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      Oxidative DNA damage in lung tissue from patients with COPD is clustered in functionally significant sequences

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          Abstract

          Lung tissue from COPD patients displays oxidative DNA damage. The present study determined whether oxidative DNA damage was randomly distributed or whether it was localized in specific sequences in either the nuclear or mitochondrial genomes. The DNA damage-specific histone, gamma-H2AX, was detected immunohistochemically in alveolar wall cells in lung tissue from COPD patients but not control subjects. A PCR-based method was used to search for oxidized purine base products in selected 200 bp sequences in promoters and coding regions of the VEGF, TGF-β1, HO-1, Egr1, and β-actin genes while quantitative Southern blot analysis was used to detect oxidative damage to the mitochondrial genome in lung tissue from control subjects and COPD patients. Among the nuclear genes examined, oxidative damage was detected in only 1 sequence in lung tissue from COPD patients: the hypoxic response element (HRE) of the VEGF promoter. The content of VEGF mRNA also was reduced in COPD lung tissue. Mitochondrial DNA content was unaltered in COPD lung tissue, but there was a substantial increase in mitochondrial DNA strand breaks and/or abasic sites. These findings show that oxidative DNA damage in COPD lungs is prominent in the HRE of the VEGF promoter and in the mitochondrial genome and raise the intriguing possibility that genome and sequence-specific oxidative DNA damage could contribute to transcriptional dysregulation and cell fate decisions in COPD.

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          Most cited references 10

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          Sequence-specific oxidative base modifications in hypoxia-inducible genes.

          Reactive oxygen species associated with hypoxic signaling in pulmonary arterial endothelial cells (PAECs) oxidatively modify specific nucleotides in the hypoxic response element (HRE) of the VEGF gene (FASEB J.19:387-394; 2005). In this study, we determined in PAECs if hypoxia caused genome-wide oxidative modifications or if they were restricted to the promoters of genes differentially regulated by hypoxia. Comet assays indicated that there were no differences between normoxic and hypoxic PAECs in terms of global DNA damage. However, a simple PCR-based method involving DNA amplification before and after treatment with formamidopyrimidine DNA glycosylase (Fpg), a bacterial DNA repair enzyme that cleaves at sites of purine base oxidation, revealed that hypoxia caused modifications in the HREs of the hypoxia-inducible VEGF, HO-1, and ET-1 genes which coincided with accumulation of their respective mRNA transcripts. Promoter sequences not involved with hypoxic induction and coding regions of these genes failed to display Fpg-sensitive sites. Oxidative modifications also were not detected in sequences of the hypoxia down-regulated ornithine decarboxylase and TFAM genes or the constitutively expressed beta-actin gene. These findings show that hypoxia-mediated oxidative DNA modifications cluster in functionally relevant promoter sequences in hypoxia-inducible genes and suggest that such oxidative modifications may be biologically significant.
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            Oxygen radical-induced mitochondrial DNA damage and repair in pulmonary vascular endothelial cell phenotypes.

            Mitochondrial (mt) DNA is damaged by free radicals. Recent data also show that there are cell type-dependent differences in mtDNA repair capacity. In this study, we explored the effects of xanthine oxidase (XO), which generates superoxide anion directly, and menadione, which enhances superoxide production within mitochondria, on mtDNA in pulmonary arterial (PA), microvascular (MV), and pulmonary venous (PV) endothelial cells (ECs). Both XO and menadione damaged mtDNA in the EC phenotypes, with a rank order of sensitivity of (from most to least) PV > PA > MV for XO and MV = PV > PA for menadione. Dimethylthiourea and deferoxamine blunted menadione- and XO-induced mtDNA damage, thus supporting a role for the iron-catalyzed formation of hydroxyl radical. Damage to the nuclear vascular endothelial growth factor gene was not detected with either XO or menadione. PAECs and MVECs, but not PVECs, repaired XO-induced mtDNA damage quickly. Menadione-induced mtDNA damage was avidly repaired in MVECs and PVECs, whereas repair in PAECs was slower. Analysis of mtDNA lesions at nucleotide resolution showed that damage patterns were similar between EC phenotypes, but there were disparities between XO and menadione in terms of the specific nucleotides damaged. These findings indicate that mtDNA in lung vascular ECs is damaged by XO- and menadione-derived free radicals and suggest that mtDNA damage and repair capacities differ between EC phenotypes.
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              Oxidants in signal transduction: impact on DNA integrity and gene expression.

              Physiological stimuli using reactive oxygen species (ROS) as second messengers caused nucleotide-specific base modifications in the hypoxic response element of the VEGF gene in lung vascular cells, with the 3' guanine of the HIF-1 DNA recognition sequence uniformly targeted. Modeling this effect by replacing the targeted guanine with an abasic site increased incorporation of HIF-1 and the bi-functional DNA repair enzyme and transcriptional coactivator, Ref-1/Ape1, into the transcriptional complex and engendered more robust reporter gene expression. Oxidants generated in the context of physiological signaling thus affect nuclear DNA integrity and may facilitate gene expression by optimizing DNA-protein interactions.
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                Author and article information

                Journal
                Int J Chron Obstruct Pulmon Dis
                International Journal of COPD
                International Journal of Chronic Obstructive Pulmonary Disease
                Dove Medical Press
                1176-9106
                1178-2005
                2011
                2011
                13 March 2011
                : 6
                : 209-217
                Affiliations
                [1 ]Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, AL, USA;
                [2 ]Program in Translational Lung Research, Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado at Denver, Aurora, CO, USA
                Author notes
                Correspondence: Mark N Gillespie, Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA, Tel +1 (251) 460-6497, Fax +1 (251) 460-6798, Email mgillesp@ 123456jaguar1.usouthal.edu
                Article
                copd-6-209
                10.2147/COPD.S15922
                3107697
                21660298
                © 2011 Pastukh et al, publisher and licensee Dove Medical Press Ltd.

                This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.

                Categories
                Original Research

                Respiratory medicine

                dna damage, vegf hypoxic response element, copd, mtdna

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