p53 Signaling Pathway Polymorphisms Associated With Emphysematous Changes in Patients With COPD

Accepted Manuscript p53 signaling pathway polymorphisms associated with emphysematous changes in COPD patients Shiro Mizuno, MD, PhD, Takeshi Ishizaki, MD, PhD, Maiko Kadowaki, MD, PhD, Masaya Akai, MD, PhD, Kohei Shiozaki, MD, PhD, Masaharu Iguchi, MD, PhD, Taku Oikawa, MD, PhD, Ken Nakagawa, MD, PhD, Kazuhiro Osanai, MD, PhD, Hirohisa Toga, MD, PhD, Jose Gomez-Arroyo, MD, PhD, Donatas Kraskauskas, DVM, Carlyne D. Cool, MD, Herman J. Bogaard, MD, PhD, Norbert F. Voelkel, MD PII:

S0012-3692(17)30377-X

DOI:

10.1016/j.chest.2017.03.012

Reference:

CHEST 994

To appear in:

CHEST

Received Date: 23 May 2016 Revised Date:

10 February 2017

Accepted Date: 1 March 2017

Please cite this article as: Mizuno S, Ishizaki T, Kadowaki M, Akai M, Shiozaki K, Iguchi M, Oikawa T, Nakagawa K, Osanai K, Toga H, Gomez-Arroyo J, Kraskauskas D, Cool CD, Bogaard HJ, Voelkel NF, p53 signaling pathway polymorphisms associated with emphysematous changes in COPD patients, CHEST (2017), doi: 10.1016/j.chest.2017.03.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1 Manuscript word count: 3248. Abstract word count: 245.

Running head: p53 and MDM2 SNPs in COPD patients.

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p53 signaling pathway polymorphisms associated with emphysematous changes in COPD patients.

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Shiro Mizuno, MD, PhD1, Takeshi Ishizaki, MD, PhD 1, Maiko Kadowaki, MD, PhD 2, Masaya Akai, MD, PhD 3, Kohei Shiozaki, MD, PhD 3, Masaharu Iguchi, MD, PhD 1, Taku Oikawa, MD, PhD 1, Ken Nakagawa, MD, PhD 1, Kazuhiro Osanai, MD, PhD 1, Hirohisa Toga, MD, PhD 1, Jose Gomez-Arroyo, MD, PhD 4, Donatas Kraskauskas, DVM 4, Carlyne D. Cool, MD 5, Herman J. Bogaard, MD, PhD 6, Norbert F. Voelkel, MD4. 1

Division of Respiratory Medicine, Kanazawa Medical University, Ishikawa, Japan. Department of Respiratory Medicine, University of Fukui, Fukui, Japan. 3 Department of Respiratory Medicine, Fukui Red Cross Hospital, Fukui, Japan. 4 Victoria Johnson Center for Obstructive Lung Diseases, Virginia Commonwealth University, Richmond, VA, USA. 5 Department of Pathology, University of Colorado Health Science Center, LTRC repository, Aurora, Colorado. 6 VU University Medical Center, Amsterdam, the Netherlands. E-mail addresses: SM: [email protected]

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Correspondence to: Shiro Mizuno, MD, PhD. 1-1 Daigaku, Uchinada, Kahoku-gun, Ishikawa, Japan.

COI disclosure: The authors declare that there are no competing interests to disclose.

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Funding: These investigations have been supported by Grants-in-Aid for Scientific Research (No. 24591144) of Japan, and funds from the Victoria Johnson Center for Lung Research of the Virginia Commonwealth University.

Abstract Background: The p53 signaling pathway may be important for the pathogenesis of emphysematous changes in the lungs of smokers. Polymorphism of p53 at codon 72 is

ACCEPTED MANUSCRIPT 2 known to affect apoptotic effector proteins, and the polymorphism of mouse double minute 2 homolog (MDM2) SNP309 is known to increase MDM2 expression. The aim of this study was to assess polymorphisms of the p53 and MDM2 genes in smokers and

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confirm the role of SNPs in these genes in the pathogenesis of pulmonary emphysema. Methods: 365 patients with a smoking history were included in this study, and the

polymorphisms of p53 and MDM2 genes were identified. The degree of pulmonary

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emphysema was determined by computed tomography scanning. SNPs, MDM2 mRNA and p53 protein levels were assessed in human lung tissues from smokers. Plasmids

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encoding p53 and MDM2 SNPs were used to transfect human lung fibroblasts (HLFs) with or without cigarette smoke extract (CSE), and effect on cell proliferation and MDM2 promoter activity were measured.

Results: The polymorphisms of p53 and MDM2 genes were associated with

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emphysematous changes in the lung, and were also associated with p53 protein and MDM2 mRNA expression in the lung tissue samples. Transfection with a p53 genecoding plasmid regulated HLFs proliferation, and the analysis of P2 promoter activity in

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MDM2 SNP309-coding HLFs showed the promoter activity was altered by CSE. Conclusions: Our data demonstrate that p53 and MDM2 gene polymorphisms are

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associated with apoptotic signaling and smoking-related emphysematous changes in the lungs from smokers.

Abbreviations list

Chronic obstructive pulmonary disease (COPD) Mouse double minute 2 homolog (MDM2)

ACCEPTED MANUSCRIPT 3 Single nucleotide polymorphisms (SNPs) Specificity protein 1 (Sp1)

Forced Vital Capacity (FVC) Forced Expiratory Volume in 1 Second (FEV1)

Lung Tissue Repository Consortium (LTRC)

Computed tomography (CT) Small interfering RNA (siRNA) Cigarette smoke extract (CSE) Percentage of the FEV1 (FEV1%)

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FEV1 percentage predicted (%FEV1)

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% Low attenuation area (LAA%)

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Global Initiative for Obstructive Lung Disease (GOLD)

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Human lung fibroblasts (HLFs)

Introduction

Chronic obstructive pulmonary disease (COPD) is a heterogeneous and multicomponent disease affecting small airways and causing parenchyma destruction.

ACCEPTED MANUSCRIPT 4 Alveolar septal cell apoptosis may contribute to the pathobiology of pulmonary emphysema and COPD. An increase in the number of apoptotic cells has been reported in human emphysematous lungs 1,2, suggesting that increased apoptosis and impaired

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regeneration of the lung alveolar structures are pathogenically important in the

pathogenesis of emphysema. Oxidative stress can activate numerous signaling cascades that lead to lung cell apoptosis. In emphysema patients, cigarette smoke generates large

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amounts of free radicals3. Even after cessation of smoking, oxidative stress persists, originating from activated macrophages and neutrophils, that are abundant in

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emphysematous lungs4,5. Oxidative stress is known to activate tumor suppressor p53, inhibiting cell cycle progression and inducing apoptosis in cells with irreparable genetic damage6. Increased levels of p53 in emphysematous lung tissues have been reported7-9; p53 protein expression was demonstrated to be significantly higher in lung tissues from

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patients with emphysema secondary to smoking when compared with tissues from smokers without emphysema or non-smokers7,9, suggesting a mechanistic relationship between p53 expression and oxidative stress associated with smoking. Importantly, the

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cellular activity of p53 is highly regulated by a product of an oncogene mouse double minute 2 homolog (MDM2). MDM2 binds directly to p53, inhibiting its ability to

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activate gene transcription, and also regulates its cellular localization, targeting p53 for proteosomal degradation10,11. Under normal conditions, p53 is maintained at very low levels because of ubiquitination by its negative regulator, MDM2, an E3 ubiquitin ligase that binds to p53 and targets it for proteasomal degradation to prevent apoptosis and senescence of the cells12. In response to cellular stress, p53 protein is stabilized and accumulates due to inactivation of p53 degradation. The p53 and MDM2 proteins are

ACCEPTED MANUSCRIPT 5 tightly regulated and linked via a feedback loop, where p53 acts as a transcriptional factor of MDM213. In addition, MDM2 is known as a p53-independent regulator of cellular growth, as it binds and inhibits the function of a retinoblastoma protein that promotes cell

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proliferation14.

