Biochemical Effects of Disrupting HSP90

说明(申请人提供)：热休克蛋白90(HSP90)是一种 在转录因子功能中起关键作用的伴侣蛋白， 丝氨酸/苏氨酸和酪氨酸激酶参与肿瘤细胞信号转导。 格尔达那霉素(GA)是一种阿萨霉素联苯喹酮，它与HSP90结合并破坏其 功能。HSP90正以GA的类似物1作为临床靶点 7-羟基氨基吉多那霉素(17AAG)。申请者实验室的努力是 为了解赤霉素A和17AAG的抗性机制， HSP90辅助伴侣和HSP90客户角色的生化表征 蛋白质在GA细胞杀伤中的作用，以及识别可以 与GA或17AAG联合使用可产生相加或协同毒性 在不增加对正常组织毒性的情况下抑制肿瘤细胞的生长。初步结果 显示肿瘤细胞对GA敏感性的100倍变化 与抑制结合相关的集落形成试验 辅助伴侣p23至HSP90，降低细胞内GA浓度，以及 增加了辅助伴侣HDJ2的水平。进一步的研究表明， 当GA与顺铂联合或联合应用时，存在协同细胞毒性 以序列依赖的方式与吉西他滨(GEM)结合。基于这些 初步研究结果：1)探讨肿瘤细胞的作用机制 对赤霉病的抗性。HSP90复合体的形成、辅助伴侣和客户蛋白 将测量水平，以确定综合体中哪些元素是关键的 用于GA抗性。此外，细胞色素P450同工酶NQO1的作用 赤霉病抗性的表达将被确定。2)确定运行机制 GA与顺铂的协同作用。顺铂对热休克蛋白90功能和细胞周期的影响 GA对铂-DNA加合物的形成和去除的影响将是 下定决心。进一步研究赤霉素A和顺铂对血管紧张素转换酶的影响 控制细胞增殖的客户蛋白将被执行。3)确定 GA与GEM的协同作用机理。创业板对热休克蛋白90的影响 将评估GA的功能和对GEM代谢的影响。其他内容 GA和GEM对客户蛋白调控作用的实验研究 将进行细胞增殖。总而言之，这些研究的结果将 更好地了解GA单独和在 结合化疗药物，找出影响因素 对这类代理人的敏感性，并将提供合理的基础 将这些药物组合在一起的进一步临床试验 化疗。

DESCRIPTION (provided by applicant): Heat shock protein 90 (HSP90) is a chaperone protein critical in the function of transcription factors, serine/threonine, and tyrosine kinases involved in tumor cell signaling. Geldanamycin (GA), an ansamycin benziquinone, binds HSP90 and disrupts its function. HSP90 is being targeted clinically with the analog of GA, 1 7-aHyl-aminogedanamycin (17AAG). Efforts from the applicant's laboratory are directed towards understanding the mechanisms of resistance to GA and 17AAG, biochemically characterizing the role HSP90 co-chaperone and HSP9O client proteins in GA cell killing, and identifying chemotherapeutic agents that can be combined with GA or 17AAG to yield additive or synergistic toxicity in tumor cells without increasing toxicity to normal tissue. Preliminary results demonstrate a 100-fold variation in tumor cell sensitivity to GA in a colony-forming assay that is correlated with inhibition of binding of the co-chaperone p23 to HSP90, decreased intracellular concentrations of GA, and increased levels of the co-chaperone HDJ2. Further studies have shown that there is a synergistic cytotoxicity when GA has been combined with cisplatin or with gemcitabine (gem) in a sequence dependent manner. Based on these preliminary results we propose to: 1) Investigate the mechanism of tumor cell resistance to GA. HSP90 complex formation, co-chaperone and client protein levels will be measured to identify which elements of the complex are critical for GA resistance. In addition, the role of cytochrome P450 isozymes NQO1 expression in GA resistance will be determined. 2) Determine the mechanism of synergy between GA and cisplatin. The effect of cisplatin on HSP90 function and the impact of GA on platinum-DNA adducts formation and removal will be determined. Further studies to establish the effect of GA and cisplatin on client proteins that control cell proliferation will be performed. 3) Determine the mechanism of synergy between GA and gem. The effect of gem on HSP90 function and the impact of GA on gem metabolism will be assessed. Additional experiments to explore the effect of GA and gem on client proteins that control cell proliferation will be performed. Together, results from these studies will provide a better understanding of GA mechanism of action alone and in combination with chemotherapeutic agents, identify the factors that affect sensitivity to this class of agents and will provide a rational basis for further clinical trials incorporating these agents into combination chemotherapy.