Cisplatin Damage Response and Cell-to-cell Communication

顺铂损伤反应和细胞间通讯

• 批准号: 7052785
• 负责人: PETER M GLAZER
• 金额: $ 28.35万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-15 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7052785
• 关键词: DNA repair; antineoplastics; athymic mouse; biological signal transduction; cell cell interaction; cell line; cisplatin; cytotoxicity; disease /disorder model; drug resistance; enzyme complex; gap junctions; gene expression; genetically modified animals; growth factor receptors; immunoprecipitation; mass spectrometry; membrane channels; membrane lipids; neoplasm /cancer chemotherapy; phosphorylation; platinum; protein kinase; protein protein interaction; protein transport; proteomics

DESCRIPTION (provided by applicant): Cisplatin is one of the most widely used chemotherapy agents, often in combination with radiation therapy. Although cells deficient in elements of the Ku/DNA-PK repair complex are extremely sensitive to ionizing radiation, we have found that such cells are markedly resistant to cisplatin. This resistance arises because these cells fail to activate a novel-signaling pathway that depends on the kinase activity of DNA-PK to generate a signal that is passed from cell to cell, inducing death in the recipient. Initial work has demonstrated that this signal is transmitted via gap junctions, since cells defective in gap junction communication are also resistant to cisplatin. These findings suggest that DNA-PK activity and gap junction communication may influence the clinical response to cisplatin in human cancers. We propose to test the hypothesis that the regulation of gap junction expression and function can influence the cellular response to cisplatin. We will investigate links between phosphorylation of connexins (the protein subunits of gap junctions) by selected kinases and cisplatin resistance. We will test the effect of factors that decrease connexin degradation (such as proteasome inhibitors) or enhance gap junction biogenesis (by altering connexin trafficking) on cell killing by cisplatin. To identify factors upstream or downstream of Ku/DNA-PK in response to cisplatin, we will probe candidate damage recognition and signaling factors, using co-immunoprecipitation techniques. We will also use mass spectrometry to identify proteins that show altered binding to Ku80 following cisplatin treatment. Using metabolic labeling and chromatography techniques, we will identify candidate molecules that may transduce the Ku/DNA-PK dependent cytotoxic signal through gap junctions. Finally, we will test the hypothesis that gap junction expression and DNA-PK function can influence cisplatin response in vivo using a series of genetically manipulated tumor models in mice. The long-term goal of this application is to elucidate the molecular mechanisms that mediate this novel cisplatin-response pathway. This work may lead to strategies to sensitize human cancers to cisplatin, providing the basis for new combined approaches to cancer therapy.

描述（由申请人提供）：顺铂是最广泛使用的化疗药物之一，通常与放射治疗联合使用。 尽管缺乏 Ku/DNA-PK 修复复合物元素的细胞对电离辐射极其敏感，但我们发现此类细胞对顺铂具有明显的耐药性。 产生这种耐药性的原因是这些细胞无法激活依赖于 DNA-PK 激酶活性的新型信号传导途径，以产生从一个细胞传递到另一个细胞的信号，从而诱导受体死亡。 初步研究表明，这种信号是通过间隙连接传递的，因为间隙连接通讯缺陷的细胞也会对顺铂产生耐药性。 这些发现表明 DNA-PK 活性和间隙连接通讯可能影响人类癌症对顺铂的临床反应。 我们建议检验间隙连接表达和功能的调节可以影响细胞对顺铂的反应的假设。 我们将研究所选激酶对连接蛋白（间隙连接的蛋白质亚基）的磷酸化与顺铂耐药性之间的联系。 我们将测试减少连接蛋白降解（例如蛋白酶体抑制剂）或增强间隙连接生物发生（通过改变连接蛋白运输）的因素对顺铂杀伤细胞的影响。 为了确定 Ku/DNA-PK 响应顺铂的上游或下游因子，我们将使用免疫共沉淀技术探测候选损伤识别和信号传导因子。 我们还将使用质谱法来鉴定顺铂治疗后与 Ku80 的结合发生改变的蛋白质。 使用代谢标记和层析技术，我们将鉴定可能通过间隙连接转导 Ku/DNA-PK 依赖性细胞毒性信号的候选分子。 最后，我们将使用一系列基因操作的小鼠肿瘤模型来检验间隙连接表达和 DNA-PK 功能可以影响体内顺铂反应的假设。 本申请的长期目标是阐明介导这种新型顺铂反应途径的分子机制。 这项工作可能会导致人类癌症对顺铂敏感的策略，为癌症治疗的新组合方法提供基础。