The most frequently studied single nucleotide polymorphisms (SNPs) in the p53

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pathway involve the p53 gene G/C polymorphism in codon 72 (rs1042522) and the

MDM2 gene G/T polymorphism located at position 309 of the first intron (rs2279744)15.

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p53 G/C polymorphism in codon 72 is important for p53 function, especially for its ability to induce apoptosis16,17. MDM2 SNP309 G/T polymorphism at position 309 serves as a transcriptional enhancer, increasing the affinity of the specificity protein 1 (Sp1) transcription factor by extending the length of a Sp1-binding site, which leads to

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increased MDM2 expression18.

Based on the current knowledge of the cellular consequences of p53 and MDM2 gene polymorphisms, we hypothesized that the polymorphisms of p53 codon 72 and

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MDM2 SNP309, either alone in or combination, are associated with lung cell apoptosis and the susceptibility to development of emphysematous lung parenchyma destruction in

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smokers. If so, COPD etiology would be attributable to both environmental (smokinginduced oxidative stress burden) and genetic (p53-dependent) factors.

Here we investigated whether p53 codon 72 and MDM2 SNP309 polymorphisms

are associated with the development of COPD/emphysema. Accordingly, our primary endpoint of this clinical study was to investigate whether the p53 codon 72 and MDM2 SNP309 polymorphisms were associated with the severity of pulmonary emphysema in

ACCEPTED MANUSCRIPT 6 smokers. The secondary endpoint was to assess whether these gene polymorphisms were associated with the severity of COPD. We first analyzed the genotype distribution of p53 codon 72 and MDM2 SNP309 polymorphisms in 365 smokers to explore the association

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of these polymorphisms with the patients’ emphysema severity. We next evaluated

whether p53 protein and MDM2 mRNA levels in the lung tissues from COPD patients

were influenced by the p53 codon 72 or MDM2 SNP309 genotypes. Finally, to address

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the mechanism of interaction between these SNPs and lung cell apoptosis induced by

smoking, we performed experiments using cultured human lung fibroblasts (HLFs) with

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plasmids coding the p53 gene with codon 72 SNP and the MDM2 gene with SNP309 SNP. Lung fibroblasts are the most common connective tissue cells in the human lung, and are known to play a role in the pathogenesis of emphysema19. Insufficiency of lung tissue repair due to the dysfunction of lung fibroblasts may contribute to the loss of

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Patients

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Materials and Methods

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alveolar elastic fibers, and the development of emphysema in COPD20,21.

From October 2011 to September 2014, 396 patients were recruited from an

outpatient department of the Division of Respiratory Medicine, Kanazawa Medical University Hospital, Department of Respiratory Medicine, University of Fukui Hospital and Division of Respiratory Medicine, Fukui Red Cross Hospital. The inclusion criteria for enrollment were: age > 40 years old and at least a 10 pack-year history of tobacco

ACCEPTED MANUSCRIPT 7 exposure. Pulmonary function tests were performed to determine forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1). COPD diagnosis was based on the patient’s history, physical examination, and spirometric data, following the

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classification of Global Initiative for Obstructive Lung Disease (GOLD)22. Among these 396 patients, 24 patients with a bronchodilator response (12% and 200 ml increase in the FEV1) were excluded because of a possible clinical asthma. We also excluded two

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patients with interstitial lung disease, two patients with pneumoconiosis, three patients with bronchiectasis. Subsequently, 254 patients with COPD and 111 patients without

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COPD were included in this study. The COPD patients were classified into three categories (mild; GOLD I, moderate; GOLD II, severe; GOLD III and IV) based on their spirometry data (Table 1). The study was approved by the Research Ethics Committee of Kanazawa Medical University, University of Fukui, and Fukui Red Cross Hospital

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Lung tissue samples

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(Protocol: NO. 0073), and all subjects gave their informed consent in writing.

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Lung tissue samples from 24 patients with > 10 pack-year smoking history were included in this study. The patient characteristics are summarized in online data supplement e-Table 1. Samples were obtained from the NIH Lung Tissue Repository Consortium (LTRC). The diagnosis of emphysema was made by an LTRC pathologist based on histological examination, and all of the lung tissue samples from COPD patients had centrilobular emphysema. All lung tissue samples were maintained at -80 °C until processing. All patients gave informed consent to have their tissues deposited at in the

ACCEPTED MANUSCRIPT 8 NIH lung tissue repository. The study was approved by the Institutional Review Board (Protocol: COMIRB 07-0269).

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Experimental protocols Determination of % low attenuation area (LAA%) from chest computed

tomography (CT) scans; cell culture; chemicals; genotyping; real-time RT-PCR analysis;

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western blot analysis; transfection with plasmids and small interfering RNA (siRNA); preparation of cigarette smoke extract (CSE); MTS assay; construction of plasmids

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coding p53 gene with codon72 C/G polymorphism and luciferase reporter assay with cells transfected with plasmids coding MDM2 gene with SNP309 G/T polymorphism are given in the online supplement.

Statistical Analysis

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The statistical significance of differences in distribution and frequency of the genotype and allele between the groups were determined by chi-squared test of

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independence and with Fisher exact test. Comparisons of age, smoking index (expressed as pack-years), pulmonary function parameters, protein levels, RT-PCR results, MTS,

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and luciferase activities were performed using ANOVA with Bonferroni corrections for multiple comparisons. Correlations were analyzed by Pearson correlation coefficient. Comparisons were considered statistically significant at p < 0.05.

Results

ACCEPTED MANUSCRIPT 9 Patient characteristics

Age, gender, smoking history, pulmonary function data and LAA% data of COPD

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patients and control smoker subjects are summarized in Table 1. No significant differences were observed in the age, gender, or smoking history between the groups.

Percent FVC was significantly increased in patients with mild COPD and decreased in

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patients with severe COPD when compared with smoker control subjects. LAA% was

significantly increased in patients with mild, moderate, and severe COPD in comparison

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with smoker control subjects, and was negatively correlated both with percentage of the FEV1 (FEV1%) and FEV1 percentage predicted (%FEV1) (Figure 1).

