HSPS, CYTOSKELETAL AND SURVIVAL RESISTANCE TO HEAT TO LOW PH ADAPTED CELLS

HSPS、细胞骨架和低 pH 适应细胞的耐热性

• 批准号: 6579386
• 负责人: RONALD Allen COSS
• 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/6579386
• 关键词: HeLa cells; acidity /alkalinity; antiport; cellular pathology; cytoskeleton; cytotoxicity; flow cytometry; fluorescence microscopy; gene expression; homeostasis; hyperthermia; intracellular; melanoma; membrane transport proteins; neoplastic cell; nucleoproteins; phosphorylation; stress proteins; western blottings

This project is designed to test if a causal relationship exists between upregulation of Heat Shock Proteins (HSPs) and heat resistance in cells grown in an acid environment. The long term objective is to develop approaches for the identification and the selective killing of tumor cells adapted to growth in low pH environments. The two hypotheses are: 1) Growth of human melanoma cells in an acidic environment induces elevated levels of HSPs, including HSP27 and HSP72, which, in turn, contribute to protection of the cytoplasm and nucleus from heat-induce protein aggregation and disruption, allowing for enhanced synthesis of HSPs and enhanced thermotolerance (TT). Cytoplasmic protection includes protection of the cytoskeleton (CSK) and proton pumps responsible for maintenance of intracellular pH (pHi). 2) The protection of the CSK, pHi homeostasis machinery and the nucleus by HSPs can be overcome by an acute reduction of pHi prior to an during hyperthermia. Specific Aims designed to test the model will examine: i) the interrelationships between levels of HSPs, the phosphorylation of HSP27, the resistance of the CSK to heat-induced collapse & the accumulation of heat-induced nuclear-associated proteins (NAPs), heat-induced synthesis of HSPs and survival in adapted cells; ii) the importance of an intact CSK for realkalinization during hyperthermia following an acid load; and iii) the sensitization of adapted cells by an acute extracellular acidification alone, and by an acute acidification of 0.3 pHe unit combined with inhibitors (DIDS, CNCn, HOE642 and mIBG) that further reduce pHi. HSP levels and phosphorylation of HSP27 will be monitored by Western blot analysis, CSK organization by morphometric and biochemical analyses, NAPs by FCM, and survival by colony formation. Project 2 will be perform all pHi measurements. A translational objective will determine if some of the endpoints will be useful as indicators of the presence of adapted cells in fresh xenograft explants and sections of the xenografts provided by Project 4. This study will contribute to our understanding of the importance of HSP27 and HSP72 for resistance of the CSK and nucleus in adapted cells during hypothermia, and the importance of an intact CSK for efficient function of proton pumps during hyperthermia. Successful accomplishment of the goals may lead to new approaches for identifying and sensitizing acidotic regions of solid tumors to thermoradiotherapy using inhibitors that reduce pHi and/or destabilize the CSK.

该项目旨在测试是否存在因果关系 热激蛋白（HSP）的上调和细胞中的耐热性 在酸环境中生长。长期目标是发展 鉴定和选择性杀死肿瘤细胞的方法 适合低pH环境中的增长。这两个假设是：1） 酸性环境中人黑色素瘤细胞的生长诱导升高 HSP的水平，包括HSP27和HSP72，反过来又有助于 保护细胞质和核免受热诱导蛋白的保护 聚集和破坏，允许增强HSP的合成和 增强的热耐（TT）。细胞质保护包括保护 负责维护的细胞骨架（CSK）和质子泵 细胞内pH（PHI）。 2）保护CSK，PHI稳态 可以通过急性减少来克服HSP的机械和核。 在热疗期间之前的PHI。旨在测试的特定目标 模型将检查：i）HSP级别之间的相互关系， Hsp27的磷酸化，CSK对热诱导的抗性 塌陷和热诱导的核相关蛋白的积累 （午睡），热诱导的HSP合成和适应细胞的存活； ii） 完整的CSK在高温期间的重素化的重要性 酸负荷； iii）通过 单独急性细胞外酸化，并通过急性酸化 0.3 PHE单元与抑制剂（DIDS，CNCN，HOE642和MIBG）结合使用 进一步减少PHI。 HSP水平和HSP27的磷酸化将为 通过Western印迹分析监控，CSK组织通过形态计量学和 生化分析，FCM的小睡和菌落形成生存。 项目2将进行所有PHI测量。翻译目标 将确定某些端点是否作为指标有用 在新鲜异种移植外植体中存在适应细胞的存在和 项目4提供的异种移植物。这项研究将有助于我们 了解HSP27和HSP72对抵抗的重要性 体温过低期间适应细胞中的CSK和核 完整的CSK，用于高温期间质子泵的有效功能。 成功实现目标可能会导致新的方法 识别和敏感实体瘤的酸性区域 使用抑制剂减少PHI和/或破坏稳定的抑制剂的热放射治疗 CSK。