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Published ahead of print on August 27, 2004, doi:10.1165/rcmb.2003-0290OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 643-649, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0290OC

Cigarette Smoke Induces Senescence in Alveolar Epithelial Cells

Takao Tsuji, Kazutetsu Aoshiba and Atsushi Nagai

First Department of Medicine, Tokyo Women's Medical University, Tokyo, Japan

Address correspondence to: Kazutetsu Aoshiba, M.D., First Department of Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: kaoshiba{at}chi.twmu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or by telomere-independent signals (stress-induced senescence). The alveolar epithelium is often injured by a variety of inhaled toxins, including cigarette smoke (CS). In the present study, we investigated whether exposure to CS induces senescence of alveolar epithelial cells. In vitro experiments showed that exposure of A549 cells or normal human alveolar epithelial cells to sublethal concentrations of aqueous CS extracts induced cellular senescence. The senescence was characterized by a dose- and time-dependent increase in senescence-associated ß-galactosidase activity, senescence-associated changes in cell morphology, an increase in cell size and lysosomal mass, accumulation of lipofuscin, overexpression of p21CIP1/WAF1/Sdi1 protein, and irreversible growth arrest. In vivo experiments in Institute for Cancer Research mice showed that inhalation of CS for 2 wk induced increases in senescence-associated ß-galactosidase activity, lipofuscin accumulation, and p21CIP1/WAF1/Sdi1 protein expression in alveolar epithelial cells. These results suggest that CS induces a phenotype that is indistinguishable from that of senescence in alveolar epithelial cells. The induction of cellular senescence by CS may contribute to impaired re-epithelialization, leading to CS-related chronic lung diseases.

Abbreviations: bromodeoxyuridine, BrdU • bovine serum albumin, BSA • cyclin-dependent kinase, CDK • CDK inhibitor, CKI • cigarette smoke, CS • CS extracts, CSE • 3,3'-diaminobenzidine, DAB • fetal calf serum, FCS • immunoglobulin, Ig • human pulmonary alveolar epithelial, HPAEpi • horseradish peroxidase, HRP • N-acetylcysteine, NAC • reactive oxygen species, ROS • phosphate-buffered saline, PBS • surfactant protein, SP • senescence-associated ß-galactosidase, SA ß-gal


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or by telomere-independent signals, such as DNA damage and oxidative stress (stress-induced senescence) (1). The state of cellular senescence is accompanied by various phenotypic changes, including a distinct, flat, and enlarged cell morphology, an increase in senescence-associated ß-galactosidase (SA ß-gal) activity, accumulation of lipofuscin, and the expression of cell cycle inhibitors, such as p16INK4a and p21CIP1/WAF1/Sdi1 (1–3). Recent evidence suggests that cellular senescence plays an important role not only in physiologic aging processes, but in pathologic disease states, such as liver cirrhosis (4) and atherosclerosis (5). However, the role of cellular senescence in lung diseases has never been examined.

The alveolar epithelium is often injured by a variety of inhaled toxins, such as SO2, O3, NO2, and cigarette smoke (CS), and when injured, it initiates repair responses. Appropriate repair responses by alveolar epithelial cells require their integrated ability to migrate, proliferate, and differentiate to cover defects that result from the injury (6). Failure of the epithelium to repair itself is assumed to be an important cause of chronic lung diseases, such as pulmonary emphysema and fibrosis (7, 8). Epithelial injury and regeneration are thought to occur continually in such diseases, and the repeated cell cycles of the epithelial cells at the site of injury may shorten the length of telomeres, thereby potentially inducing replicative senescence. Inhaled toxins also generate oxidative stress and DNA damage in epithelial cells, which may cause stress-induced senescence. Once epithelial cells reach the senescence stage, they can no longer proliferate. The repair responses by alveolar epithelial cells may cease as a result, and the cessation of the repair responses may in turn result in architectural and functional disruptions in the alveolar epithelium that may allow lung diseases to progress.

Cigarette smoking is the most important risk factor for pulmonary emphysema and fibrosis. In fact, cigarette smoking is thought to be the cause of recurrent epithelial injury and impaired repair. For example, previous studies have shown that CS causes death of alveolar epithelial cells (9, 10), and that CS inhibits epithelial repair responses, such as chemotaxis, proliferation, and contraction of three-dimensional collagen gels (11, 12). We hypothesized that CS also induces senescence of alveolar epithelial cells and prevents their proliferating to repair injured epithelium. In the present study, we therefore investigated whether CS induces the senescence phenotypes of alveolar epithelial cells in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Treatment and CS Exposure
All exposure and handling of mice was approved by the Institutional Animal Care and Use Committee at the animal facility of Tokyo Women's Medical University. Eight-week-old male Institute for Cancer Research (ICR) mice (n = 6) were purchased from Japan SLC, Inc. (Shizuoka, Japan). For the experiments, three mice were placed in a vented plastic cage (27 x 27 x 18 cm) with a narrow orifice connected with a stopcock through which CS was delivered. Commercial plain-tipped (nonfiltered) cigarettes (Peace; Japan Tobacco Inc., Tokyo, Japan) yielding 24 mg tar and 2.4 mg nicotine under a standard smoking regimen were used in this study. For CS exposure, mainstream smoke puffs from 3 cigarettes (45 puffs/cigarette; 20 ml/puff; 1 s duration/puff in 10 s intervals) were delivered over 1 h with 1-min intervals at the end of every 20 min to refresh the air. The CS was allowed to escape through four exhaust holes (1 cm) on the side panels. During exposure, the animals were given access to water and food ad libitum in the exposure cage. After the exposure periods, the cage was refilled with room air. Control mice (n = 3) were placed in the cage and allowed to inhale room air. CS exposure was repeated daily on Days 1–5 each week for 2 consecutive weeks. At 6 h after the final CS exposure, mice were killed by intraperitoneal injection of pentobarbital (100 mg/kg), and the lungs were removed. The right lung was used for immunoblot analysis, and the left lung was inflated by manual instillation with 50% optimal cutting temperature compound, quickly frozen, and sectioned (3 µm).

