Cip1 in large airway epithelium in smokers with and without COPD

Biochimica et Biophysica Acta 1832 (2013) 1473–1481

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The role of p21 Waf1/Cip1 in large airway epithelium in smokers with and without COPD Giuseppina Chiappara a, Mark Gjomarkaj a,⁎, Alessia Virzì a, Serafina Sciarrino a, Maria Ferraro a, Andreina Bruno a, Angela Marina Montalbano a, Patrizio Vitulo b, Marta Ida Minervini c, Loredana Pipitone b, Elisabetta Pace a a b c

Istituto di Biomedicina e Immunologia Molecolare (I.B.I.M.)-Consiglio Nazionale delle Ricerche (CNR), Palermo, Italy Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (ISMETT), Palermo, Italy University of Pittsburgh, Department of Pathology, Pittsburgh, PA, USA

a r t i c l e

i n f o

Article history: Received 2 February 2013 Received in revised form 27 March 2013 Accepted 22 April 2013 Available online 29 April 2013 Keywords: Apoptosis Proliferation Metaplasia Epithelium COPD

a b s t r a c t Airway epithelium alterations, including squamous cell metaplasia, characterize smokers with and without chronic obstructive pulmonary disease (COPD). The p21 regulates cell apoptosis and differentiation and its role in COPD is largely unknown. Molecules regulating apoptosis (cytoplasmic p21, caspase-3), cell cycle (nuclear p21), proliferation (Ki67/PCNA), and metaplasia (survivin) in central airways from smokers (S), smokers-COPD (s-COPD) and non-smokers (Controls) were studied. The role of cigarette smoke extracts (CSE) in p21, survivin, apoptosis (caspase-3 and annexin-V binding) and proliferation was assessed in a bronchial epithelial cell line (16HBE). Immunohistochemistry, image analysis in surgical samples and flow-cytometry and carboxyfluorescein succinimidyl ester proliferative assay in 16HBE with/without CSE were applied. Cytoplasmic and nuclear p21, survivin, and Ki67 expression significantly increased in large airway epithelium in S and in s-COPD in comparison to Controls. Caspase-3 was similar in all the studied groups. p21 correlated with epithelial metaplasia, PCNA, and Ki67 expression. CSE increased cytoplasmic p21 and survivin expression but not apoptosis and inhibited the cell proliferation in 16HBE. In large airway epithelium of smokers with and without COPD, the cytoplasmic p21 inhibits cell apoptosis, promotes cell proliferation and correlates with squamous cell metaplasia thus representing a potential pre-oncogenic hallmark. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The bronchial epithelium has a central role in coordinating the main responses seen in chronic obstructive pulmonary disease (COPD) [1]. The major risk factor for COPD is cigarette smoke. Cigarette smoke contributes to the development of the disease by a variety of mechanisms: it affects chemotaxis, proliferation, and remodeling of extra-cellular matrix in epithelial cells derived from normal human airways [2]. In this context, alterations in epithelial repair may be important contributors to the architectural changes that can lead to airflow limitation in COPD [3]. Alterations of airway epithelium, including squamous cell metaplasia, goblet and basal cell hyperplasia, are often present in smokers with and without COPD [4,5] and are considered by the World Health Organization as pre-invasive precursor lesions for lung cancer [6]. Abbreviations: COPD, chronic obstructive pulmonary disease; S, smokers; CSE, cigarette smoke extracts; PCNA, proliferating cell nuclear antigen; IAPs, inhibitor of apoptosis proteins; CFSE, carboxyfluorescein succinimidyl ester ⁎ Corresponding author at: Istituto di Biomedicina e Immunologia Molecolare-Consiglio Nazionale delle Ricerche, Via Ugo La Malfa, 153-90146 Palermo, Italy. Tel.: +39 091 680 9125; fax: +39 091 680 9122. E-mail address: [email protected] (M. Gjomarkaj). 0925-4439/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbadis.2013.04.022

