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

项目摘要 胃食道反流病(GERD)可并发于Barrett‘s食道， 它是一种化生的肠型粘膜，取代了受损的食道鳞状粘膜 被GERD。GERD和Barrett‘s食道都是食管腺癌的危险因素，食管腺癌是一种致命的 几十年来发病率迅速增加的癌症。慢性胃十二指肠反流病致癌 Barrett‘s食管炎、氧化应激及DNA氧化损伤的改变 化生的粘膜。GERD的现代医学治疗几乎完全针对减少 使用质子泵抑制剂(PPI)等药物产生胃酸，这些药物在 控制反流性食管炎。然而，PPI并不能消除胃酸分泌，它们只是减少胃酸分泌。 它，而且它们对纠正潜在的反流素质毫无作用。因此，PPI并不能防止弱反流 酸性物质和胆盐，两者都会对食道造成氧化损伤。这也许可以解释 为什么食管腺癌的发病率在广泛使用PPI的情况下继续上升。至 为了预防Barrett‘s癌症，需要新的治疗方法来最大限度地减少回流诱导的氧化基因组损伤。 最近的数据表明，食管腺癌的发生是GERD的直接结果- 巴雷特化生诱导的DNA氧化损伤。如果不加以修复，这种DNA损伤会导致基因组 不稳定和致癌。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。这种基因经常被灭活 然而，巴雷特食道的致癌作用。在某些p53缺失的细胞类型中，p38可以扮演 基因组稳定性的“守护者”。在早期的研究中，我们发现食管酸灌流 活体患者非发育异常巴雷特粘膜中p38的活化，体外巴雷特细胞 对胆汁酸引起的DNA损伤非常敏感。我们还建立了巴雷特上皮细胞系 它忠实地概括了酸和胆盐在初级组织中诱导的分子事件。我们有 在这些独特的细胞系中的一些失活的p53，我们建议在研究中使用它来概括 巴雷特癌变的早期阶段。 我们有初步数据表明，弱酸性胆盐诱导巴雷特上皮细胞 产生可导致DNA氧化损伤的活性氧物种(ROS)。这种氧化损伤会导致 在p53完整的巴雷特细胞中，磷酸化p38的适度、短暂的增加，而氧化的DNA损伤触发了 P53基因缺失的巴雷特细胞中磷酸化p38表达持续增强。我们发现对p38的抑制会损害 巴雷特细胞清除无嘌呤/脱嘧啶(AP)部位的能力，这是氧化的早期表现 通常被碱基切除修复蛋白AP内切酶-1(APE-1)消除的DNA损伤。我们 已发现酸性胆盐导致Barrett细胞表达增加，并使其核定位增加 核磷蛋白1(NPM1)，一种增强APE-1功能效率的蛋白质；这些事件还包括 P38抑制作用减弱。基于这些发现，我们假设p38通路在 由回流诱导的氧化应激引起的巴雷特细胞是一种重要的癌症预防机制，它通过 上调NPM1以提高APE-1修复DNA氧化损伤的效率。我们的建议 研究旨在阐明p38激活调节NPM1增强APE-1的机制。 修复回流引起的巴雷特细胞氧化DNA损伤的效率，并证明急性 Barrett病患者的反流性食管炎与p38激活和增强的标志物相关 APE-1在其Barrett化生中的作用这些研究将阐明早期的细胞和分子 推动Barrett‘s食道肿瘤进展的事件，从而为 新的医疗方法，以防止我们的退伍军人患者患上致命的巴雷特癌症。

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.