MDM2 and p53 genotype frequencies in patients with COPD and smoker control subjects

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The results of the comparison of polymorphisms of p53 codon 72 and MDM2 SNP309 are summarized in Table 2 and 3. Although the frequency of MDM2 SNP309

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genotype was not significantly different between COPD groups and smoker controls, we found significant differences in the MDM2 SNP309 allele frequency between the severe

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COPD group and smoking controls. p53 codon 72 CG and GG genotypes and the G allele occurred more frequently in patients with severe COPD than in smoker control subjects (Tables 2 and 3). The LAA% in patients with MDM2 SNP309 TT genotype was significantly greater compared with patients with the GG genotype. Similarly to the p53 polymorphism, the LAA% in patients with p53 codon 72 GG genotype was significantly greater than in patients with the CC genotype (Figure 2A and 2B). However, there were

ACCEPTED MANUSCRIPT 10 no significant differences in the smoking status between both the various p53 and MDM2 genotypes (Tables 4 and 5).

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Since MDM2 and p53 expression are closely related, we next examined the existence of a synergistic effect of p53 and MDM2 polymorphisms on the degree of

emphysema, using p53 CC and MDM2 GG genotype carriers as a reference. We found

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that patients with either the p53 CG and MDM2 TT genotype, p53 GG and MDM2 GT genotype, and p53 GG and MDM2 TT genotype were at a greater risk of developing

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pulmonary emphysema than the p53 CC and MDM2 GG genotype patients. Patients with the p53 GG and MDM2 TT genotype had the highest degree of emphysema (Figure 2C).

p53 protein and MDM2 mRNA expression in lung tissue samples from COPD patients

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Next, we measured MDM2 mRNA and p53 protein levels in lung tissue samples from COPD patients to examine the effects of MDM2 polymorphisms on the p53 protein

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expression. The profiles of patients from whom the lung tissue samples were obtained are summarized in online data supplement e-Table 1. All patients had a smoking history and

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all tissue samples from COPD patients showed centrilobular emphysema.

We found that the p53 protein expression was increased in patients with moderate

to severe COPD, as previously described7. The MDM2 mRNA expression was significantly increased in lung tissues from patients with the MDM2 SNP309 GG genotype when compared with tissues of patients with the TT genotype (Figure 3A), and p53 protein expression was increased in the lung tissues from patients with the TT

ACCEPTED MANUSCRIPT 11 genotype when compared with tissues of patients with the GG genotype (Figure 3B). The p53 protein expression was correlated with the MDM2 mRNA expression in the lung

Effects of MDM2 and p53 gene transfection in HLFs

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tissues from COPD patients (Figure 3C).

To investigate whether p53 polymorphism was pathogenically important, we used

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plasmids coding p53 gene with codon 72 C or G polymorphism to transfect HLFs, and

measured cell survival with the MTS assay. The expression of p53 protein was induced in

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cells transfected with both plasmids (Figure 4A), however, the MTS assay revealed that transfection with a plasmid carrying p53 codon 72 allele G significantly suppressed cell proliferation when compared with transfection with a plasmid carrying p53 codon 72 C allele (Figure 4B).

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We also examined the transcriptional activity of the MDM2 promoter using plasmids encoding MDM2 SNP309 G or T allele in cultured HLFs. We found that CSE

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significantly decreased HLFs survival starting with a concentration of 0.3% (Figure 5A), and we verified that CSE induced the synthesis of both p53 and Sp1 proteins starting with

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a concentration of 1%, which regulate the MDM2 P2 promoter activity14 (Figure 5B). Furthermore, Sp1 protein expression was suppressed by siRNA-silencing of p53 gene expression (Figure 5C). When we cultured HLFs transfected with plasmids encoding the MDM2 SNP309 polymorphisms with 1% CSE for 24 hours, luciferase reporter activity measurements revealed that CSE exposure significantly increased MDM2 transcriptional activity. In addition, the MDM2 promoter activity induced by CSE exposure in cells transfected with a plasmid encoding the MDM2 SNP309 G allele was significantly higher

ACCEPTED MANUSCRIPT 12 than in cells transfected with a plasmid encoding the MDM2 SNP309 T allele (Figure

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5D).

Discussion

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In the present study, we examined the polymorphisms of p53 codon 72 and

MDM2 SNP309 in smokers, and we found that these plymorphisms were associated with

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smoking-related emphysematous changes. We also found that MDM2 SNP309 polymorphisms are associated with the MDM2 mRNA and p53 protein expression in lungs tissues from COPD patients. Cell culture studies confirmed that p53 and MDM2 polymorphisms have an impact the CSE-induced MDM2 promoter activity and p53 protein abundance, which may both be associated with smoking-related lung cell

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apoptosis and emphysematous changes in the human lungs.

The SNPs in the p53 pathway have been thus far most extensively studied in

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cancer, since p53-mediated responses are crucial both in reducing cancer frequency and in mediating the response to cancer therapies18, and the p53 mutational status can serve as

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an independent prognostic indicator in some cancer types23. Recently, Ren et al., reported that p53 and MDM2 polymorphisms synergistically increase the risk of lung adenocarcinoma in Chinese women. They demonstrated that patients with the p53 codon 72 CC genotype and the MDM2 SNP309 GG genotype were at a higher risk of developing lung cancer when compared with patients with the p53 codon 72 GG and MDM2 SNP309 TT genotype. Cooking oil fumes, fuel smoke, and passive smoking

ACCEPTED MANUSCRIPT 13 history may increase the risk of lung adenocarcinoma in these patients24. Interestingly, the risk of lung cancer in the Chinese study and the risk of emphysema in our present study show a completely opposite pattern of p53 and MDM2 gene polymorphisms. One

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could postulate that p53-related apoptosis of the lung cells might prevent the survival of DNA damaged cells, which could decrease the risk of lung cancer. Indeed, conflicting

reports exist regarding the risk of lung cancer in COPD patients, although a significant

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association between the presence of emphysema and lung cancer risk has been reported25. However, uncertainty still surrounds the relationship between the severity of emphysema

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and lung cancer risk. Wilson et al., reported that lung cancer risk is lower in moderate to severe emphysema compared with mild emphysema26. Likewise, Li et al., reported that the risk of lung cancer increased up to 3.80 fold in patients with 5% of more LAA, while the cancer risk did not increase in patients with 10% or more LAA%27. Further studies

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and long term follow-up of patients will be required to clarify the association between the severity of emphysema and lung cancer risk in the COPD patients identified as carrying

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p53 gene-related polymorphisms.