Differentiation of multiple tissue types, including squamous epithelia, is associated to an increased p21Waf1 expression. p21, acting as a sensor of cellular stress, might have a role in lung responses triggered by cigarette smoke [7]. In particular, the intracellular distribution of p21 (nuclear or cytoplasmic) may have different functional consequences. Nuclear p21 induces cell cycle arrest and differentiation and it blocks DNA replication by binding to proliferating cell nuclear antigen (PCNA) [8,9]. In oral squamous cell carcinoma, detectable nuclear p21 is associated with higher cell proliferation metaplastic markers such as nuclear Ki67 [10]. Membranous and cytoplasmic Ki67 immunoreactivities have been observed in a number of histopathological entities, including epithelial alterations in asthma [11] but the frequency and the relationship to COPD have not been described yet [12]. Cytoplasmic p21 activity may be both pro-apoptotic and antiapoptotic depending also from the different cell phenotypes. Accordingly, during monocyte differentiation cytoplasmic p21 inhibits the apoptosis of these cells [8,13] and previous findings indicate that p21 plays a fundamental role in the protection against cytotoxic stimulation of certain cell types. However, the mechanism by which p21 antagonizes apoptosis is still unknown [13]. Apoptosis progression relies on the interplay between pro-survival factors and pro-apoptotic factors, and this balance crucially decides whether a cell under stress lives or dies. Pro-survival proteins,

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including survivin, a member of inhibitor of apoptosis proteins (IAPs), are able to inhibit the apoptotic executors: i.e. cytoplasmic caspases [14]. Survivin levels are increased in smokers in comparison to non-smokers and survivin expression in exhaled breath condensate in patients with lung cancer positively correlates with the number of cigarettes smoked [15]. The aims of our study are to assess whether cytoplasmic and nuclear p21, Ki67, PCNA and survivin expression are altered in large airway epithelium in smokers and in smokers with COPD and to investigate whether these alterations contribute to the development of airway epithelial squamous metaplasia.

assessed as positive epithelial cells/mm basement membrane. Results were expressed as medians and 25–75 percentiles [18]. Survivin immunolocalization was assessed using a composite score [19,20]. Staining intensities were scored as no staining (0), weak staining [1], moderate staining [2], or strong staining [3]. The percentage of staining area was classified as 0 (0%); 1 (1–10%); 2 (11–50%); and 3 (51–100%). Composite scores of 1–3 were defined as having low survivin protein expression, and scores of 4–9 were considered to be high expression of survivin (Supplemental materials).

2.3. Metaplastic bronchial epithelium analysis 2. Materials and methods 2.1. Study population The study population of this study was recruited at ISMETT in Palermo, Italy. All recruited patients underwent surgery for lung cancer. The study protocol was approved by the ISMETT Ethic Committee (IRRB07/2008) and informed written consent was obtained from each patient. The patients were classified into the following groups as previously reported [16]: 1) non smoking patients (Controls) without chronic pulmonary underlying events (n = 11); 2) smoking patients (>15 pack/year) without COPD (S) (n = 12); 3) smoking patients (>15 pack/year) with COPD (s-COPD) (n = 17). COPD patients were treated with bronchodilators and were classified on the basis of preoperative lung function: FEV1 less than 80% of reference, FEV1/FVC less than 70%, and broncho-dilatation reversibility less than 12%. The following variables at admission were recorded: age, gender, smoking habits (Table 1) (Supplemental materials).

Metaplastic epithelium analysis was performed by Image Analysis software (Leica Quantimet system) [4]. The length of the basement membrane was traced of all intact non-squamous metaplastic epithelium (A), intact squamous metaplastic epithelium (B), and damaged epithelium (C), in order to calculate the % of intact epithelium [(A + B) / (A + B + C)] and the % of metaplastic epithelium [B / (A + B)]. In addition, the presence of metaplastic epithelium was also scored as absent (0) or present (Supplemental materials).

2.4. Preparation of cigarette smoke extracts (CSE) Each cigarette (Marlboro) was smoked for 5 min in 25 ml phosphatebuffered saline (PBS) and cigarette smoke solution was prepared as described previously [21]. The CSE–PBS solution was filtered through a 0.22-μm pore filter, adjusted to pH 7.4 and used within 30 min of preparation. This solution was considered to be 100% CSE (Supplemental materials).

