Reflux-Induced Oxidative DNA Damage Repair Early in Barrett's Carcinogenesis

项目摘要 胃食管反流病（GERD）可并发巴雷特食管， 一种化生的、膀胱型的粘膜替代了受损的食管鳞状粘膜 的GERD。GERD和Barrett食管都是食管腺癌的危险因素， 癌症的发病率在几十年来迅速上升。慢性胃食管反流病会导致恶性胃食管反流病， 通过引起炎症、氧化应激和氧化DNA损伤来转化巴雷特食管 化生性粘膜GERD的现代医学治疗几乎完全是为了减少 质子泵抑制剂（PPI）等药物对胃酸产生非常有效， 控制反流性食管炎然而，PPI并不能消除胃酸分泌， 它，而且他们没有采取任何措施来纠正潜在的反流素质。因此，PPI不能防止弱 酸性物质和胆汁盐，这两者都可以对食管造成氧化损伤。这或许可以解释 为什么尽管PPI广泛使用，食管腺癌的发生率仍持续上升。到 为了预防巴雷特癌症，需要新的治疗方法来最大限度地减少反流诱导的氧化基因组损伤。 最近的数据表明，食管腺癌的发展是GERD的直接后果， 导致巴雷特化生中的氧化性DNA损伤。如果不修复，这种DNA损伤会导致基因组 不稳定性和致癌作用。维持基因组的完整性需要适当的细胞反应， 氧化损伤，通常由p53基因提供。这种基因在哺乳期内经常失活。 Barrett食管的致癌作用。在一些p53缺陷型细胞中，p38可以承担以下作用： 基因组稳定性的“守护者”。在早期的研究中，我们发现食管酸灌注特异性地 在体内患者的非发育异常Barrett粘膜中激活p38，体外Barrett细胞 对胆汁酸诱导的DNA损伤特别敏感。我们还建立了巴雷特上皮细胞系 它忠实地再现了原代组织中由酸和胆汁盐诱导的分子事件。我们有 在这些独特的细胞系中，我们建议使用这些细胞系进行研究， 巴雷特癌症形成的早期阶段 我们有初步的数据表明，弱酸性胆盐诱导巴雷特上皮细胞， 产生活性氧（ROS），导致氧化性DNA损伤。这种氧化损伤导致 在p53完整的Barrett细胞中，磷酸化p38的适度短暂增加，而氧化性DNA损伤则引发了一种新的细胞凋亡。 在p53缺陷型Barrett细胞中，磷酸化p38持续增加。我们发现，抑制p38损害 Barrett细胞去除脱嘌呤/脱嘧啶（AP）位点的能力，氧化性炎症的早期表现， 通常由AP内切核酸酶-1（APE-1）（一种碱基切除修复蛋白）消除的DNA损伤。我们 已经发现酸性胆汁盐导致巴雷特细胞增加其表达和核定位， 核磷蛋白1（NPM 1），一种增强APE-1功能效率的蛋白质;这些事件也是 p38抑制作用。基于这些发现，我们假设p38通路的激活与肿瘤细胞的凋亡有关。 Barrett细胞通过回流诱导的氧化应激是一种重要的癌症预防机制， 上调NPM 1以增强APE-1修复氧化性DNA损伤的效率。我们提出的 研究旨在阐明p38激活调节NPM 1以增强APE-1的机制 在Barrett细胞中修复反流诱导的氧化DNA损伤的效率，并证明急性 Barrett患者的反流性食管炎与p38激活和增强的 APE-1在Barrett化生中的作用。这些研究将阐明早期的细胞和分子 导致Barrett食管肿瘤进展的事件，从而为Barrett食管的发展提供基础。 新的医学治疗方法来预防我们退伍军人患者的致命巴雷特癌症。

Project Summary Gastroesophageal reflux disease (GERD) can be complicated by Barrett’s esophagus, the condition in which a metaplastic, intestinal-type mucosa replaces esophageal squamous mucosa that has been damaged by GERD. Both GERD and Barrett’s esophagus are risk factors for esophageal adenocarcinoma, a deadly cancer whose incidence has been increasing rapidly for decades. Chronic GERD contributes to the malignant transformation of Barrett’s esophagus by causing inflammation, oxidative stress and oxidative DNA damage in the metaplastic mucosa. The modern medical treatment of GERD is directed almost exclusively at decreasing gastric acid production with medications such as proton pump inhibitors (PPIs), which are very effective in controlling reflux esophagitis. However, the PPIs do not eliminate gastric acid secretion, they merely decrease it, and they do nothing to correct the underlying reflux diathesis. Thus, PPIs do not prevent the reflux of weakly acidic material and bile salts, both of which can inflict oxidative injury on the esophagus. This might explain why the frequency of esophageal adenocarcinoma continues to rise despite the widespread use of PPIs. To prevent Barrett’s cancers, new treatments are needed to minimize reflux-induced, oxidative genomic damage. Recent data suggest that esophageal adenocarcinomas develop as a direct consequence of GERD- induced oxidative DNA damage in Barrett’s metaplasia. Left unrepaired, this DNA damage leads to genomic instability and carcinogenesis. Maintenance of genomic integrity requires an appropriate cellular response to oxidative injury, which normally is provided by the p53 gene. This gene is inactivated frequently during carcinogenesis in Barrett’s esophagus, however. In some p53-deficient cell types, p38 can assume the role of “guardian” of genomic stability. In earlier studies, we showed that esophageal acid perfusion specifically activated p38 in the non-dysplastic Barrett’s mucosa of patients in vivo, and that Barrett’s cells in vitro were uniquely susceptible to bile acid-induced DNA damage. We also have established Barrett’s epithelial cell lines that faithfully recapitulate molecular events induced by acid and bile salts in primary tissues. We have inactivated p53 in some of these unique cell lines, which we propose to use in studies that recapitulate the early stages of Barrett’s carcinogenesis. We have preliminary data demonstrating that weakly acidic bile salts induce Barrett’s epithelial cells to generate reactive oxygen species (ROS) that cause oxidative DNA damage. This oxidative injury results in a modest, brief increase in phospho-p38 in p53-intact Barrett’s cells, while oxidative DNA damage triggers a strong, sustained phospho-p38 increase in p53-deficient Barrett’s cells. We show that inhibition of p38 impairs the ability of Barrett’s cells to remove apurinic/apyrimidinic (AP) sites, the early manifestations of oxidative DNA damage that ordinarily are eliminated by AP endonuclease-1 (APE-1), a base-excision-repair protein. We have found that acidic bile salts cause Barrett’s cells to increase their expression and nuclear localization of nucleophosmin 1 (NPM1), a protein that enhances the functional efficiency of APE-1; these events also are impaired by p38 inhibition. Based on these findings, we hypothesize that activation of the p38 pathway in Barrett’s cells by reflux-induced oxidative stress is an important cancer-preventive mechanism that works by upregulating NPM1 to enhance the efficiency of APE-1 in repairing oxidative DNA damage. Our proposed studies are designed to elucidate mechanisms whereby p38 activation regulates NPM1 to enhance APE-1 efficiency in repairing reflux-induced oxidative DNA damage in Barrett’s cells, and to demonstrate that acute reflux esophagitis in Barrett’s patients is associated with p38 activation and with markers of enhanced efficiency of APE-1 in their Barrett’s metaplasia. These studies will elucidate early cellular and molecular events that drive neoplastic progression in Barrett’s esophagus, thereby providing the basis for development of new medical treatments to prevent deadly Barrett’s cancers in our Veteran patients.