Sp1 is a ubiquitously expressed DNA-binding protein that was first identified

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based on its ability to interact with GC-rich motifs28. Our data from experiments with cultured HLFs show that CSE treatment increased nuclear Sp1 protein expression (Figure 5B), and the effect was suppressed by p53 gene silencing (Figure 5C). Previous studies suggest that oxidative stress increases Sp1 expression, which is implicated in a variety of cellular events associated with transactivation of numerous genes28,29. We suggest that CSE exposure results in enhanced MDM2 P2 promoter activity as a consequence of increased Sp1 expression (Figure 5D). In our in vitro study, we verified that CSE indeed

ACCEPTED MANUSCRIPT 14 induces Sp1 and p53 expression in HLFs (Figure 5B). It has been appreciated that Sp1 plays an important role in activating the MDM2 P2 promoter, affecting MDM2 promoter activity, and MDM2 gene expression. A T-to-G mutation SNP at nucleotide 309 in the P2

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promoter of MDM2 (MDM2 SNP309) results in an increased affinity for the

transcriptional activator Sp1, leading to increased MDM2 mRNA and protein levels and subsequent attenuation of the p53 pathway18. Our finding that the MDM2 SNP309

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polymorphism is associated with MDM2 mRNA and p53 protein expression in the lungs from COPD patients (Figure 3) is in line with the findings from our cell culture studies

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showing that the MDM2 SNP309 regulates CSE-induced MDM2 promoter activity. The analysis of the P2 promoter activity in MDM2 SNP309 background in HLFs showed that the MDM2 promoter activity was altered by CSE (Figure 5D). These results suggest the possibility that MDM2 promoter polymorphism can alter MDM2 expression in the

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presence of CSE via p53 and Sp1 expression. This, in turn, may affect p53-dependent apoptosis in the lungs from COPD patients, and our lung tissue data show that p53 protein expression is higher in patients with the MDM2 SNP309 TT genotype compared

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with patients with the GG genotype (Figure 3). Furthermore, these results are compatible with our clinical data that showed a more extensive emphysema, closely related with lung

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cell apoptosis, in patients with the MDM2 SNP309 TT genotype compared with patients with the GG genotype (Figure 2). Taken together, we conclude that the susceptibility to lung cell apoptosis and to development of pulmonary emphysema induced by oxidative stress from cigarette smoking may be altered by the gene polymorphisms in p53 codon 72 and MDM2 SNP309.

ACCEPTED MANUSCRIPT 15 It is possible that the CSE differs from the cigarette smoke inhaled into the lung, and it should be apparent that the lung fibroblasts are not the only cells which are affected by the cigarette smoke. Some volatile components may be lost from the CSE that are

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present in the inhaled cigarette smoke, and the relative concentrations of components

may be different between CSE and inhaled cigarette smoke. We also consider that some lung cells may be more or less susceptible than HLFs when it comes to the toxicity of

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cigarette smoke. Nevertheless, p53-mediated cell apoptosis induced by cigarette smoke

has been reported for a variety of lung cell types, including pulmonary endothelial cells30,

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alveolar epithelial cells31,32, and cells of the terminal bronchioles33. CSE induces apoptosis accompanied by p53 protein expression in various primary cells, such as human umbilical venous endothelial cells34, pulmonary endothelial cells30, lung fibroblasts35-38 and alveolar epithelial cells39. The reaction of cells to CSE in the present

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study could comprise a universal reaction of mammalian cells that express non-mutated p53 gene. We believe that CSE-triggered oxidative stress is primary responsible for the observed effects. Toxic effects of the highly aggressive aldehyde acrolien, are also

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likely40. However, CSE contains more than 4,000 compounds41. Because of the numerous target compounds, it will be extremely difficult to identify the responsible compound in

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this study. We wish to identify some of the responsible compound of CSE in a future study.

In our present study, we observed a significant association between the severity of

emphysema and both p53 and MDM2 polymorphisms (Figure 2A and 2B). The association between pulmonary function impairment and these polymorphisms was significant, but weak when compared with the severity of emphysema (Tables 2 and 3).

ACCEPTED MANUSCRIPT 16 Although pulmonary function impairment and the extent of emphysema are significantly correlated, we found a large range of FEV1 values at each LAA% (Figure 1) and it is

early stage emphysema is concerned42.

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widely-accepted that lung function studies are rather insensitive when the diagnosis of

In conclusion, polymorphisms of p53 codon 72 and MDM2 SNP 309 are

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associated with smoking-related emphysematous changes and we suggest that the

analysis of p53 and MDM2 polymorphisms may provide a predictive biomarker of

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susceptibility to pulmonary emphysema in smokers. In addition, we have provided experimental evidence for a role of the p53 and MDM2 polymorphisms of in CSE-

Acknowledgments

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induced lung cell apoptosis.

S. M. designed and organized the experiments. S.M, M.K, M.A, K.S, M.I, T.O,

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K.N, K.O, J.G., and D.K. participated in the laboratory measurements and data analysis. C.D.C., and T.I, H.T, H.J.B., and N. F. V. participated in the design of the study and

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supervised the study. S.M, N.F.V, and H.J.B were involved in writing of the manuscript. We would like to thank Vita Kraskauskiene and Makoto Kobayashi for their

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ACCEPTED MANUSCRIPT 19 Schäfer G, Cramer T, Suske G, Kemmner W, Wiedenmann B, Höcker M. Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements. J Biol Chem 2003;278(10):8190–8198.

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Di YP, Zhao J, Harper R. Cigarette smoke induces MUC5AC protein expression through the activation of Sp1. J Biol Chem 2012;287(33):27948–27958.

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Damico R, Simms T, Kim BS, et al. p53 mediates cigarette smoke-induced apoptosis of pulmonary endothelial cells: inhibitory effects of macrophage migration inhibitor factor. Am J Respir Cell Mol Biol 2011;44(3):323–332.

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Shetty SK, Bhandary YP, Marudamuthu AS, et al. Regulation of airway and alveolar epithelial cell apoptosis by p53-Induced plasminogen activator inhibitor-1 during cigarette smoke exposure injury. Am J Respir Cell Mol Biol 2012;47(4):474–483.

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Tiwari N, Marudamuthu AS, Tsukasaki Y, Ikebe M, Fu J, Shetty S. p53- and PAI1-mediated induction of C-X-C chemokines and CXCR2: importance in pulmonary inflammation due to cigarette smoke exposure. Am J Physiol Lung Cell Mol Physiol 2016;310(6):L496–506.

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Wu C-H, Lin H-H, Yan F-P, Wu C-H, Wang C-J. Immunohistochemical detection of apoptotic proteins, p53/Bax and JNK/FasL cascade, in the lung of rats exposed to cigarette smoke. Arch Toxicol 2006;80(6):328–336.

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Wang J, Wilcken DE, Wang XL. Cigarette smoke activates caspase-3 to induce apoptosis of human umbilical venous endothelial cells. Mol Genet Metab 2001;72(1):82–88.

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Kim S-Y, Lee J-H, Huh JW, et al. Cigarette smoke induces Akt protein degradation by the ubiquitin-proteasome system. J Biol Chem 2011;286(37):31932–31943.

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Kim S-Y, Lee J-H, Kim HJ, et al. Mesenchymal stem cell-conditioned media recovers lung fibroblasts from cigarette smoke-induced damage. Am J Physiol Lung Cell Mol Physiol 2012;302(9):L891–908.

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Nyunoya T, Monick MM, Klingelhutz A, Yarovinsky TO, Cagley JR, Hunninghake GW. Cigarette smoke induces cellular senescence. Am J Respir Cell Mol Biol 2006;35(6):681–688.

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D'Anna C, Cigna D, Costanzo G, et al. Cigarette smoke alters cell cycle and induces inflammation in lung fibroblasts. Life Sci 2015;126:10–18.

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Bhandary YP, Shetty SK, Marudamuthu AS, et al. Plasminogen activator inhibitor1 in cigarette smoke exposure and influenza A virus infection-induced lung injury. PLoS ONE 2015;10(5):e0123187.