2.2. Histology Bronchial rings were taken from lung tissues, away from the tumor site, fixed (10% neutral buffer formalin) and embedded in paraffin wax. A pathologist examined the bronchial rings and confirmed that they were tumor free and surrounded by tumor free tissue. Deparaffinized sequential sections (4 μm thick) were stained with hematoxylin and eosin (HE) and used for immunohistochemistry [17]. The following antibodies were used: monoclonal mouse anti-p21 (sc-817; Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:10), monoclonal mouse anticaspase-3, specific for the active form, (Am-65; Calbiochem-Merck KGaA, Darmstadt, Germany) (1:5), monoclonal mouse anti-Ki67 antigen (M 7240 Clone MIB-1; DAKO, Glostrup, Denmark) (1:25), monoclonal mouse anti-PCNA (PC10-M0879; DAKO) (1:5), and rabbit polyclonal anti-survivin (NB500-201; Novus Biologicals, Littleton, CO, USA) (1:200); (heat pre-treatment at 92 °C; overnight incubation at room temperature). The reaction was revealed by using the avidin– biotin technique (AP-LSAB2 KIT; DAKO). An antibody diluent (DAKO) and irrelevant rabbit or mouse antibodies (Santa Cruz Biotechnology) of the same isotype were used as controls. The cell nuclei were stained for 1 min with Hematoxylin (DAKO). The immunoreactivity in the epithelium of large airways (internal perimeter >6 mm) was evaluated blindly by two independent investigators (G.C. and A.V.) and was Table 1 Demographic characteristics.

Gender (M/F) Age (years) Means ± SD Packs/year FEV1% of predicted Means ± SD FEV1/FVC % of predicted Means ± SD

Controls = 11

Smokers = 12

s-COPD = 17

9/2 60 ± 13

10/2 63 ± 9

15/2 64 ± 8

– 89 ± 16

59 ± 21 77 ± 12

70 ± 31 68 ± 15

83 ± 6

80 ± 9

60 ± 7

Fig. 1. Cytoplasmic p21 expression in large airways. Immunohistochemistry for cytoplasmic p21 in surgical samples from Controls (n = 11), S (n = 12), and s-COPD (n = 17) subjects. Cells were stained with an anti-p21 antibody (see Materials and methods for details). Data are expressed as the number of positive epithelial cells/mm basement membrane. Results are expressed as medians and 25th–75th percentiles. *p b 0.05. Representative immunostaining in a Control, in a Smoker, and in a s-COPD at 400× magnification is shown.

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Table 2 Cytoplasmic p21, nuclear p21, caspase-3, Ki67 and PCNA expression in bronchial epithelium. Subjects group

Control (C)

Smokers (S)

Smokers-COPD (s-COPD)

(C) vs. (S)

(C) vs. (s-COPD)

(S) vs. (s-COPD)

Cytoplasmic p21 Nuclear p21 Caspase-3 Ki67 PCNA

3.1 (2.2–3.2) 2.3 (1.5–2.8) 5 (0.8–2.9) 2.7 (1.8–2.9) 4.3 (4.1–4.7)

25.5 7.9 0.5 11.1 0.2

28 (20–44.2) 5.8 (4.2–7.4) 3.9 (0.5–4.5) 15 (11.2–21.4) 0.3 (0.14–0.3)

pb pb p= pb pb

pb pb p= pb pb

p p p p p

(16.9–80) (6.01–14.2) (0.35–3.85) (10.2–13.1) (0.15–0.23)

0.0001 0.0001 0.1 0.0001 0.0001

0.0001 0.0001 0.2 0.0001 0.0001

= = = = =

0.6 0.1 0.2 0.1 0.1

Results are expressed in medians; 25th–75th percentile values are shown in parenthesis. Control (C), Smokers (S), Smokers-COPD (s-COPD). A non-parametric Mann–Whitney test was then applied between two groups as the initial Kruskal–Wallis test was significant.

2.5. 16HBE cell line The SV40 large T antigen-transformed 16HBE cell line is a cell line that retains the differentiated morphology and function of normal human airway epithelia [22]. 16HBE cells were plated in a 6 well plate (4 × 10 5 cells/ml) (Dulbecco's modified Eagle's medium/FBS), maintained at 37 °C in a humidified atmosphere (5% CO2) and when at 70–80% confluence incubated with/without 20% CSE for 24 h (Supplemental materials). The concentration of 20% of CSE was selected on the basis of preliminary cytofluorimetric experiments testing different CSE concentrations (5%, 10%, 20%) (Fig. S1—Supplemental materials) on the expression of p21 and survivin. 2.6. Cell apoptosis by flow-cytometry Cell apoptosis was evaluated with and without CSE (20%), by staining with Annexin V-Fluorescein Isothiocyanate and Propidium Iodide using a commercial kit (Bender MedSystem, Vienna, Austria) following the manufacturer's directions. Cells were analyzed using a FACStar Plus (Becton Dickinson, Mountain View, CA, USA) as previously described [21] (Supplemental materials).

vivo evaluations. Results are expressed as medians and 25th–75th percentiles. *p b 0.05. Correlations were determined with a Spearman rank correlation test. For survivin, correlations were performed using staining intensities (0, 1, 2, 3). A p value of less than 0.05 was considered statistically significant. Results are expressed as means ± SD and unpaired t tests for in vitro experiments.