Preparation of CS Extract Solution
The aqueous CS extract (CSE) was prepared according to a previously described method with a modification (13). Mainstream smoke was generated with one cigarette (Peace; Japan Tobacco Inc.) immediately before use by drawing consecutive puffs into a 20-ml plastic syringe with a stopcock connected through one port to a glass vessel containing 3 ml of dimethyl sulfoxide. A 20-ml puff drawn in 1 s was obtained at 10 s intervals. Each puff was held for 3 s and bubbled through the dimethyl sulfoxide in 5 s. One cigarette yielded an average of 45 puffs by this procedure. To prepare gas-phase CSE, the smoke was drawn into the syringe through a 0.22 µm pore-size filter (Milex-HA; Nihon Milipore, Ltd., Tokyo, Japan) rated to remove the particle phase of CS. The aqueous CSE was diluted in culture medium before use, and the CSE solution was prepared by the same person (T.T.) by exactly the same method and used within 3 min of preparation.

Cell Culture and In Vitro CS Exposure
Alveolar type II–like epithelial cell line A549 (American Type Culture Collection #CCL-185) was maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS). The cells (104 cells/cm2) between passages 27 and 35 were plated onto 8-well chamber slides or 100-mm tissue culture plates precoated with type I collagen (Vitrogen; Cohesion Technologies, Palo Alto, CA) and grown in the above medium. Once the cells reached 50% confluence, they were rinsed three times with phosphate-buffered saline (PBS) and incubated in Dulbecco's modified Eagle's medium containing 0.1% FCS with or without 0.0500–0.0001 volume % of aqueous CSE, in the presence or absence of N-acetylcysteine (NAC, 500 µM; Sigma-Aldrich Japan, Tokyo, Japan), ascorbic acid (500 µM, Sigma-Aldrich Japan), or catalase (500 U/ml, Sigma-Aldrich Japan).

Normal human pulmonary alveolar epithelial (HPAEpi) cells comprised of alveolar type I and type II cells were obtained from ScienCell Research Laboratories (San Diego, CA). HPAEpi cells are isolated from human lung tissue and cryoserved at primary culture and delivered frozen. Before the experiments, the cells were thawed, plated at a density of 104 cells/cm2 onto 8-well chamber slides precoated with type I collagen, and grown in the alveolar epithelial cell medium (ScienCell Research Laboratories) containing 5% FCS. Once the cells (passage 1) had reached 50% confluence, they were incubated in alveolar epithelial cell medium containing 0.1% FCS, with or without aqueous CSE.

Analysis for Cell Proliferation and Death
A549 cells or HPAEpi cells in 8-well chamber slides were incubated for 36 h with medium containing 0.1% FCS with or without CSE. After 24 h, the cells were pulse-labeled with 10 µM of bromodeoxyuridine (BrdU) for 1 h, and cells that had incorporated BrdU were detected immunocytochemically, as described below. For analysis of cell death, cells were stained with 5 µg/ml Hoescht33342 (Sigma-Aldrich Japan) and 5 µg/ml propidium iodide (Sigma-Aldrich Japan) and visualized by epifluorescence microscopy. Viable (normal, blue nuclei), apoptotic (condensed, blue nuclei), and necrotic (normal, red nuclei) cells were counted.

SA ß-gal Staining
SA ß-gal staining was performed according to a previously described method (14), with slight modifications. Cell samples in 8-well chamber slides were fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS for 5 min at room temperature. Frozen lung tissue sections were fixed with 0.2% glutaraldehyde in PBS for 5 h at 4°C. The slides were rinsed with PBS and incubated with an SA ß-gal staining solution containing 40 mM sodium citrate (pH 6.0), 150 mM NaCl, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, and 1 mg/ml of 5-bromo-4-chloro-3-indoyl ß-D galactoside (X-gal). After SA ß-gal staining, some slides were processed for immunostaining.

Schmorl Reaction
Lipofuscin was stained with the Schmorl reaction (15). Tissue sections were fixed with 3% paraformaldehyde in PBS for 10 min at room temperature, rinsed in PBS, and incubated for 5 min at room temperature in a solution containing 0.75% ferric chloride and 0.1% potassium ferricyanide. Some sections were then processed for immunostaining.

Immunohistochemistry and Immunocytochemistry
Tissue sections stained for SA ß-gal were incubated with 0.3% hydrogen peroxide for 10 min at room temperature to inhibit endogenous peroxidase activity before blocking the nonspecific binding sites with 3% bovine serum albumin (BSA) and 2% normal goat serum. The sections were then incubated at room temperature with rabbit polyclonal anti–surfactant protein (SP)-A (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h. The primary antibody was reacted with biotinylated anti-rabbit immunoglobulin (Ig) G conjugated with horseradish peroxidase (HRP)–labeled polymer (EnVison+ peroxidase; DAKO Japan, Kyoto, Japan). Immunoreactants were visualized with 3,3'-diaminobenzidine (DAB) and counterstained with nuclear fast red solution.

