Somatic DNA alterations in lung epithelial barrier cells in COPD patients

https://doi.org/10.1016/j.pupt.2009.12.001Get rights and content

Abstract

Background

Instability of the Microsatellite DNA Instability (MSI) and Loss of Heterozygosity (LOH) have been previously detected in sputum cells of COPD patients. However, the particular cell subpopulation exhibiting genetic instability in COPD was uncertain. The aim of this study was to determine which cell type expresses Microsatellite DNA Instability in sputum and BALF samples from COPD patients.

Methods

Thirty-five COPD patients and 30 non-COPD smokers were studied. Sputum was induced from 20 COPD patients and 20 non-COPD smokers and BALF was obtained from 15 COPD patients and 10 non-COPD smokers. The sputum cell pellet and BALF samples were processed using immunomagnetic technology to separate antibody-specific cell subpopulations, using CD45 + for leukocytes, Epithelial enrich (MACS) for sputum epithelial cells and HEA-human epithelial antigen-(Dynal) for BAL epithelial cells. Microsatellite DNA amplification was performed using specific primers, namely G29802, D6S2223, D6S344, D6S263, D5S207, D13S71, RH70958, and D17S250. The presence of MSI and/or LOH was analyzed with LI-COR Saga GT Microsatellite Analysis Software.

Measurements and main results

None of the non-COPD smokers exhibited any genetic alteration. MSI and LOH were found in 15 cases (8 MSI and 7 LOH) in sputum and BAL samples. MSI and/or LOH were revealed only in the epithelial barrier cells. LOH was detected in D5S207, D6S344, G29802 and D17S250 microsatellite markers, while MSI in D13S71, D5S207 and D6S344. The entire leukocyte subpopulation exhibited no genetic alteration.

Conclusions

Our results support the hypothesis that chronic inflammation and oxidative burden in COPD can lead to DNA damage of the lung epithelial barrier cells, detected at the Microsatellite DNA level. Further studies are required to investigate the significance of these findings in the pathogenesis of COPD.

Introduction

Chronic obstructive pulmonary disease (COPD) is a major health problem worldwide with increasing prevalence, morbidity and mortality. Although chronic obstructive pulmonary disease (COPD) is a common disease and its primary cause, cigarette smoking, is well known, a large number of questions remain to be elucidated regarding disease pathogenesis involving genetics, environmental and epigenetic factors [1], [2]. The genetic background of COPD has been the focus of many recent studies [3]. The role of acquired somatic mutations in the pathogenesis of COPD has been shown to be important [4], [5]. Acquired somatic mutations are considered as sporadic changes in genes or gene regulatory regions that occur spontaneously and rarely. However, they dramatically increase in frequency in tissues exposed to repeated exogenous mutagenic insults. Although the susceptibility to acquiring such mutations might be controlled by inherited genes, somatic mutations do not affect the germ line and are not heritable [4].

Microsatellites are unique to every individual and identical in cells from different tissues in the same individual [6]. They are highly polymorphic markers used for genome mapping in many organisms including humans and are often located in or near important gene loci, allowing them to be used as markers for disease and providing information about individual gene status [7]. The assessment of microsatellite DNA instability in peripheral tissue samples has offered a reliable means for the study of acquired mutations. This is accomplished with the use of microsatellite markers targeting specific chromosomal loci near or in genes that are known or suspected to be implicated in the pathogenesis of a disease [4], [7]. Loss of heterozygosity and microsatellite DNA instability are genetic alterations that have been initially reported in a number of human cancers and in precancerous and malignant lesions of smokers [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. However, during recent years such phenomena have also been detected in various benign pulmonary and extra-pulmonary diseases [5], [7], [18], [19], [20]. We have recently detected microsatellite DNA instability (MSI) and/or loss of heterozygosity (LOH) in sputum cells of COPD and asthmatic patients. [5], [7], [18], [19], [20]. To our knowledge MSI and/or LOH have not yet been detected in BAL samples from COPD patients. However the particular sputum and/or BAL cell population exhibiting this genetic instability has not yet been identified. Thus, in continuation of our previous work [5], [18], [19], [20], the specific aims of this study were to examine whether airway and bronchoalveolar cells are prone to somatic acquired genetic alterations by evaluating sputum and BALF cells and to determine which specific cell population is affected by these somatic acquired genetic alterations. The identification of the susceptible cell population to somatic DNA damage in COPD may elucidate an important component of disease pathogenesis. The natural ability of the injured lung in COPD to shut down persisting inflammation and initiate proper tissue repair is dependent on intact DNA auto-repair mechanisms. Thus, our study could answer whether increased inflammatory burden on COPD patients results in acquired somatic DNA mutations of lung epithelia.

Section snippets

Subjects

A total of 35 COPD patients and 30 control subjects (non-COPD smokers) were studied. All patients were diagnosed with COPD, GOLD stage II. All COPD patients were ex-smokers more than 6 months, whereas the control group consisted of current and ex-smokers. However, all current smokers had withdrawn smoking at least 48 h prior to bronchoscopy or sputum induction.

Patients were allocated in two groups: the first group (A) consisted of 20 COPD patients who underwent sputum induction, 18 males and 2

Results

Immuno- magnetic separation was performed in sputum and BALF samples, in order to isolate two specific cell populations: (a) leukocytes and (b) epithelial cells. This was verified by flow cytometry using anti-cytokeratin-FITC (MACS Technology, Miltenyi Biotec, Germany). The purity of epithelial cells ranged from 78% to 93% (Fig. 1). Then, each cell subpopulation was processed for genomic analysis. None, of the healthy control subjects, exhibited any genetic alteration in the eight

Discussion

The main finding of this study was that somatic DNA alterations in sputum and BALF, was exclusively exhibited in the epithelial cells. This might represent a significant observation taking into account the central role of epithelial cells in COPD pathogenesis [22]. We have used magnetic beads technology and microsatellite DNA analysis in order to identify the cellular subpopulation susceptible to somatic DNA damage in COPD patients. This is the first study to separate sputum and BAL cellular

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

KS recruited the study subjects, carried out sputum induction and processing, performed the immunomagnetic separation studies, and drafted the manuscript. ET conceived of the study, participated in its design and coordination and drafted the manuscript. EN carried out the molecular genetic studies. AK participated in the recruitment of the subjects. IL carried out bronchoscopy and BALF. NT carried out bronchoscopy and BALF and performed the statistical analysis. NS conceived of the study, and

Acknowledgements

The present study was supported by an unrestricted and competitive research grant from the National Ministry of Education of Greece (Project “Pythagoras II”).

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