3. Results 3.1. Demographic characteristics of the subjects The demographic characteristics and the functional evaluations of the studied groups are shown in Table 1. All recruited patient groups were similar with regard to age. The number of packs/year was similar between S and s-COPD (Table 1).

2.7. Expression of p21, caspase 3 and survivin by flow-cytometry To evaluate the expression of p21, caspase-3, and survivin, 16HBE cells were permeabilized using a commercial fix-perm cell permeabilization kit (Caltag Laboratories, Burlingame, CA, USA), incubated in the dark (30 min, 4 °C) with specific antibodies (see above) followed by a Fluorescein Isothiocyanate-conjugated goat anti-mouse (DAKO) or anti rabbit immunoglobulin G and then evaluated by flow cytometry. Mouse or rabbit negative control immunoglobulins (DAKO) were used for negative controls. Data are expressed as Geo-mean fluorescence intensity. 2.8. Immunocytochemistry 16HBE cells were cultured with/without CSE 20% for 24 h and were assessed for the expression of p21 and caspase-3 by immunocytochemistry avidin–biotin technique (AP-LSAB2 KIT DAKO). 2.9. Cell proliferation assay 16HBE cell proliferation was measured using carboxyfluorescein succinimidyl ester (CFSE) labeling assay [23]. CSE (20%) stimulated 16HBE were incubated with CFSE (Molecular Probes, Inc., Eugene, OR) (at a final concentration of 5 μM) at 37 °C for 10 min. Cell proliferation was assessed by flow-cytometry (Supplemental materials). 2.10. Statistical analysis A non-parametric Mann–Whitney test was then applied between two groups as the initial Kruskal–Wallis test was significant for ex

Fig. 2. Nuclear p21 expression in large airways. Nuclear p21 in surgical samples from Controls (n = 11), S (n = 12), and s-COPD (n = 17) subjects. Cells were stained with an anti-p21 antibody (see Materials and methods for details). Data are expressed as the number of positive epithelial cells/mm basement membrane. Results are expressed as medians and 25th–75th percentiles. *p b 0.05. Representative immunostaining in a Control, in a Smoker, and in a s-COPD at 400× magnification is shown. Arrows indicate nuclear p21 expression in S and s-COPD.

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in S and in s-COPD in comparison to Controls (Fig. 1) (Table 2). The p-21 immunoreactivity was present both in the basal and in columnar epithelial cells and was concentrated in the cytoplasm of metaplastic epithelial cells. The nuclear p-21 immunoreactivity was significantly increased in S and in s-COPD in comparison to control subjects (Fig. 2) (Table 2). The immunoreactivity was present both in the basal and in columnar epithelial cells (Fig. 2). 3.3. Immunoreactivity of caspase-3 in bronchial epithelium The caspase-3 immunoreactivity in bronchial epithelium was similar in all study groups (Fig. 3) (Table 2). The immunoreactivity was present both in the basal and in columnar epithelial cells (Fig. 3). 3.4. Immunoreactivity of survivin in bronchial epithelium The cytoplasmic survivin immunoreactivity was present in the basal and in columnar epithelial cells and higher expression of survivin (score 4–9) was observed in the cytoplasm of metaplastic epithelium rather than in the non metaplastic epithelium. The higher expression of survivin (score 4–9) was present in the majority of S and of s-COPD but only in one of the controls (Fig. 4) (Table 2). 3.5. Immunoreactivity of PCNA in bronchial epithelium Fig. 3. Caspase-3 expression in large airways. Immunohistochemistry for caspase-3 in surgical samples from Controls (n = 11), S (n = 12), and s-COPD (n = 17) subjects. Cells were stained with an anti-caspase-3 antibody (see Materials and methods for details). Data are expressed as the number of positive epithelial cells/mm basement membrane. Results are expressed as medians and 25th–75th percentiles. Representative immunostaining in a Control, in a Smoker, and in a s-COPD at 400× magnification is shown.