Tissue sections stained for lipofuscin with the Schmorl reaction were incubated with 3% BSA and 2% normal goat serum. The sections were then incubated at room temperature with rabbit polyclonal anti–SP-A (1:100; Santa Cruz Biotechnology, Inc.) for 1 h. The primary antibody was reacted with biotinylated anti-rabbit IgG conjugated with alkaline phosphatase–labeled polymer (EnVison+ alkaline phosphatase; DAKO Japan). Immunoreactants were visualized with the chromogen fast red/naphtol phosphate.

For double-staining for SP-A and p21CIP1/WAF1/Sdi1, tissue sections were first stained with anti–SP-A and then incubated with biotinylated anti-rabbit IgG conjugated with alkaline phosphatase–labeled polymer (EnVison+ alkaline phosphatase) and NBT/BCIP. The immunoreactants were removed by immersing the slides in a 0.1 M glycine buffer solution (pH 2.2). Next, the slides were stained with rabbit polyclonal anti-P21CIP1/WAF1/Sdi1 (1:100; Santa Cruz Biotechnology, Inc.) and incubated with biotinylated anti-rabbit IgG conjugated with HRP-labeled polymer (EnVison+ peroxidase; DAKO Japan) and a DAB solution. We found that replacement of the primary antibodies with the same concentration of control IgG showed no positive staining.

Tissue sections double-stained for SA ß-gal activity and SP-A antigen, for lipofuscin and SP-A, or for p21CIP1/WAF1/Sdi1 and SP-A antigens were assessed semiquantitatively. For each slide, 10 microscopic fields were randomly selected and viewed at a magnification of 200x. For each field, the number of cells positive for SP-A, the number of cells positive for SP-A with SA ß-gal activity, the number of cells positive for SP-A and lipofuscin, and the number of cells positive for SP-A and p21 were counted. The average percentages of cells positive for SP-A and SA ß-gal activity, of cells positive for SP-A and lipofuscin, and of cells positive for both SP-A and p21, in the population of cells positive for SP-A, were calculated.

For immunocytochemistry, cell samples in 8-well chamber slides were incubated with 0.3% H2O2 for 5 min and then incubated with 3% BSA in PBS for 30 min. The slides were reacted with anti–SP-A or anti-p21CIP1/WAF1/Sdi1 followed by the secondary antibody, as described above. Immunoreactants were visualized by a DAB reaction. For immunostaining with anti-BrdU, the slides were pretreated with 3 N HCl for 30 min, neutralized with 0.1 M borax buffer, pH 8.5, for 10 min, and then reacted with anti-BrdU antibody (1:100; Chemicon International, Temecula, CA).

Immunoblot Analysis
Cell lysates were solubilized in RIPA buffer (0.15 M NaCl, 50 mM Tris-Cl, pH 7.4, 0.5% NP40, and 0.1% sodium dodecyl sulfate) containing 10 µg/ml and 1 mM sodium vanadate. The samples were then fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and probed with mouse monoclonal anti-p21 antibody (1:400; Santa Cruz Biotechnology, Inc.) or rabbit polyclonal anti-actin (1:2500; Sigma-Aldrich Japan). The primary antibodies were detected with an HRP-conjugated antibody, which in turn was visualized by enhanced chemiluminescence (SuperSignal; Pierce, Rockford, IL).

Lysosomal Mass Analysis
Morphologic examination of the lysosomal mass was performed as previously described (16). Briefly, cells in 8-well chamber slides were incubated with 5 µg/ml of acridine orange (Sigma-Aldrich) for 10 min. After washing with PBS, the coverslips were mounted on slides and viewed immediately under an Olympus BX60 microscope with an Olympus longpass filter set comprised of a bandpass 450–490 nm exciter, a 510 nm dichroic mirror, and a long pass 520 nm emitter (Olympus, Tokyo, Japan).

Statistical Analysis
All data are expressed as means ± SEM. Differences were tested for significance by an analysis of variance and Scheffe's test, or by a two-tailed, unpaired Student t test, as appropriate. All calculations were performed using StatView 1.0 for Macintosh (Abacus Concepts, Inc., Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exposure to Aqueous CSE Induces Cellular Senescence in A549 Cells and Normal Human Alveolar Epithelial Cells
To determine whether exposure to CS induces cellular senescence in alveolar epithelial cells, we first exposed alveolar type II–like epithelial (A549) cells to aqueous CSE. Because trypan blue staining showed that CSE solution at 0.1 vol/vol% or higher caused the death of more than 20% of the A549 cells, we exposed the cells with CSE to sublethal concentrations of 0.05 vol/vol% or lower. Evaluation of cell death by staining with Hoechst33342 and propidium iodide and fluorescence microscopy demonstrated that most of the cell death induced by CSE at 0.1 vol/vol% and higher was necrosis, as recently reported (17). For example, culturing cells with 0.1 vol/vol% of CSE for 36 h caused the death of 22.8 ± 2.1% of cells (mean ± SEM of the results for four samples). A mean of 97.6 ± 2.2% of the dead cells underwent necrosis, whereas only 2.4 ± 2.2% (the rest of the population) underwent apoptosis.

