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: 6663968
• 负责人: GEORGE E. ILIAKIS
• 金额: $ 22.84万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6663968
• 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作为一种手段 使肿瘤细胞对高温敏感。它的设计目的是测试 热诱导细胞杀伤中的增强作用实现的假说 在急性酸化后，部分是通过细胞的减少来调节的 通常由电池中的热损伤激活的循环检查点 在正常pH下。重点放在分子表征上。 高温暴露的细胞中，检查点在S期激活。 尽管人们很早就知道细胞暴露在高温下 引起强烈的DNA复制抑制，我们的实验室是第一个 以提供证据证明这种抑制与进程有关 相当于在S阶段激活一个检查站。它一般都是 认为取消检查站会加剧有毒后果 诱导性损害。本实验设计针对的是分子 S相关卡的特征及对急性关卡的评估 酸化作为克服它的一种手段。新的初步数据允许我们 建立受热条件下DNA复制调控模型 细胞，第一组特定的目标被设计成系统地 测试一下它的假设。第二套具体目标旨在 从分子水平上刻画其废除的基础 酸化，并将这一信息应用于治疗人类 使用特定于该程序的协议的肿瘤。在建议中的关键角色 实验为真核细胞DNA复制提供了一种体外分析方法 基于猴病毒40(SV40)的体外DNA复制试验。我们建议 首先测试一个假设热诱导染色质损伤的模型 结构诱导的调控通路相当于检查点 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启动和复制水平上进行的 被认为独立于通过顺式作用过程的直接抑制 由染色质损伤引起，或由热诱导引起的抑制 放松对酶机械的管制。后一种过程被认为是 主要是为了抑制链的延长。建议的研究结果 预计实验将阐明检查站的作用 热敏活性及其分子机制的研究 决定因素，以及急性废除它的机制 酸化。这些研究最终将增进我们对 在酸性环境中的热敏化，并可能允许 生理学干预新靶点的确定 对肿瘤的修饰，或通过阻止激活的药物 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作用。