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.