ACCEPTED MANUSCRIPT 20 Kitaguchi Y, Taraseviciene-Stewart L, Hanaoka M, Natarajan R, Kraskauskas D, Voelkel NF. Acrolein induces endoplasmic reticulum stress and causes airspace enlargement. PLoS ONE 2012;7(5):e38038.

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Vu T, Jin L, Datta PK. Effect of Cigarette Smoking on Epithelial to Mesenchymal Transition (EMT) in Lung Cancer. J Clin Med 2016;5(4):44.

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Gurney JW, Jones KK, Robbins RA, et al. Regional distribution of emphysema: correlation of high-resolution CT with pulmonary function tests in unselected smokers. Radiology 1992;183(2):457–463.

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Figure Legends

ACCEPTED MANUSCRIPT 21 Figure 1. Correlation between %low attenuation area (LAA%) and pulmonary function The figure shows correlation analysis between LAA% measured from chest

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computed tomography (CT), and FEV1% (A) and %FEV1 (B). The LAA% was

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significantly correlated with both FEV1% (R = 0.609) and %FEV1 (R = 0.482).

Figure 2. LAA% analysis with p53 and MDM2 polymorphisms

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The figure shows analysis of LAA% in smoking patients with different p53 codon 72 genotypes (A), MDM2 SNP309 genotypes (B), and as a function of different combined p53 and MDM2 genotype (C). LAA% was significantly higher in patients with p53 codon 72 GG genotype compared with patients with p53 codon 72 CC genotype, and

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higher in patients with MDM2 SNP309 TT genotype compared with patients with MDM2 SNP309 GG genotype. Combination analysis of both genotypes revealed that LAA% was significantly higher in patients with p53 codon 72 GG and MDM2 SNP309

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TT genotype, p53 codon 72 GG and MDM2 SNP309 GT genotype, and p53 codon 72 CG and MDM2 SNP309 TT genotype compared with patients with the p53 codon 72 CC

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and MDM2 SNP309 TT genotype. Data are expressed as means ± SD.

Figure 3. MDM2 mRNA and p53 protein levels in lung tissues from COPD patients The figure contains data on MDM2 mRNA and p53 protein levels in COPD patients with different MDM2 SNP309 genotypes. MDM2 mRNA expression was significantly lower in patients with the MDM2 SNP309 TT genotype compared with

ACCEPTED MANUSCRIPT 22 patients with GG genotype (A). p53 protein expression was significantly higher in patients with the TT genotype of MDM2 SNP309 than in patients with the GG genotype of MDM2 SNP309 (B). p53 protein levels were significantly correlated with MDM2

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mRNA expression (C).

(D) Representative images of western blot analysis of p53 and β-actin levels in

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lung tissue samples from COPD patients. Data are expressed as means ± SD.

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Figure 4. Transfection of human lung fibroblasts (HLFs) with a p53 codon 72 gene construct

(A) Western blot analysis of p53 protein levels in cultured HLFs transfected with a plasmid coding p53 gene codon 72 C or G allele. The p53 protein was strongly expressed in cells transfected with the plasmid coding p53 gene, but not in cells

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transfected with an empty vector (MOCK).

(B) MTS assay showed that transfection of cells with a plasmid coding p53 G

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allele significantly suppressed cell proliferation when compared with transfection with a plasmid coding p53 codon 72 C allele.

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Data are expressed as means ± SD (n = 6).

Figure 5. The effect of cigarette smoke extract (CSE) on HLFs (A) MTS assay that shows CSE significantly decreased the survival of cultured HLFs dose-dependently starting with a concentration of 0.3%. (B) Western blot analysis of Sp1 and p53 protein levels in cultured HLFs treated with various concentrations of

ACCEPTED MANUSCRIPT 23 CSE. Sp1 and p53 protein expression was significantly increased by the CSE treatment starting with a concentration of 1%. The upper panel shows representative images of western blot analysis of Sp1, lamin B, p53, and β-actin levels in cultured HLFs treated

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with various concentrations of CSE. Data are expressed as means ± SD (n = 4). (C) Western blot analysis of Sp1 and p53 protein levels in p53-silenced cultured HLFs

treated with 1% CSE. The Sp1 protein expression was suppressed after transfection with

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p53 siRNA. The left panel shows representative images of western blot analysis of

Sp1,lamin B, p53, and β-actin levels in cultured HLFs treated with CSE, with or without

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control or p53 siRNA. Data are expressed as means ± SD (n = 4). (D) The graph shows luciferase reporter activity in HLFs transfected with plasmids encoding either MDM2 SNP309 G or T allele, cultured in the presence or absence of 1% CSE. The MDM2 promoter activity was significantly increased by CSE and decreased in cells transfected

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with a plasmid coding the MDM2 SNP309 T allele compared with cells transfected with

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4).

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a plasmid coding the MDM2 SNP309 G allele. Data are expressed as means ± SD (n =

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Table 1, Characteristics of patients Smoker control

Mild COPD

Moderate COPD

Severe COPD

GOLD

…

Stage I

Stage II

Stage III/IV

NO. of patients

111

70

104

80 (64/16)

Age, year

70.8 ± 9.3

72.2 ± 9.3

72.5 ± 8.7

73.9 ± 8.7

Sex, male (female)

107 (4)

66 (4)

99 (5)

76 (4)

Smoking, pack-year

60.5 ± 38.0

57.4 ± 33.7

61.4 ± 31.2

63.4 ± 29.2

Current smoker, NO.

42

22

FVC, % predicted

97.8 ± 14.6

113.8 ± 14.7 *

FEV1/FVC, %

77.3 ± 6.1

64.5 ± 5.1 *

FEV1, % predicted

96.6 ± 13.9

BDR, %

5.0 ± 4.6

LAA, %

5.2 ± 6.0

obstructive

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16

97.2 ± 16.2

75.7 ± 18.1 *

53.0 ± 9.2 *

39.3 ± 10.4 *

96.4 ± 13.2

64.7 ± 8.8 *

37.5 ± 8.1 *

5.1 ± 3.1

7.6 ± 7.2

9.7 ± 8.8

10.1 ± 10.5 *

15.6 ± 11.2 *

23.9 ± 14.9 *

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Chronic

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Characteristics

pulmonary

disease

(COPD),

Global

Initiative

for

Obstructive Lung Disease (GOLD), Forced Vital Capacity % predicted (FVC, % predicted), Forced Expiratory Volume in 1 Second % predicted (FEV1 %

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predicted), Bronchodilator response (BDR)

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Values are expressed as means ± SD. *P<0.05 versus smoker control.

