S-PHASE CHECKPOINT ABROGATION BY ACIDIFICATION IN HEATED CELLS

通过加热细胞中的酸化消除 S 相检查点

• 批准号: 6300442
• 负责人: GEORGE E. ILIAKIS
• 金额: $ 18.41万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6300442
• 关键词: DNA replication; acidity /alkalinity; biological signal transduction; calorimetry; cell cycle; chromatin; cytotoxicity; genetic regulatory element; hyperthermia; neoplastic cell; posttranslational modifications; protein denaturation; protein structure; simian virus 40; stress proteins; temperature; tissue /cell culture; transcription factor

The present Project contributes to the fundamental goal of the Program to exploit the acidified microenvironment of the tumor as a means for sensitizing tumor cells to hyperthermia. It is designed to test the hypothesis that the potentiation in heat-induced cell killing achieved after acute acidification is mediated in part by an abrogation of cell cycle checkpoints that are normally activated by heat damage in cells kept under normal pH. The focus is on the molecular characterization of a checkpoint activated in S-phase in cells exposed to high temperatures. Although it has long been known that exposure of cells to hyperthermia causes a strong inhibition of DNA replication, our laboratory is the first to provide evidence that this inhibition is associated with processes equivalent to the activation of a checkpoint in S-phase. It is generally thought that abrogation of checkpoints potentiates the toxic consequences of the inducing damage. The experimental design aims at the molecular characterization of the S-phase checkpoint and the evaluation of acute acidification as a means to overcome it. Novel preliminary data allow us to formulate a model for the regulation of DNA replication in heated cells, and the first set of Specific Aims is designed to systematically test its postulates. The second set of Specific Aims is designed to characterize t the molecular level the basis of its abrogation by acute acidification, and to apply this information to the treatment of human tumors using protocols specific to the Program. Key role in the proposed experiments plays an in vitro assay for eukaryotic DNA replication, the Simian virus 40 (SV40) based in vitro DNA replication assay. We propose to test first a model postulating that heat-induced damage in chromatin structures induces regulatory pathways equivalent to check point activation that inhibit DNA replication by transacting-processes. This inhibition of DNA replication is at the level of replicon initiation and is considered independent of direct inhibition via cis-acting processes resulting from chromatin damage, or inhibition caused by heat-induced deregulation of the enzymatic machinery. The latter processes are thought to inhibit predominantly chain elongation. The results of the proposed experiments are expected to shed light on the role of checkpoint activation in heat sensitivity and to elucidate its molecular determinants, as well as the mechanism of its abrogation by acute acidification. The studies will ultimately enhance our understanding of heat sensitization in an acidotic environment, and may allow the definition of new targets for intervention by means of physiological modifications in the tumor, or by means of drugs preventing the activation of these checkpoints. Such intervention may further improve the efficacy of hyperthermia in the management of human tumors.

本项目有助于实现该方案的基本目标， 利用肿瘤的酸化微环境， 使肿瘤细胞对高温敏感。它旨在测试 假设热诱导细胞杀伤的增强实现了 急性酸化后，部分由细胞的破坏介导， 循环检查点，通常由细胞中的热损伤激活， 在正常的pH值。重点是对分子表征的一个 在暴露于高温的细胞中，检查点在S期被激活。 尽管人们早就知道细胞暴露在高温下 导致DNA复制的强烈抑制，我们的实验室是第一个 以提供证据证明这种抑制作用与 相当于S期检查点的激活。一般 我认为取消检查站会加剧有毒后果， 的诱导性损害。实验设计针对分子 S期检查点的表征和急性 酸化作为克服它的一种手段。新的初步数据使我们能够 建立了一个加热环境中DNA复制调控的模型， 细胞，第一套具体目标的目的是系统地 测试其假设。第二套具体目标旨在 在分子水平上表征其被急性 酸化，并将此信息应用于人类的治疗 使用该计划特定的方案治疗肿瘤。在拟议的 实验在真核DNA复制的体外测定中起作用， 基于猿猴病毒40（SV40）的体外DNA复制试验。我们建议 首先测试一个假设染色质中热诱导损伤的模型， 结构诱导相当于检查点的调节途径 通过交易过程抑制DNA复制的激活。这 DNA复制的抑制是在复制子起始的水平， 被认为不依赖于通过顺式作用过程的直接抑制 由于染色质损伤，或抑制引起的热诱导 酶机制的失调。后者被认为是 以主要抑制链伸长。建议的结果 实验有望揭示检查点的作用， 热敏感性的活化，并阐明其分子机制 决定因素，以及通过急性 酸化这些研究最终将提高我们对 酸环境中的热致敏作用，并可能导致 确定新的干预目标， 修饰的肿瘤，或通过药物阻止激活 这些检查站。这种干预可能会进一步提高疗效 热疗在人类肿瘤治疗中的应用。