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: 6269549
• 负责人: GEORGE E. ILIAKIS
• 金额: $ 14.43万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6269549
• 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计划的基本目标 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。它旨在测试 假设热诱导细胞杀伤效果增强 急性酸化部分由细胞消除介导后 通常由细胞热损伤激活的周期检查点被保留 在正常pH值下。重点是a的分子表征 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 对DNA复制产生强烈的抑制作用，我们实验室是第一个 提供证据证明这种抑制与过程有关 相当于S期检查点的激活。一般是 认为取消检查站会加剧毒性后果 的诱发损害。实验设计旨在分子 S 期检查点的特征和急性期的评估 酸化作为克服它的手段。新颖的初步数据使我们能够 建立加热条件下 DNA 复制调控模型 细胞，第一组具体目标旨在系统地 检验其假设。第二组具体目标旨在 在分子水平上表征其被急性废除的基础 酸化，并将这些信息应用于人类的治疗 使用该计划特定的协议来治疗肿瘤。在提议中发挥关键作用 实验对真核 DNA 复制进行了体外测定， 基于猿猴病毒 40 (SV40) 的体外 DNA 复制测定。我们建议 首先测试一个假设染色质热诱导损伤的模型 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水平上进行的 被认为独立于顺式作用过程的直接抑制 由染色质损伤或热诱导抑制引起 酶机制的放松管制。后面的过程被认为 主要抑制链伸长。拟议的结果 实验有望揭示检查点的作用 热敏性激活并阐明其分子 决定因素，以及其被急性废除的机制 酸化。这些研究最终将增强我们对 在酸中毒环境中热敏化，并可能允许 通过生理学手段确定新的干预目标 改变肿瘤，或通过药物阻止激活 这些检查点。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。