When the A549 cells were exposed to CSE at 0.01 vol/vol% for 36 h, they exhibited a phenotype that is typical of cellular senescence (i.e., an increase in SA ß-gal activity) (Figure 1B), a distinct, flat, and enlarged morphology (Figure 1D), and an increase in lysosomal mass (Figure 1F) (1, 14, 16). When A549 cells were exposed to 0.01 vol/vol% of CSE solution before growth stimulation with 10% FCS, cellular uptake of BrdU was reduced to half the uptake by the control cells, suggesting that irreversible growth arrest had occurred (Figure 1G). When cells were exposed to CSE at 0.01 vol/vol% for 36 h, washed with PBS, and then stimulated with 10% FCS for a long period of time (i.e., 5 d), a reduction of BrdU uptake was still observed (28.1 ± 2.7% of BrdU uptake by control cells; n = samples), confirming the irreversibility of CSE-induced growth arrest. Quantitative analyses for cellular senescence showed that exposure of A549 cells to CSE caused a dose-dependent and time-dependent increase in SA ß-gal activity (Figures 2A and 2B), with ~ 60% of the A549 cells expressing SA ß-gal activity after exposure to 0.05 vol/vol% of CSE solution for 36 h.



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  		Figure 1. Senescent phenotype of A549 cells exposed to CSE. A549 cells were (B, D, and F) or were not exposed (A, C, and E) to CSE solution (0.01 vol/vol%) for 36 h and examined for markers of cellular senescence. (A and B) Staining for SA ß-gal activity (original magnification, x200). (C and D) Cell morphology, characterized by flat, enlarged cells, after exposure to CSE (D) (x200). (E and F) Lysosome staining with acridine orange shows a marked increase in the number and size of the lysosomes (orange fluorescence) in cells after treatment with CSE (F) (x200). The green fluorescence is emitted by the binding of acridine orange to nucleic acids. (G) Incorporation of BrdU by A549 cells. A549 cells were exposed to or not exposed to 0.01 vol/vol% of CSE solution for 36 h, rinsed with PBS, and then incubated with 10% FCS for 24 h. During the final 60 min of incubation, the cells were pulse-labeled with 10 µM of BrdU. Cells that had incorporated BrdU were detected by immunocytochemistry. **P < 0.01 versus control cells not treated with CSE. All data are the means ± SEM of the results for 4 samples.

 


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  		Figure 2. Quantitative analyses of SA ß-gal activity in A549 cells exposed to CSE. A549 cells were or were not exposed to various concentrations of CSE for 36 h (A and C), or exposed (closed circles) or not exposed (open circles) to 0.01 vol/vol% of CSE for various time periods (B). *P < 0.05 and **P < 0.01 versus cells not exposed to CSE. All data are means ± SEM of the results for four samples.

 
Exposure of A549 cells to CSE also increased the total number of cells expressing SA ß-gal (Figure 2C), indicating that the increase in the percentage of cells expressing SA ß-gal was not merely due to a decrease in overall cell number that may have occurred after cell death.

An immunoblot analysis also showed that exposure of the A549 cells to CSE caused a dose-dependent increase in the expression of p21CIP1/WAF1/Sdi1 protein, a cyclin-dependent kinase inhibitor that mediates cellular senescence in many types of cells (Figure 3) (18, 19). In accordance with the results of the immunoblot analysis, immunocytochemistry demonstrated that exposure to CSE enhanced cellular levels of p21CIP1/WAF1/Sdi1 expression (photographs not shown). The enhanced level of p21CIP1/WAF1/Sdi1 expression was evident even after a period of 5 d of incubation with 10% FCS, after 36 h of pretreatment with CSE (17.6 ± 3.0% immunopositive cells in CSE-pretreated cells versus 5.8 ± 2.7% immunopositive cells in untreated cells; n = 4, P < 0.01).



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  		Figure 3. Immunoblot analysis of p21CIP1/WAF1/Sdi1 protein in A549 cells after treatment with CSE. A549 cells were exposed to various concentrations of CSE or 50 µg/ml of bleomycin (positive control) for 36 h. The cell lysate (50 µg per lane) was then analyzed by immunoblotting with anti-p21 or anti-actin antibodies.

 
Because A549 cells are a lung cancer cell line that may not reflect the senescence properties of normal alveolar epithelial cells, we performed experiments using alveolar type II cells isolated from normal human lungs (HPAEpi cells). When exposed to CSE, HPAEpi cells also exhibited a flat, enlarged morphology (Figure 4B), an increase in SA ß-gal activity (Figure 4C), and irreversible growth arrest (Figure 4D), suggesting that CS induces senescence of normal alveolar epithelial cells.



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  		Figure 4. Senescent phenotype of normal HPAEpi cells exposed to CSE. HPAEpi cells were or were not exposed to CSE solution (0.01 vol/vol%) for 36 h and examined for markers of cellular senescence. (A and B) Cell morphology, characterized by flat, enlarged cells, after exposure to CSE (B), compared with cells not exposed to CSE (A) (x200). (C) SA ß-gal activity. Cells were stained for SA ß-gal activity and then immunostained with anti–SP-A. The number of cells positive for both SA ß-gal activity and anti–SP-A immunostaining was calculated as a percentage of the total number of cells positive for anti–SP-A immunostaining. The photomicrograph shows representative cells that stained positive for both SA ß-gal activity and SP-A (original magnification, x1000). (D) Incorporation of BrdU by HPAEpi cells. HPAEpi cells were or were not exposed to 0.01 vol/vol% CSE solution for 36 h, rinsed with PBS, and then incubated with 10% FCS for 24 h. During the final 60 min of incubation, the cells were pulse-labeled with 10 µM of BrdU. Cells were immunostained for BrdU and SP-A, and the number of cells positive for both BrdU and SP-A was calculated as a percentage of the total number of cells positive for SP-A. **P < 0.01 versus control cells not exposed to CSE. All data are means ± SEM; n = 4.