3.2. Immunoreactivity of cytoplasmic and nuclear p-21 in bronchial epithelium We evaluated the expression of p-21 in bronchial epithelium. The cytoplasmic p-21 immunoreactivity was significantly increased

The PCNA immunoreactivity was significantly decreased in S and in s-COPD in comparison to Controls (Fig. 5) (Table 2). The immunoreactivity was present in the basal and in columnar epithelial cells and was localized in cytoplasm and nuclei (Fig. 5). 3.6. Immunoreactivity of Ki67 in bronchial epithelium The Ki67 immunoreactivity in bronchial epithelium was significantly increased in S and in s-COPD in comparison to control subjects (Fig. 6) (Table 2). The immunoreactivity was present in the basal and in columnar epithelial cells and was mainly concentrated in the cytoplasm (Fig. 6).

Fig. 4. Survivin expression in large airways. Immunohistochemistry for survivin in surgical samples from Controls (n = 11), S (n = 12), and s-COPD (n = 17) subjects. Cells were stained with an anti-survivin antibody (see Materials and methods for details). The intensity and percentage scores were multiplied to give a composite score of [1–9] for each specimen (see Materials and methods for details). Representative immunostaining in a Control, in a Smoker, and in a s-COPD at 400× magnification is shown.

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3.7. Morphometric analysis in the epithelium of central airways Morphometric analysis in central airways of all recruited subjects showed an increased percentage of metaplastic epithelium in S and in s-COPD in comparison to control subjects (Fig. 7).

3.8. Correlations Correlations between the number of cytoplasmic and nuclear p21, PCNA and Ki67 positive cells and metaplastic squamous bronchial epithelium in central airways of S and s-COPD were assessed. A positive correlation was found between cytoplasmic or nuclear p21 and metaplastic squamous bronchial epithelium in central airways of S and s-COPD (Table 3). A negative correlation was found between nuclear p21 and PCNA while a positive correlation was observed between cytoplasmic p21 and Ki67 in metaplastic squamous bronchial epithelium in central airways of S and s-COPD (Table 3). Furthermore, a positive correlation was found between Ki67 and metaplastic squamous bronchial epithelium in central airways of S and s-COPD (Table 3). Cytoplasmic and nuclear p21, Ki67 and survivin positively correlated while PCNA negatively correlated with packs/year of smoke (Table 3).

3.9. Effects of CSE on the apoptosis and proliferation of bronchial epithelial cells (16HBE) Fig. 5. PCNA expression in central airways. Immunohistochemistry for PCNA in surgical samples from Controls (n = 11), S (n = 12), and s-COPD (n = 17) subjects. Cells were stained with an anti-PCNA antibody (see Materials and methods for details). Data are expressed as the number of positive epithelial cells/mm basement membrane. Results are expressed as medians and 25th–75th percentiles. *p b 0.05. Representative immunostaining in a Control, in a Smoker, and in a s-COPD at 400× magnification is shown.

The effects of CSE in a bronchial epithelial cell line were tested. CSE were able to increase at higher extent cytoplasmic than nuclear p21 in 16HBE (Fig. 8) but did not induce caspase-3 expression or cell apoptosis (Annexin V binding) in these cells (Fig. 9). Moreover, CSE increased survivin expression, but reduced cell proliferation in 16HBE (CFSE assay) (Fig. 10).

Fig. 6. Ki67 expression in large airways. Immunohistochemistry for Ki67 in surgical samples from Controls (n = 11), S (n = 12), and s-COPD (n = 17) subjects. Cells were stained with an anti-ki67 antibody (see Materials and methods for details). Data are expressed as the number of positive epithelial cells/mm basement membrane. Results are expressed as medians and 25th–75th percentiles. *p b 0.05. Representative immunostaining in a Control, in a Smoker, and in a s-COPD at 400× magnification is shown.

Fig. 7. Morphometric analysis in large airways. Squamous cell metaplasia in smokers and s-COPD is higher than control (smokers vs. Controls p = 0.0008 and s-COPD vs. Controls p = 0.0004). Representative immunostaining in a Control, in a Smoker, and in a s-COPD at 100× magnification is shown.