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Table 2, p53 and MDM2 genotype frequencies in COPD patients p53 codon 72 genotype CG

GG

Smoker Control

26 (23.4%)

46 (41.4%)

39 (35.1%)

Mild COPD

10 (14.3%)

34 (48.6%)

26 (37.1%)

Moderate COPD

17 (16.3%

48 (46.2%)

39 (37.5%)

Severe COPD

7 (8.8%)

37 (46.3%)

36 (45.0%)

GG

GT

TT

P value

Smoker Control

37 (33.3%)

50 (45.0%)

24 (21.6%)

Reference

Mild COPD

18 (25.7%)

35 (50.0%)

17 (24.3%)

0.5546

Moderate COPD

22 (21.2%)

55 (52.9%)

27 (26.0%)

0.135

Severe COPD

17 (21.3%)

38 (47.5%)

25 (31.3%)

0.1261

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Reference 0.3097 0.4274

0.0274

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P value

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Table 3, p53 and MDM2 allele frequencies in COPD patients p53 codon 72 allele P value

Smoker Control

44.1%

55.9%

Reference

Mild COPD

38.6%

61.4%

0.3257

Moderate COPD

39.4%

60.6%

0.3298

Severe COPD

31.9%

68.1%

0.019

G

T

P value

1.259 (0.818-1.938)

1.214 (0.827-1.783)) 1.689 (1.104-2.584)

Odds ratio (95% CI)

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Odds ratio (95% CI)

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G

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C

55.9%

44.1%

Reference

Mild COPD

50.7%

49.3%

0.3866

1.230 (0.805-1.880))

Moderate COPD

47.6%

52.4%

0.1007

1.393 (0.953-2.037)

Severe COPD

45.0%

55.0%

0.0385

1.546 (1.027-2.328)

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Table 4, Characteristics of patients of categorized by p53 codon 72 genotype p53 codon 72 CG

p53 codon 72 GG

NO. of patients

60

165

140

Age, year

72.63 ± 9.3

73.0 ± 9.3

Smoking, pack-year

65.0 ± 30.1

58.7 ± 30.8

22

54

Sex, male (female)

58 (2)

157 (8)

133 (7)

FVC, % predicted

99.7 ± 20.4

93.8 ± 20.2

96.7 ± 19.7

FEV1/FEV1, %

63.4 ± 13.9

59.7 ± 16.0

57.8 ± 17.5

FEV1, % predicted

82.5 ± 24.6

73.0 ± 25.8 *

73.9 ± 27.4 *

Current smoker,

61.5 ± 37.4

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71.2 ± 8.5

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NO.

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p53 codon 72 CC

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Table 5, Characteristics of patients of categorized by MDM2 SNP309 genotype MDM2 SNP309 GT

MDM2 SNP309 TT

NO. of patients

94

178

93

Age, y

72.5 ± 9.4

71.5 ± 9.3

Sex, male (female)

91 (3)

166 (12)

Smoking, pack-year

62.9 ± 38.4

60.4 ± 32.2

34

55

FVC, % predicted

95.5 ± 17.3

96.6 ± 21.0

94.9 ± 21.2

FEV1/FEV1, %

62.5 ± 14.7

59.4 ± 16.6

57.0 ± 17.2

FEV1, % predicted

77.2 ± 23.4

74.7 ± 26.7

71.5 ± 28.6

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NO.

73.5 ± 8.1 91 (2)

59.6 ± 30.2

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Current smoker,

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MDM2 SNP309 GG

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  e-Appendix 1. Detailed methods and results

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Determination of %low attenuation area (LAA%) from chest CT scans

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CT scans of 286 patients from Kanazawa Medical University were acquired with Somatom Definition FLAH, CT scans of 36 patients from the University of Fukui were acquired with Somatom Definition AS+, and CT scans of 43 patients from the Fukui Red Cross Hospital were acquired with Brilliance 64. All scans were acquired with a slice thickness of less than 2 mm. We calculated LAA% using a threshold of -960 HU at a 2 mm slice thickness to assess emphysematous changes and the total lung volume with LungVisonTM version 2.1 (Cybernet Systems CO.LTD, Tokyo, Japan). We also performed a multivariate analysis of LAA between institution/scanner types on the effect of p53 and MDM2 SNP, and found almost the same association as with data from Kanazawa Medical University. The average and trend of LAA% changes in patients from University of Fukui and Fukui Red Cross Hospital were very comparable with the data from Kanazawa Medical University, however, the number of patients from these institutions was too small to analyze (e-Figure 1). In addition, we analyzed correlation between DLco/VA and LAA% in each institution (99 patients from Kanazawa Medical University, 16 patients from University of Fukui, and 21 patients from Fukui Red Cross Hospital), and found a strong correlation between %DLco/VA and LAA% in the data from all institution, and also found a similar pattern of correlation among the three groups (e-Figure 2). Cell culture

Chemicals

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Human lung fibroblasts (HLFs; purchased from Lonza) were isolated from human peripheral lung tissue, and used at 6th passage for all experiments. They were cultured in DMEM supplemented with 10% FBS (Lonza). The cells were cultured in 175-cm2 tissue culture flasks in a cell-culture incubator (37˚C, 5% CO2, and 95% air) and used in all the experiments after trypsinization. After reaching confluence, media were changed, and after addition of various concentrations of CSE, the cells were cultured for another 24 hours and used for MTS assay (0.1% ~ 10% of CSE) and Western blot analysis (0.1% ~ 3% of CSE). Some cells were transfected with p53 siRNAs or p53-expressing plasmids coding p53 gene with codon 72 C/G polymorphisms, or luciferase reporter plasmids coding MDM2 gene with SNP309 G/T polymorphisms, and cultured for 48 hours after transfection. In additional experiments, cells transfected with p53 siRNA or plasmids coding MDM2 gene with SNP309 G/T polymorphisms were cultured for additional 24 hours with or without 1% of CSE. After incubation, the cells were harvested and used for luciferase assays, and western blot analysis. The concentration of CSE (1%) was determined by MTS assay and examining dose-dependent effect of CSE on protein expression of p53 and Sp1 in HLFs (Figure 5A and B).

Chemicals and materials were obtained from the following sources: RNA later-ICE kit frozen tissue transition solution was from Ambion Inc. (Austin, TX); high capacity cDNA reverse transcription kit was from Applied Biosystems Inc. (Foster City, CA); ECL system (Western Lightening and Western Lightening plus-ECL) was from PerkinElmer (Waltham, MA); high-capacity cDNA archive kit and the power SYBR Green PCR master mix were from Applied Biosystems (Foster City, CA); NE-PER nuclear and cytoplasmic extraction reagents was from Thermo Scientific (Rockford, IL); 4–12% Bis-Tris Nupage gels, and MES-SDS running buffer were from Invitrogen Online supplements are not copyedited prior to posting and the author(s) take full responsibility for the accuracy of all data.

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(Carlsbad, CA, USA); the polyvinylidene difluoride (PVDF) membrane was from Bio-Rad Laboratories (Richmond, CA); protease inhibitor cocktail tablets were from Roche Applied Science (Indianapolis, IN); miRNeasy mini kit, miScript reverse transcription kit and miScript SYBR Green dye were from Qiagen (Valencia, CA); mouse anti-HIF-1α antibody, mouse anti-p53 antibody, and goat anti-lamin B antibody were from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA); All other chemicals were purchased from Sigma (St. Louis, MO). Genotyping

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Genomic DNA from peripheral blood was extracted and stored in PAX gene collection tubes (Qiagen, Valencia, CA, USA) at -20 °C using PAXgene blood DNA kit (Qiagen, Valencia, CA, USA). Genomic DNA from lung tissue samples was extracted using DNeasy blood & tissue kit (Qiagen).