 
Because CS contains reactive oxygen species (ROS) (20), we investigated whether various antioxidants would prevent the cellular senescence induced by CS. As shown in Figure 5A, the CS-induced expression of SA ß-gal activity by A549 cells was significantly inhibited by NAC, suggesting that the CS-induced cellular senescence is mediated, at least in part, by oxidative stress. Removing the particle (i.e., tar) component from whole smoke partially reduced the expression of SA ß-gal activity (Figure 5B), suggesting that constituents present in both the gas-phase and tar-phase components of CS have a capacity for inducing cellular senescence. Senescence of A549 cells was not induced by nicotine (10–6 M), a major tar component of CS (data not shown).



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  		Figure 5. Effect of antioxidants and removal of the particle component from whole smoke on CS-induced enhancement of SA ß-gal activity. (A) A549 cells were or were not exposed to 0.01 vol/vol% CSE for 36 h in the presence or absence of catalase (500 U/ml), ascorbic acid (500 µM), and NAC (500 µM). (B) A549 cells were or were not exposed to 0.01 vol/vol% whole or gas-phase CSE for 36 h. *P < 0.05 versus cells exposed to whole CSE. {dagger}{dagger}P < 0.01 versus control cells not exposed to CSE. All data are means ± SEM; n = 4.

 
CS Induces Alveolar Epithelial Senescence In Vivo
To determine whether CS also induces senescence of alveolar epithelial cells in vivo, mice were exposed to the smoke from 3 cigarettes for 1 h daily on Days 1–5 each week for 2 wk. Except for the alveolar macrophages, which are enriched with lysosomal ß-galactosidase (21), the SA ß-gal staining of alveolar epithelial cells was almost completely absent in the lungs of mice that were not exposed to CS (Figure 6A), whereas alveolar epithelial cells that stained positive for SA ß-gal activity were frequently observed in the lungs of mice exposed to CS for 2 wk (Figure 6B). The SA ß-gal staining was most intense in the alveolar cuboidal epithelial cells in the alveolar corners, which were identified as type II cells both by their shape and location and by positive immunostaining for SP-A antigen (Figure 6D). Tissue sections were also stained for cellular accumulation of lipofuscin, another marker of cellular senescence (15, 22). The Schmorl reaction showed that lipofuscin was accumulated in type II cells in the lungs of mice exposed to CS for 2 wk (Figure 6L), whereas lipofuscin was almost absent in the lungs of mice that were not exposed to CS (Figure 6K). Immunohistochemistry for p21CIP1/WAF1/Sdi1, a senescence-associated, cyclin-dependent kinase inhibitor, showed overexpression of p21CIP1/WAF1/Sdi1 protein in the lungs of mice exposed to CS (Figure 6F). The immunosignal was most intense in the cuboidal alveolar epithelial cells in the alveolar corners. These cells were identified as type II cells on the basis of their shape and location (Figure 6F) and by their positive immunostaining for SP-A antigen (Figure 6H). The p21CIP1/WAF1/Sdi1 immunosignal appeared to be somewhat diffuse and was not localized to the nucleus of the type II cells. We believe that there are two possible explanations for this: first, p21CIP1/WAF1/Sdi1 protein has recently been shown to be present in the cytoplasm as well as the nucleus (23), and, second, CS may also induce p21 expression in endothelial cells and fibroblasts located in the alveolar wall, thereby perpetuating the presence of diffuse immunosignals. We found that replacement of anti-p21CIP1/WAF1/Sdi1 antibody with the same concentration of control IgG showed no positive staining (Figures 6I and 6J). An immunoblot analysis confirmed the immunohistochemical findings showing that p21CIP1/WAF1/Sdi1 protein is overexpressed in the lungs of mice exposed to CS for 2 wk (Figure 6M). When the SA ß-gal staining was analyzed semiquantitatively, mice exposed to CS showed a 5-fold increase in the incidence of type II cells positive for SA ß-gal activity over the control mice (Figure 7A). Similarly, mice exposed to CS showed a 5-fold increase in the incidence of type II cells positive for lipofuscin accumulation over the control mice (Figure 7B). Furthermore, mice exposed to CS also showed a 4-fold increase in the incidence of type II cells positive for p21CIP1/WAF1/Sdi1 protein (Figure 7C). These results suggest that CS induces senescence phenotypes in alveolar epithelial cells in vitro and in vivo.



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  		Figure 6. SA ß-gal activity (A–D), p21CIP1/WAF1/Sdi1 protein expression (E–H), and lipofuscin accumulation (K and L) in lung tissue sections from mice killed after inhalation of CS (B, D, F, H, J, and L) or clean air (A, C, E, G, I, and K) for 2 wk. (A–D) SA ß-gal staining (green color) followed either by nuclear staining with a fast red solution (red color, A and B) or by anti–SP-A immunostaining to identify type II epithelial cells (brown color, C and D). The inset in D shows a higher magnification of a type II epithelial cell that stained positive for both SA ß-gal and SP-A. (E–H) Cells were immunostained for anti-p21 (brown color). (G and H) Cells that had been immunostained for anti-p21 were further stained for anti–SP-A (purple color). The inset in F shows a higher magnification of type II epithelial cells (i.e., alveolar cuboidal epithelial cells located in an alveolar corner) and stained positive for p21CIP1/WAF1/Sdi1. The inset in H shows a higher magnification of type II epithelial cells that stained positive for both SP-A and p21CIP1/WAF1/Sdi1. (I and J) Results of replacement of anti-p21CIP1/WAF1/Sdi1 antibody with the same concentration of control IgG after anti–SP-A immunostaining. (L) Accumulation of lipofuscin detected by the Schmorl reaction, which specifically stains lipofuscin-containing granules (greenish blue color) (15). The inset in L shows a type II epithelial cell that has stained positive by both the Schmorl reaction and anti–SP-A immunostaining (red color). Original magnification: A, B, E, F, and I–L, x200; C, D, G, and H, x400; insets in D, F, H, and L, x1,000. (M) Results of immunoblot analyses for p21CIP1/WAF1/Sdi1 protein and actin in lung tissue obtained from mice killed after inhalation of CS (lanes 1–3) or clean air (lanes 4–6) for 2 wk. The optical density of each band was determined, and p21/actin ratio is shown below each lane.

