocally evoked by the bleomycin treatment in all of the tissues as determined by the ISNEL index. The lung cells belong to the conditional renewal system, which is quiescent under normal physiological conditions, and the enterocytes of the villi are nonreplicating resting cells. On the other hand, the crypt epithelial cells belong to the renewal system, which constantly proliferates and turns over. The mode of response to DNA damage and the main pathway mediated by p53 appear to differ among these epithelial systems. That is, in the renewal system, either p53-dependent or p53-independent apoptotic cell death and cell cycle arrest play a major role, but in the conditional renewal system and in the resting cells as well, other mechanism(s) mediated by p53 may carry out an essential role, particularly under conditions where cell turnover and proliferation are not enhanced. In this context, it would be of interest to elucidate whether the cycling cells in the lung undergo either p53-dependent or p53-independent apoptotic cell death in response to bleomycin treatment, as do the crypt cells of the small intestine. The frequency of lung cells expressing p53 in the bleomycin-treated wild-type mice was much higher than that of Ki-67-labeled cells. Identification of the type of cells that expressed p53 was not easy, but both replicating cells (Clara cells, basal cells, and type 2 alveolar cells) and nonreplicating cells (ciliated cells) appeared to be labeled by p53 immunostaining. Although the possibility cannot be ruled out that the cycling cells, comprising a very small fraction of the lung cells, undergo apoptotic cell death in response to bleomycin treatment, we were unable to obtain any evidence of apoptotic cell death in the lung by the methods used in the current study.
p53 is known to have a variety of functions in the response to DNA damage and other stimuli.1,3 Several recent studies demonstrated that p53 directly binds to and repairs damaged DNA by catalyzing DNA renaturation and strand transfer.4,41,42 Such a function of p53 is presumed to be essential to the response to DNA damage in the conditional renewal system, including the lung cells, and in the resting cell populations, such as the enterocytes of the small intestinal villi, although further study on the response to DNA damage and the roles of p53 and other factors in noncycling cells is needed to confirm this possibility.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant from the Smoking Research Foundation, Japan.
Accepted for publication June 30, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiation. Oncogene 1997, 14:2759-2766[Medline]
ß B: ten years after. Cell 1996, 87:13-20[Medline]
-induced NF-ß activation and IL-8 release in A549 cells with the proteasome inhibitor MG132. Am J Respir Cell Mol Biol 1998, 19:259-268ß. Oncogene 1998, 17:1821-1826[Medline]
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