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Genotyping of p53 codon 72 and MDM2 SNP309 was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Briefly, PCR was performed with 100 ng of genomic DNA and specific oligonucleotide primers using Type-it Mutation Detect PCR kit (Qiagen) in GeneAmpTM PCR System thermocycler (Applied Biosystems). The sequence of forward primer for p53 was 5’- CATGGGACTGACTTTCTGCT -3’ and the sequence of reverse primer was 5’GGTGTGATGGGATGGATAAA -3’. The sequence of forward primer for MDM2 was 5’CGGGAGTTCAGGGTAAAGGT -3’ and the sequence of reverse primer was 5’CGGAACGTGTCTGAACTTGA -3’. Polymorphism of p53 gene codon 72 was analyzed by BstUI (New England Biolab; Beverly, MA) digestion of PCR products for 4 h at 60 °C. The G allele, but not the C allele, has a single BstUI site within the amplified fragment; the fragments were resolved by electrophoresis on 1.5% agarose gel with ethidium bromide staining. MDM2 gene SNP309 polymorphism was analyzed by MspA1I (New England Biolab; Beverly, MA) digestion of PCR products for 12 h at 37 °C. The T allele, but not the G allele, has a single MspA1I site within the amplified fragment; the fragments were resolved by electrophoresis on 2.0% agarose gel with ethidium bromide staining.

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Real-time RT-PCR analysis of mRNA levels from lung tissue samples

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Fresh frozen lung tissue samples were immersed in RNA later-ICE kit solution according to the manufacturer’s instructions. After soaking overnight, the tissue samples were homogenized using FastPrep-24 (MP Biomedicals, Solon, OH). Isolation of total RNA from lung tissues was performed using a miRNeasy mini kit according to the manufacture’s protocol. Total RNA (1 µg) was reverse-transcribed using random primers and MultiScribe RT (High-Capacity cDNA Archive Kit) for mRNA analysis and miScript reverse transcription kit for miRNA analysis. PCR was performed with the obtained reverse transcribed products using specific oligonucleotide primers. The sequence of forward primer for MDM2 was 5’- CGACAAAGAAAACGCCACAA -3’ and the sequence of reverse primer was 5’- TCCTGATCCAACCAATCACC -3’. The sequence of forward primer for ß-actin was 5’- GCAAGCAGGAGTATGACGAG -3’ and the sequence of reverse primer was 5’CAAATAAAGCCATGCCAATC -3’. All PCR reactions were performed with a LightCyclerTM PCR system (Roche Diagnostics, Meylan, France) using DNA binding SYBR Green dye for mRNA analysis and miScript SYBR Green dye for miRNA analysis of PCR products. The ß-actin gene was used as a reference of mRNA.

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  Western blot analysis

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Cytoplasmic and nuclear proteins from the lungs and cultured cells were prepared using NEPER Nuclear and Cytoplasmic Extraction Reagents based following the manufacturer’s protocol. Protein extracts were analyzed for protein content using a Bradford method. Each sample was quantified, and then 40 µg of protein (cytoplasmic protein) or 20 µg of protein (nuclear protein) was loaded into each lane of a 4–12% Bis-Tris Nupage gel with MES SDS running buffer, according to the manufacturer’s protocol. The proteins were transferred to a PVDF membrane by electrophoresis, and the membrane was then probed with the primary and secondary antibodies. The ECL system was used for protein detection of the proteins. Lamin B was used as a reference of nuclear protein, and ß-actin protein was used as a reference of cytosolic protein.

Cell transfection with plasmids and small interfering RNA (siRNA)

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p53 and control siRNAs were designed and synthesized by Invitrogen. The p53 and control RNA target sequences were 5’-GCGCACAGAGGAAGAGAAU-3’ and 5’CCUAGAACCUAAGACCCUU-3’, respectively. HLFs were seeded into 6-cm dishes and incubated until they had reached about ~60% confluence. After rinsing, the cells were incubated with liposome solution containing Opti-MEM medium, 10 µL/mL of Lipofectamine 3000 (Invitrogen), and 100 nM siRNA, and 10% FBS. After 8 hours of incubation, an equivalent amount of Opti-MEM medium containing 10% FBS was added to the dishes and the incubation was continued for additional 16 hours. After 24 hours of transfection, the liposome solutions were replaced with DMEM containing 10% FBS and cultured for additional 24 hours. After incubation, MTS assay and Western blot analysis were performed following the incubation.

Preparation of Cigarette Smoke Extract (CSE)

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CSE was prepared as previously reported1. Briefly, one cigarette without a filters (Marlboro; Philip Morris International Inc; New York City, New York) was burned, and the smoke was passed, using a vacuum pump with a flowmeter at a rate of 300 mL/min, through a glass Cambridge filter (Cambridge Filter Japan, Ltd; Tokyo, Japan) with 0.20-µm pores, to remove particles and bacteria, into a vessel containing phosphate-buffered saline (PBS) (1 mL per one cigarette). The CSE-PBS solution was prepared fresh for each set of experiments, and the effect of CSE on cell viability was checked by MTS assay each time.

MTS assay MTS assay was performed using a CellTiter 96® AQueous One Solution cell proliferation assay kit in accordance with the manufacturer’s protocol. Briefly, HLFs were seeded in 24-well culture plates, cultured until confluence, and transfected with p53 siRNA or p53-expressing plasmid, in the presence or absence of various concentrations (0.1 to 10%) of CSE. The cells were then incubated with 20 µL of CellTiter 96® AQueous One Solution Reagent for 15 min, and absorbance at 450 nm was measured. Online supplements are not copyedited prior to posting and the author(s) take full responsibility for the accuracy of all data.

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Construction of plasmids coding p53 gene with codon72 C/G polymorphism and luciferase reporter assay with cells transfected with plasmids coding MDM2 SNP309 G/T polymorphism

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pCMV-p53 (plasmid coding wild-type p53 genes) and pCMV β-galactosidase-encoding plasmid were purchased from Clontech (San Jose, CA, USA). pCMV-p53 plasmid coding p53 gene with codon 72 C polymorphism was created from wild-type pCMV-p53 using KOD-Plus-Mutagenesis Kit (Toyobo CO., LTD, Osaka, Japan) by inverse PCR, according to the manufacture’s protocol (eFigure 3). Luciferase reporter constructs coding MDM2 gene with SNP309 G/T polymorphism were generated from pGL3-luciferase reporter vector (Promega, Madison, WI, USA) and a synthesized DNA containing MDM2 SNP309 G/T promoter region (919bp). Synthesized DNA fragments were inserted into the pGL3-luciferase reporter vector, as described previously2 (e-Figure 4). All plasmid constructs were verified by PCR-RFLP.

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All plasmids were purified using Qiagen plasmid midi and maxi kits. HLFs were seeded into 6-cm dishes and incubated in DMEM containing 10% FBS for 24 hours, until they had reached about ~70% confluence. After rinsing, the cells were incubated with 2.5 mL of liposome solution containing serum-free Opti-MEM medium, Lipofectamine 3000 (10 µL/plate) and plasmid constructs (2 µg/plate), and β-galactosidase-encoding plasmid (0.5 µg/plate). After 5 hour of incubation, the same amount of Opti-MEM medium containing 20% FBS was added to the dishes and the incubation was continued for another additional 19 hours. After 24 hours of transfection, the cells were washed twice with PBS, and the liposome solution was replaced with DMEM containing 10% FBS. After 48 h of the transfection, luciferase reporter activity was measured in MDM2 SNP309 G/T plasmid-transfected cells using luciferase assay system (Promega) for gene transcription activity. β-galactosidase activity was assayed to normalize the data. Cells transfected with p53 gene codon 72 C/G plasmids were analyzed by MTS assay and Western blotting.