 


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  		Figure 7. Semiquantitative analyses of SA ß-gal activity (A), lipofuscin accumulation (B), and p21CIP1/WAF1/Sdi1 protein expression (C) in lung tissue sections from mice that inhaled CS or clean air. The numbers of cells positive for both SA ß-gal activity and anti–SP-A, both lipofuscin accumulation and anti–SP-A, or both anti-p21CIP1/WAF1/Sdi1 and anti–SP-A, were calculated as a percentage of the total number of cells positive for anti–SP-A immunostaining. Data shown are means ± SEM; n = 3. **P < 0.01 versus mice that inhaled clean air.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study demonstrate that sublethal concentrations of CS induce senescence phenotypes in alveolar epithelial cells both in vitro and in vivo. Previous studies have shown that CS has cytotoxic effects on alveolar epithelial cells (e.g., high concentrations of CS have been reported to ultimately cause the death of A549 cells by either apoptosis or necrosis) (24). To the best of our knowledge, our study provides the first evidence that CS induces the senescence of alveolar epithelial cells at sublethal concentrations.

The mechanism by which CS induces the senescence of alveolar epithelial cells remains uncertain. In the present study, however, we found that the CS-induced senescence of alveolar epithelial cells was inhibited by the addition of the antioxidant NAC, suggesting that oxidative stress is involved in signaling pathways mediating the CS-induced senescence. In fact, CS is a rich source of ROS and ROS inducers, and ROS, particularly at low levels, has been shown to induce senescence in some types of cells. In the present study, however, the inhibition of the CS-induced senescence of alveolar epithelial cells by NAC was only partial, and other antioxidants, such as ascorbic acid and catalase, had no significant effect on the CS-induced senescence. We therefore suspect that mechanisms other than oxidative stress may also contribute to the CS-induced senescence of alveolar epithelial cells.

Our findings also show that the CS-induced senescence of alveolar epithelial cells is associated with the accumulation of p21CIP1/WAF1/Sdi1 protein. Recent studies have shown that two families of cyclin-dependent kinase inhibitors (CKIs) are involved in the intracellular signaling pathways mediating cellular senescence (19). The first family, inhibit cyclin-dependent kinase (INK) 4, includes p15INK4b, p16INK4a, p18INK4c, and p19INK4 d,which bind specifically to cyclin-dependent kinase (CDK) 4 and CDK6 and prevent the formation of cyclin D–CDK complexes. p21CIP1/WAF1/Sdi1, p27KIP1, p57KIP2 form the second family, CIP/KIP, which binds to CDK4-cyclin D, CDK6-cyclin D, CDK2-cyclin E, and CDK6-cyclin D complexes (19). Our finding of an enhanced level of p21CIP1/WAF1/Sdi1 protein in A549 cells after CS exposure corroborates that of a previous study showing that exposure to either CSE or H2O2 increases the level of p21CIP1/WAF1/Sdi1 mRNA in those cells (25). However, because the two families of CKIs have been shown to interact with each other, we cannot exclude the possibility that other members of the CKI families are also involved in the signaling pathway that mediates the CS-induced senescence of alveolar epithelial cells.

The first limitation of this study was that we used A549 cells, which are immortalized cancer cells that lack the proliferative controls found in nontransformed cells. However, we found that normal human alveolar epithelial cells exposed to CSE also exhibited senescence phenotypes, including SA ß-galactosidase activity, a distinct, flat, and enlarged morphology, and irreversible growth inhibition. We also found that the alveolar epithelial cells of mice that inhaled CS showed an increase in SA ß-gal activity, suggesting that the response of A549 cells to CS is not very different from that of normal alveolar epithelial cells.

The second limitation of this study was that we used a SA ß-gal staining method to identify senescent cells in lung tissues. This method has been widely used by many investigators to identify senescent cells both in vitro and in vivo (4, 5, 18, 26–29). However, SA ß-gal activity may not be specific for senescence in all tissues, as it may also represent the expression of normal endogenous lysosomal acid ß-galactosidase, and may not distinguish senescent cells from quiescent or terminally differentiated cells (16, 30, 31). Determination of telomere length by in situ hybridization would be an alternative method of detecting senescent cells in vivo, but it is not suitable for the detection of telomere-independent stress-induced senescence, which is presumably the case in CS exposure. In the present study, an enhanced level of lipofuscin accumulation in type II cells after CS exposure supports the results obtained by the SA ß-gal staining method. However, because no absolute markers enabling specific identification of cellular senescence in vivo are yet available, we can not completely rule out the possibility that SA ß-gal activity might also reflect cell conditions other than senescence.

The third limitation of this study arose from the CS exposure experiments. First, although in vitro exposure to CSE is a standard procedure, its relevance to the in vivo state remains unclear. Second, the CS exposure in vivo in the present study seemed to be very intense. Third, because mice are obligatory nasal breathers, some toxic products that would normally be inhaled by humans may have been deposited in the nasal passages of the mice. Although these limitations must be taken into account when interpreting the results of this study in relation to cigarette smokers, the results of the present study demonstrate that CS has the ability to induce senescence of alveolar epithelial cells.