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Table 3 Correlations. Spearman correlation Cytoplasmic p21 vs. metaplastic epithelium Nuclear p21 vs. metaplastic epithelium Nuclear p21 vs. PCNA Ki67 vs. metaplastic epithelium Cytoplasmic p21 vs. Ki67 p21 cytoplasmic vs. packs/years Active-caspase-3 vs. packs/year Ki67 vs. packs/year PCNA vs. packs/year p21 nuclear vs. packs/year Survivin vs. packs/year

Rho Rho Rho Rho Rho Rho Rho Rho Rho Rho Rho

= = = = = = = = = = =

0.789 0.69 −0.572 0.638 0.78 0.686 −0.19 0.7 −0.5 0.4 0. 855

pb pb p= pb pb pb p= pb p= p= pb

0.0001 0.0001 0.0006 0.0001 0.0001 0.0001 0. 3 0.0001 0.064 0.005 0.0001

4. Discussion Injury to superficial airway epithelium is normally accompanied by mitotic activity in the remaining cells and rapid restoration of the denuded surface. Tobacco smoke is the major etiological factor for the development of COPD and is a potent stimulus of DNA damage by oxidant injury. Tobacco smoke may affect the expression and activity of proteins associated with cell proliferation and cell apoptosis thus altering physiological reparative mechanisms. Smoking contributes to COPD occurrence and progression accelerating the rate of decline in lung function and this decline was proportional to pack/years smoked [24]. The present study demonstrates for the first time that in large airway epithelium of smokers and s-COPD: 1) the cytoplasmic p21,

nuclear p-21 and survivin immunoreactivity is increased; and 2) the PCNA immunoreactivity is decreased. Furthermore, a negative correlation was observed between nuclear p21 and PCNA while a positive correlation was observed between cytoplasmic p21 and Ki67 in bronchial metaplastic squamous epithelium. In vitro experiments, using a bronchial epithelial cell line, demonstrated that CSE increase survivin expression and, at higher extent, cytoplasmic than nuclear p21. Differently, CSE do not induce caspase-3 expression or cell apoptosis (Annexin V binding) and reduce cell proliferation. The starting hypothesis of our study was that cigarette smoke could lead to an imbalance between apoptosis and proliferation of bronchial epithelial cells affecting the expression and the sub-cellular distribution of p21. p21 belongs to the Cip/kip family, proteins inhibiting a variety of cyclin-dependent kinase activities [25]. p21 is highly responsive to oxidative stress and may play a role in the cellular processes involving the imbalance of cell proliferation/apoptosis [26]. On the basis of its sub-cellular distribution, nuclear or cytoplasmic p21 may affect both cell proliferation and cell apoptosis. Cytoplasmic p21 plays an important role in regulating apoptotic processes. It is well known that oxidative stress induced by cigarette smoke may contribute to chronic airway inflammation through a reduction of apoptosis in alveolar macrophages (AMs) and bronchial epithelial cells [27]. In this regard, it has been previously demonstrated that alveolar macrophages and bronchial epithelial cells from smokers have reduced cell death as well as increased cytoplasmic p21 expression [27]. p21 is a substrate for executive caspases such as caspase-3 and this kind of degradation can be reversed by specific caspase inhibitors [28]. Indeed, cytoplasmic p21 activity may be both pro-apoptotic and anti-apoptotic [13] in different cell phenotypes [8]

Fig. 8. Effects of CSE in p21 expression of 16HBE. p21 expression was assessed by flow cytometry (A–B) and by immunohistochemistry LSAB technique (C) in 16HBE at baseline and upon CSE 20% stimulation (n = 3). A. Data are expressed as Geo-mean fluorescence intensity ± SD. *p b 0.05. B. Representative histogram plots of the p21 expression with overlay. Arrows indicate curves relative to baseline and to CSE 20%. C. Representative immunostaining at 400× and at 1000× magnification. Arrows indicate cytoplasmic and nuclear p21 expression.