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Protocol

Principal Investigator (PI): Shiro Mizuno, MD. PhD.

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Country: Japan

Kanazawa Medical University, 1-1 Daigaku, Uchinada-cho, Kahoku-gun Ishikawa, Japan, 920-0265.

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Funding: Grants-in-Aid for Scientific Research of Japan (No. 24591144)

Planned number of centers: 3 centers 1. 2. 3.

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Title of study: p53 signaling pathway polymorphisms and emphysematous changes in COPD patients

Division of Respiratory Medicine, Kanazawa Medical University, Ishikawa, Japan. Department of Respiratory Medicine, University of Fukui, Fukui, Japan. Department of Respiratory Medicine, Fukui Red Cross Hospital, Fukui, Japan.

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Primary and secondary objectives with the associated endpoints 1. Primary objective and endpoint:

To investigate whether p53 codon 72 and MDM2 SNP309 polymorphisms are associated with the severity of pulmonary emphysema in smokers.

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(Association between p53 and MDM2 polymorphisms (rs1042522, rs2279744) and levels of LAA% measured from chest HRCT in non-COPD smokers and COPD patients.)

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2. Secondary objective and endpoint:

To assess whether these gene polymorphisms are associated with the severity of COPD. (Distribution and frequency of the genotype and allele of p53 and MDM2 polymorphisms between non-COPD smokers and COPD patients categorized by GOLD classification.)

Population and patients selection: Total number of patients: 400 patients

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  Timelines and Study duration: Start date: October 2011

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End date: September 2014

Inclusion and Exclusion Criteria Non-COPD smokers

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Inclusion criteria

Capable of providing informed consent Men and women, age more than 40 Current or former smokers with more than 10 pack-year Normal lung function test based on post-bronchodilator spirometry All individuals have chest HRCT (slice less than 2 mm) Good overall health without history of chronic lung disease, including asthma Normal routine laboratory evaluation, including general hematologic studies, general serologic studies, and urine analysis 8. Women - not pregnant 9. Willingness to participate in the study

Exclusion criteria

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1. 2. 3. 4. 5. 6. 7.

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1. Unable to meet the inclusion criteria 2. Positive for bronchodilator test (12% and 200mL increase in FEV1) 3. Evidence of any lung diseases in chest HRCT except pulmonary emphysema and bronchial wall thickness in chest HRCT 4. Current active infection or acute illness of any kind 5. Evidence of malignancy within the past 5 years

Smokers with COPD Inclusion criteria 1. 2. 3. 4. 5. 6. 7. 8.

Capable of providing informed consent Men and women, age more than 40 Current or former smokers with more than 10 pack-year Meeting GOLD stages I-IV criteria for chronic obstructive lung disease (COPD) based on postbronchodilator spirometry All individuals have chest HRCT (slice less than 2 mm) Normal routine laboratory evaluation, including general hematologic studies, general serologic studies, and urine analysis Women - not pregnant Willingness to participate in the study

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  Exclusion criteria

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1. Unable to meet the inclusion criteria 2. Positive for bronchodilator test (12% and 200mL increase in FEV1) 3. Evidence of any lung diseases except pulmonary emphysema and bronchial wall thickness in chest HRCT 4. Current active infection or acute illness of any kind 5. Evidence of malignancy within the past 5 years

Sample collection and preparation

Method for determining LAA%

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1. Blood samples will be taken from each patients of Non-COPD smoker and COPD smokers using Paxgene blood DNA tube for genotype and allele analysis 2. DNA tubes will be stored at -80C until the time of extraction of DNA. 3. Genomic DNA will be extracted from Paxgene blood DNA tube and used for genotyping of MDM2 and p53 related polymorphisms (rs1042522, rs2279744) by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) analysis.

Statistical Analysis

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CT scans will be acquired with either a 64 or 128 multidetector CT scanner with a slice thickness of less than 2 mm. %low attenuation area (LAA%) will be measured using a threshold of −960 HU to assess emphysematous change and total lung volume using a computer software LungVisionTM version 2.1 (Cybernet Systems CO. LTD., Tokyo, Japan.).

References

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The statistical significance of differences in distribution and frequency of the genotype and allele between the groups will be determined by chi-squared test of independence and with Fisher exact test. Comparisons of age, smoking index (expressed as pack-years), pulmonary function parameters will be performed using ANOVA with Bonferroni corrections for multiple comparisons. Comparisons were considered statistically significant at p < 0.05.

1.

Hanaoka M, Droma Y, Chen Y, et al. Carbocisteine protects against emphysema induced by cigarette smoke extract in rats. Chest 2011;139(5):1101–1108.

2.

Chien W-P, Wong R-H, Cheng Y-W, Chen C-Y, Lee H. Associations of MDM2 SNP309, transcriptional activity, mRNA expression, and survival in stage I non-small-cell lung cancer patients with wild-type p53 tumors. Ann Surg Oncol 2010;17(4):1194–1202.

Online supplements are not copyedited prior to posting and the author(s) take full responsibility for the accuracy of all data.

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Sex, male (female)

16 (8)

Age, year

66.6 ± 10.7

Smoking, pack-year

50.2 ± 37.5

FVC, % predicted

81.2 ± 25.3

FEV1/FVC %

48.8 ± 20.1

FEV1, % predicted

55.7 ± 33.3

GOLD (Stage I/II/III/IV)

6/9/0/9

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24

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Number of lung tissue samples

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e-Table 1, characteristics of patients of lung tissue samples

Forced Vital Capacity (FVC), Forced Expiratory Volume in 1 Second (FEV1), Global Initiative for Obstructive Lung Disease (GOLD)

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Values are expressed as means ± SD.

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e-Figure 1 Multivariate analysis of LAA% association with p53 and MDM2 polymorphisms in patients from different institutions.

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e-Figure 2

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Correlation between LAA% and %DLco/VA in patients from different institutions (99 patients from Kanazawa Medical University, 16 patients from University of Fukui, and 21 patients from Fukui Red Cross Hospital).

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e-Figure 3 Construction of pCMV-p53 plasmid coding p53 gene with codon 72 C/G polymorphism. Online supplements are not copyedited prior to posting and the author(s) take full responsibility for the accuracy of all data.

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e-Figure 4 Construction of luciferase reporter vectors coding MDM2 gene with SNP309 G/T polymorphism. Online supplements are not copyedited prior to posting and the author(s) take full responsibility for the accuracy of all data.

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e-Figure 5

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Flowchart depicting how the analytical samples were obtained.

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BDR: Bronchodilator response (12% and 200 mL increase in FEV1).

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Online supplements are not copyedited prior to posting and the author(s) take full responsibility for the accuracy of all data.