Recent evidence indicates that pulmonary emphysema, an important cigarette-smoking–related disorder, is a dynamic disease associated with increased turnover of alveolar cells. For example, apoptosis of alveolar epithelial and endothelial cells has been shown to be enhanced in the lungs of patients with pulmonary emphysema (7, 32, 33). To repair the alveolar architecture, the loss of alveolar cells resulting from apoptosis must be offset by proliferation of the remaining alveolar cells. In fact, we previously found that both the apoptosis and proliferation of alveolar epithelial cells are enhanced in emphysema patients, showing that the turnover of alveolar epithelial cells is elevated in emphysematous lungs as a result of recurrent epithelial death and proliferation (7). The results of the present study show that CS induces the senescence of alveolar epithelial cells. The induction of senescence may prohibit epithelial cells from proliferating to repopulate the loss of epithelial cells resulting from apoptosis in emphysematous lungs.

Based on the above observations, we propose that cellular senescence may be involved in the CS-related pulmonary diseases associated with chronic epithelial damage. As shown by the results of the present study, cigarette smoking may induce the senescence of alveolar epithelial cells. In addition, the continuous proliferation of alveolar epithelial cells required to regenerate their loss as a result of apoptosis or necrosis accelerates telomere shortening, which in turn leads to the senescence of alveolar epithelial cells. When the alveolar epithelial cells reach the senescence stage, epithelial proliferation ceases, and the alveolar damage in cigarette smokers is no longer repaired. This model provides a plausible explanation for the chronic nature of CS-related pulmonary diseases, such as pulmonary emphysema and fibrosis, which evolve slowly over many years.

Recent studies have also shown that senescent cells produce higher levels of matrix metalloproteinases, such as collagenases and stromelysin, and profibrotic cytokines, such as interleukin-1ß and transforming growth factor-ß, than those produced by normal cells (34–36). Thus, the cellular senescence induced by CS may contribute not only to the failure of epithelial regeneration but also to inflammatory responses and tissue remodeling.

In conclusion, we found that CS induces senescence of alveolar epithelial cells both in vitro and in vivo. A more detailed characterization of cellular senescence in relation to cigarette smoking should shed light on the mechanism of CS-induced lung diseases.


   Acknowledgments
 
The authors are very grateful to Masayuki Shino and Yoshimi Sugimura for their technical assistance. This work was supported by a grant from the Respiratory Failure Research Group of the Ministry of Health, Labor, and Welfare of Japan.

