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学疗法剂之一，通常与放射治疗结合使用。 尽管缺乏KU/DNA-PK修复复合物元素的细胞对电离辐射非常敏感，但我们发现此类细胞对顺铂明显抗性。 出现这种电阻是因为这些细胞无法激活一种依赖于DNA-PK的激酶活性来产生从细胞到细胞传递的信号的新颖信号途径，从而引起受体死亡。 最初的工作表明，由于间隙连接通信中有缺陷的细胞也对顺铂具有抗性，因此该信号是通过间隙连接传输的。 这些发现表明，DNA-PK活性和间隙连接通信可能会影响人类癌症中对顺铂的临床反应。 我们建议检验以下假设：间隙连接表达和功能的调节会影响对顺铂的细胞反应。 我们将研究通过选定的激酶和顺铂耐药的连接蛋白（间隙连接蛋白亚基）的磷酸化（间隙连接的蛋白质亚基）之间的联系。 我们将测试降低连接蛋白降解（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）对顺铂杀死细胞杀死的因素的影响。 为了确定响应于顺铂的KU/DNA-PK上游或下游的因素，我们将使用共免疫沉淀技术探测候选损伤识别和信号传导因素。 我们还将使用质谱法来鉴定顺铂处理后与Ku80结合改变的蛋白质。 使用代谢标记和色谱技术，我们将确定可以通过间隙连接传递KU/DNA-PK依赖性细胞毒性信号的候选分子。 最后，我们将检验以下假设：间隙连接表达和DNA-PK功能可以使用小鼠中的一系列遗传操纵肿瘤模型影响体内的顺铂反应。 该应用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。 这项工作可能导致使人类癌症对顺铂的策略，从而为新的癌症治疗方法提供了基础。