Abstract
Experiments in this thesis were designed to investigate the upstream signaling of GA-induced stress response and elucidate the inductive mechanisms of Hsp90 upon GA treatment. Geldanamycin (GA), an ansamycin-derivative benzoquinone compound, was originally isolated as a natural product with anti-fungal activity. GA specifically inhibits the essential ATPase activity of Hsp90 results in inactivation, destabilization, and degradation of Hsp90 client proteins, including a wide variety of signal-transducing proteins that regulate cell growth and differentiation, such as protein kinases and steroid hormone receptors. Recently, several reports have shown that treatment with GA induces a heat-shock response and intracellular calcium may involve in the process. In this study, we found changes in upstream signaling mediators, including HSF1 and calcium, as well as possible involvement of protein kinase in human non-small cell lung cancer H460 cells treated with Hsp90 binding agents (i.e., GA and radicicol). We further found that GA was able to provoke a rapid calcium influx and thereby resulted in an instant induction of Hsp70. Our results demonstrated that calcium mobilization, a calcium dependent and H7-sensitive protein kinase, along with HSF1 activation by phosphorylation, were all involved in the Hsp70 induction process triggered by the drug. On the other hand, treatment of cells with GA also induced the expression level of Hsp90 other than Hsp70. In mammals, two major cytoplasmic isoforms of Hsp90, known as Hsp90alpha and Hsp90beta, have been identified and found to be highly conserved among different species. In order to examine the expression of Hsp90 isoforms, the levels of mRNA and nascent protein of Hsp90alpha and Hsp90beta in GA-treated rat brain tumor 9L cells were quantitatively examined using real-time qPCR and metabolic labeling analyses. It is found that Hsp90alpha was more inducible compared to Hsp90beta after GA treatment. Interestingly, the hsp90alpha mRNA was, on the contrary, lower than that of hsp90beta. The relative mRNA level of hsp90beta was respectively about 8.8-fold and 3.7-fold that of hsp90alpha under normal condition and upon GA treatment. Furthermore, we examine whether distinct expression of Hsp90alpha and□Hsp90beta is a result of post-transcriptional regulation. The translational efficiency of Hsp90 isoforms employing in vitro translation was further measured. We found that hsp90alpha mRNA revealed highly effective translation. The translational efficiency of hsp90alpha mRNA was respectively about 8.9-fold and 5.8-fold that of hsp90beta under normal condition and upon GA treatment. Our results indicate that differential expression of Hsp90alpha and Hsp90beta is a consequence involving both the distinct mRNA profiles and the differential translation processes. In conclusion, treatment with GA was able to activate HSF1 and bring about the induction of Hsp70. Intracellular calcium mobilization and related protein kinase activities are involved in signaling for GA. The primary effect of GA on expression of Hsp90 isoforms is according to promote the transcription of both hsp90 genes. The differential expression of□Hsp90alpha and□Hsp90beta is a consequence of both distinct mRNA profiles and differential translation processes.