Received in original form August 5, 2003

Received in final form August 5, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hayflick, L., and P. S. Moorhead. 1961. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 25:585–621.[CrossRef][Medline]
  2. Marcotte, R., and W. Wang. 2002. Replicative senescence revisited. J. Gerontol. 57A:B257–B269.
  3. Hornsby, P. J. 2002. Cellular senescence and tissue aging in vivo. J. Gerontol. 57A:B251–B256.
  4. Wiemann, S. U., A. Satyanarayana, M. Tsahuridu, H. L. Tillmann, L. Zender, J. Klempnauer, P. Flemming, S. Franco, M. A. Blasco, M. P. Manns, and K. L. Rudolph. 2002. Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis. FASEB J. 16:935–942.[Abstract/Free Full Text]
  5. Minamino, T., H. Miyauchi, T. Yoshida, Y. Ishida, H. Yoshida, and I. Komuro. 2002. Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation 105:1541–1544.[Abstract/Free Full Text]
  6. Rennard, S. I. 1999. Inflammation and repair processes in chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 160:s12–s16.
  7. Yokohori, N., K. Aoshiba, and A. Nagai. 2004. Increased levels of cell death and proliferation in alveolar wall cells in patients with pulmonary emphysema. Chest 125:626–632.[Abstract/Free Full Text]
  8. Selman, M., T. E. King, and A. Pardo. 2001. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann. Intern. Med. 134:136–151.[Abstract/Free Full Text]
  9. Lannan, S., K. Donaldson, D. Brown, and W. MacNee. 1994. Effect of cigarette smoke and its condensates on alveolar epithelial cell injury in vitro. Am. J. Physiol. 266:L92–L100.
  10. Hoshino, Y., T. Mio, S. Nagai, H. Miki, I. Ito, and T. Izumi. 2001. Cytotoxic effects of cigarette smoke extracts on an alveolar type II cell derived cell line. Am. J. Physiol. 281:L509–L516.
  11. Cantral, D. E., J. H. Sisson, T. Veys, S. I. Rennard, and J. R. Spurzem. 1995. Effects of cigarette smoke extract on bovine bronchial epithelial cell attachment and migration. Am. J. Physiol. 268:L723–L728.
  12. Wang, H., X. Liu, T. Umino, C. M. Skold, Y. Zhu, T. Kohyama, J. R. Spurzem, D. J. Romberger, and S. I. Rennard. 2001. Cigarette smoke inhibits human bronchial epithelial cell repair process. Am. J. Respir. Cell Mol. Biol. 25:772–779.[Abstract/Free Full Text]
  13. Aoshiba, K., A. Nagai, and K. Konno. 1994. Nicotine prevents a reduction in neutrophil filterability induced by cigarette smoke exposure. Am. J. Respir. Crit. Care Med. 150:1101–1107.[Abstract]
  14. Dimri, G. P., X. Lee, G. Basile, M. Acosta, G. Scott, C. Roskelley, E. E. Medrano, M. Linskens, I. Rebelj, O. Pereira-Smith, M. Peacocke, and J. Campisi. 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92:9363–9367.[Abstract/Free Full Text]
  15. Bancroft, J. D. 1967. An Introduction To Histochemical Techniques. Butterworths, London. 148–175.
  16. Kurz, D. J., S. Decary, Y. Hong, and J. D. Erusalimsky. 2000. Senescence-associated ß-glactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J. Cell Sci. 113:3613–3622.[Abstract]
  17. Wickenden, J. A., M. C. H. Clarke, A. G. Rossi, I. Rahman, S. P. Faux, K. Donaldson, and W. MacNee. 2003. Cigarette smoke prevents apoptosis through inhibition of caspase activation and induces necrosis. Am. J. Respir. Cell Mol. Biol. 29:562–570.[Abstract/Free Full Text]
  18. Wang, Y., G. Blandino, and D. Givol. 1999. Induced p21waf expression in H1299 cell line promotes cell senescence and protects against cytotoxic effect of radiation and doxorubicin. Oncogene 18:2643–2649.[CrossRef][Medline]
  19. Marcotte, R., and E. Wang. 2002. Replicative senescence revisited. J. Gerontol. A Biol. Sci. Med. Sci. 57:B257–B269.[Abstract/Free Full Text]
  20. Pryor, W. A., and K. Stone. 1993. Oxidants in cigarette smoke radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann. N. Y. Acad. Sci. 686:12–27.[Medline]
  21. Skudlarek, M. D., and R. T. Swank. 1979. Biosynthesis of two lysosomal enzymes in macrophages: evidence for a precursor of beta-galactosidase. J. Biol. Chem. 254:9939–9942.[Abstract/Free Full Text]
  22. Nakano, M., F. Oenzil, T. Mizuno, and S. Gotoh. 1995. Age-related changes in the lipofuscin accumulation of brain and heart. Gerontology 41:69–79.
  23. Puddicombe, S. M., C. Torres-Lozano, A. Richter, F. Bucchieri, J. L. Lordan, P. H. Howarth, B. Vrugt, R. Albers, R. Djukanovic, S. T. Holgate, S. J. Wilson, and D. E. Davies. 2003. Increased expression of p21(waf) cyclin-dependent kinase inhibitor in asthmatic bronchial epithelium. Am. J. Respir. Cell Mol. Biol. 28:61–68.[Abstract/Free Full Text]
  24. Hoshino, Y., T. Mio, S. Nagai, H. Miki, I. Ito, and T. Izumi. 2001. Cytotoxic effects of cigarette smoke extract on an alveolar type II cell–derived cell line. Am. J. Physiol. Lung Cell. Mol. Physiol. 281:L509–L516.[Abstract/Free Full Text]
  25. Marwick JA, Kirkham P, Gilmour PS, Donaldson K, MacNee W, Rahman I. 2002. Cigarette smoke–induced oxidative stress and TGF-beta1 increase p21waf1/cip1 expression in alveolar epithelial cells. Ann. N.Y. Acad. Sci. 973:278–283.[Abstract/Free Full Text]
  26. Robles, S. J., and G. R. Adami. 1998. Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts. Oncogene 16:1113–1123.[CrossRef][Medline]
  27. te Poele, R. H., A. L. Okorokov, L. Jardine, J. Cummings, and S. P. Joel. 2002. DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res. 62:1876–1883.[Abstract/Free Full Text]
  28. Serrano, M., A. W. Lin, M. E. McCurrach, D. Beach, and S. W. Lowe. 1997. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INKa. Cell 88:593–602.[CrossRef][Medline]
  29. Bodnar, A. G., M. Ouellette, M. Frolkis, S. E. Holt, C. Chiu, G. B. Morin, C. B. Harley, J. W. Shay, S. Lichtsteiner, and W. E. Wright. 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279:349–352.[Abstract/Free Full Text]
  30. Coates, P. J. 2002. Markers of senescence? J. Pathol. 196:371–373.[CrossRef][Medline]
  31. Krishna, D. R., B. Sperker, P. Fritz, and U. Klotz. 1999. Does pH 6 beta-galactosidase activity indicate cell senescence? Mech. Ageing Dev. 109:113–123.[CrossRef][Medline]
  32. Kasahara, Y., R. M. Tuder, C. D. Cool, D. A. Lynch, S. C. Flores, and N. F. Voelkel. 2001. Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am. J. Respir. Crit. Care Med. 163:737–744.[Abstract/Free Full Text]
  33. Segura-Valdez, L., A. Pardo, M. Gaxiola, B. D. Uhal, C. Becerril, and M. Selman. 2000. Upregulation of gelatinases A and B, collagenases 1 and 2, and increased parenchymal cell death in COPD. Chest 117:684–694.[Abstract/Free Full Text]
  34. Sottile, J., D. M. Mann, V. Diemer, and A. J. Millis. 1989. Regulation of collagenase and collagenase mRNA production in early- and late-passage human diploid fibroblasts. J. Cell. Physiol. 138:281–290.[CrossRef][Medline]
  35. Kumar, S., A. J. Millis, and C. Baglioni. 1992. Expression of interleukin 1–inducible genes and production of interleukin 1 by aging human fibroblasts. Proc. Natl. Acad. Sci. USA 89:4683–4687.[Abstract/Free Full Text]
  36. Ding, G., N. Franki, A. A. Kapasi, K. Reddy, N. Gibbons, and P. C. Singhal. 2001. Tubular cell senescence and expression of TGF-beta1 and p21 (WAF1/CIP1) in tubulointerstitial fibrosis of aging rats. Exp. Mol. Pathol. 70:43–53.[CrossRef][Medline]



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