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Fig. 9. Effects of CSE in caspase 3 expression and annexin V binding of 16 HBE. Expression of caspase 3 (A–B) and cell apoptosis by PI/Annexin V binding method (D) was evaluated by flow-cytometry in 16HBE at baseline and upon CSE 20% stimulation (n = 3). Expression of caspase 3 was also assessed by immunohistochemistry LSAB technique (C). A. Data are expressed as Geo-mean fluorescence intensity ± SD. B. Representative histogram plots of the caspase 3 expression with overlay. C. Representative immunostaining at 400× magnification. D. Representative dot plots for assessing cell apoptosis by PI/Annexin V method. The propidium and Annexin V double-positive cells (i.e. late apoptotic cells) were present in the upper right quadrant and the single Annexin-V-positive cells (i.e. early apoptotic cells) were present in the lower right quadrant.

Fig. 10. Survivin expression and CFSE assay by flow cytometry in 16HBE. Survivin expression (A) and cell proliferation (B) using carboxyfluorescein succinimidyl ester (CFSE) labeling assay was evaluated by flow cytometry in 16HBE at baseline and upon CSE 20% stimulation. A. Data are expressed as Geo-mean fluorescence intensity ± SD. *p b 0.05. Representative histogram plot of the survivin expression with overlay. Arrows indicate curves relative to baseline and to CSE 20%. B. Data are expressed as percentage of CFSE positive cells ± SD. Representative histogram plots for cell proliferation assay.

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or on the basis of the presence of additional pro-survival proteins. The family of evolutionary conserved pro-survival proteins, IAPs, is essential in protecting cells from entering apoptosis by negative regulation of downstream caspases. Survivin is the smallest member of the IAP family and exhibits anti-apoptotic characteristics since its cytoplasmic localization, supporting the pro-caspase 3/p21 complex, leads to anti-apoptosis [14]. In the present study, in large airway epithelium of S and s-COPD, the cytoplasmic p21 and survivin immunoreactivity was increased while the activated caspase-3 immunoreactivity was similar in all study groups, thus supporting the concept that these molecular events lead to a protection of the epithelium from cell apoptosis. Alterations observed in large airways [16,29] do not always reflect those present in small airways and future experiments are needed to clarify whether the alterations observed in the present study in large airways are also present in small airways. Furthermore, these alterations are specifically related to smoke exposure and not to the presence of the cancer since all the recruited patients including the control group were affected by lung cancer. Further evidences have been provided by the in vitro experiments which demonstrate that cigarette smoke induces cytoplasmic p21 and survivin without inducing caspase 3 or cell apoptosis in bronchial epithelial cells. On the other hand, nuclear p21, mainly involved in regulating cell proliferation, is also increased in the large airway epithelium of S and s-COPD. The intracellular nuclear accumulation of p21 may be one of the mechanisms by which cigarette smoke induces inhibition of cell proliferation. In the present study, in vitro, CSE inhibit the proliferation of bronchial epithelial cells according to previous results on alveolar type II epithelial cells [30]. p21 inhibits cyclin-dependent kinases and interacts with PCNA blocking its activities and its interactions with other PCNA binding proteins. PCNA, originally characterized as a DNA polymerase accessory protein, interacting with multiple partners is involved in DNA repair, DNA methylation and chromatin assembly. Recent findings revealed that p21 undergoes proteosomal degradation when it directly engages PCNA [31]. This PCNA mediated degradation pathway of p21 seems to be linked only to chromatin bound PCNA and not to free PCNA [31]. Here, nuclear and cytoplasmic p-21 immunoreactivity was significantly increased while PCNA immunoreactivity was significantly decreased in basal and in columnar epithelial cells of S and of s-COPD in comparison with control subjects. It is conceivable that PCNA decrease, leading to a reduced degradation of p21, may contribute to the increase in p21 in S and in s-COPD observed in the present study. Differently, Ki67 immunoreactivity, another proliferation marker, was significantly increased in epithelial cells of S and of s-COPD in comparison with control subjects. In this regard, it has been demonstrated that cigarette smoke appears to elicit a dose-related proliferative response in the bronchial epithelium of active smokers measured by the Ki-67 proliferation index [32]. Molecular changes (allelic loss and genomic instability) in the bronchial epithelium may persist long after smoking cessation [33]. Ki-67 is an antigen associated with nuclear proliferation, and it is expressed during the growth and synthesis phases of the cell cycle, but not during the resting phase [34]. This antigen provides information about the proportion of active cells in the cell cycle. Ki-67 is significantly increased in subjects with intestinal metaplasia [35], in bronchial epithelium of severe asthma [36] and after allergen exposure in bronchial epithelium of atopic asthmatics in relation to bronchial epithelial metaplasia [11]. Ki-67 expression is increased in smokers, is higher in male than in female and increases with increasing levels of histologic dysplasia [37]. In oral squamous cell carcinoma, detectable nuclear p21 is associated with higher Ki-67 expression [10] and differentiation of multiple tissue types, including squamous epithelia, is associated to an increased p21 expression [26]. It is well known that there is a relation between epithelial cell proliferation and differentiation. Ki-67+ cell numbers and the squamous cell metaplasia were positively associated [4]. According to these data, we now here show a correlation between

