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: 6300441
• 负责人: RONALD Allen COSS
• 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/6300441
• 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.

该项目旨在测试之间是否存在因果关系 上调热休克蛋白 (HSP) 和细胞耐热性 在酸性环境中生长。长期目标是发展 识别和选择性杀死肿瘤细胞的方法 适应低pH环境生长。这两个假设是：1） 人类黑色素瘤细胞在酸性环境中生长会导致 HSP 水平，包括 HSP27 和 HSP72，这反过来又有助于 保护细胞质和细胞核免受热诱导蛋白的影响 聚集和破坏，从而增强 HSP 的合成和 增强耐热性（TT）。细胞质保护包括保护 细胞骨架（CSK）和质子泵负责维持 细胞内 pH（pHi）。 2) 保护CSK、pHi稳态 热休克蛋白的机械和细胞核可以通过急剧减少来克服 热疗之前和期间的 pHi。旨在测试的具体目标 模型将检查： i) HSP 水平之间的相互关系， HSP27 的磷酸化，CSK 对热诱导的抵抗力 热诱导核相关蛋白的塌陷和积累 (NAP)、热诱导的 HSP 合成和适应细胞的存活；二） 完整的 CSK 对于热疗期间重新碱化的重要性 酸负荷后； iii) 适应细胞的致敏作用 单独急性细胞外酸化，以及通过急性细胞外酸化 0.3 pHe 单位与抑制剂（DIDS、CNn、HOE642 和 mIBG）组合， 进一步降低pHi。 HSP 水平和 HSP27 磷酸化将 通过蛋白质印迹分析监测，通过形态计量学和 CSK 组织 生化分析、FCM 的 NAP 以及集落形成的存活率。 项目 2 将执行所有 pHi 测量。转化目标 将确定某些端点是否可用作指标 新鲜异种移植外植体和切片中存在适应细胞 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 对于耐药性的重要性 低温期间适应细胞中的 CSK 和细胞核，以及其重要性 完整的 CSK，可确保质子泵在热疗过程中高效运行。 目标的成功实现可能会带来新的方法 识别实体瘤的酸中毒区域并使其敏感 使用降低 pHi 和/或破坏稳定的抑制剂的热放射治疗 CSK。