the increase of Ki67 expression and squamous cell metaplasia in central airways of smokers and of s-COPD. Unfortunately, in our study the recruited subjects were predominantly men and this limitation did not allow to assess whether the alterations of the studied markers were present in women, a population more susceptible to smoke exposure than male [38]. Moreover, in bronchial biopsies from smokers with bronchitis or COPD, in which goblet cell hyperplasia and squamous metaplasia are often seen, emerging data indicate that the mitotic response may be suppressed [39]. The altered cell differentiation and the development of cell metaplasia may be related not only to altered cell proliferation but also to altered cell apoptosis as confirmed here by the findings that higher expression of survivin is present in the metaplastic epithelium and that cytoplasmic or nuclear p21 correlate with metaplastic epithelium. In this regard, it has been demonstrated that survivin is not expressed in normal bronchial epithelium and that its levels are increased in smokers in comparison to non-smokers prevalently in metaplastic areas of the bronchi [40]. Nicotine increases the transcription of the survivin gene in non-small cell lung cancer cells and we now confirm that cigarette smoke extracts increase survivin in a bronchial epithelial cell line [41]. Furthermore, squamous cell metaplasia is considered by the World Health Organization as a pre-invasive precursor lesion for lung cancer [6]. In conclusion, our study shows that, in bronchial airways of current smokers with and without COPD, cigarette smoke, altering expression and sub-cellular distribution of p21, may play an important role in the imbalance between apoptosis, proliferation, and differentiation of bronchial epithelial cells leading to squamous metaplasia, an alteration associated with increased airway obstruction of COPD and with increased risk of lung cancer in smokers [42]. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbadis.2013.04.022. Acknowledgements This work was supported by the Italian National Research Council and by a GlaxoSmithKline grant for the Center of Excellence in COPD (CDS014). Giuseppina Chiappara designed the study, performed the statistical analysis of the data, wrote the manuscript and declares that she has had access to and takes responsibility for the integrity of the data and the accuracy of the data analysis. Alessia Virzì, Serafina Sciarrino, Maria Ferraro, Andreina Bruno and Angela Marina Montalabano performed all the experiments of the study and participated in the interpretation of the data. Marta Ida Minervini, Patrizio Vitulo and Loredana Pipitone contributed to the patient selection, collected and managed surgical samples. Elisabetta Pace and Mark Gjomarkaj contributed to the interpretation of the data and to the writing out of the manuscript. This work is dedicated to the memory of Antonio Maurizio Vignola. References [1] Edith Puchelle, Jean-Marie Zahm, Jean-Marie Tournier, Christelle Coraux, Airway epithelial repair, regeneration, and remodeling after injury in chronic obstructive pulmonary disease, Proc. Am. Thorac. Soc. 3 (2006) 726–733. [2] Hangjun Wang, Xiangde Liu, Takeshi Umino, C. Magnus Sköld, Yunkui Zhu, Tadashi Kohyama, John R. Spurzem, Debra J. Romberger, Stephen I. Rennard, Cigarette smoke inhibits human bronchial epithelial cell repair processes, Am. J. Respir. Cell Mol. Biol. 25 (2001) 772–779. [3] Christelle Coraux, Jacqueline Roux, Thomas Jolly, Philippe Birembaut, Epithelial cell–extracellular matrix interactions and stem cells in airway epithelial regeneration, Proc. Am. Thorac. Soc. 5 (2008) 689–694. [4] Thérèse S. Lapperre, Jacob K. Sont, Annemarie van Schadewijk, Margot M.E. Gosman, Dirkje S. Postma, Ingeborg M. Bajema, Wim Timens, Thais Mauad1, Pieter S. Hiemstra, the GLUCOLD Study Group 7, Smoking cessation and bronchial epithelial remodelling in COPD: a cross-sectional study 26 November, Respir. Res. 8 (2007) 85.

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