| Summary: | 碩士 === 國立清華大學 === 生命科學系 === 90 === The cellular activity of several regulatory and signal transduction proteins, which depend on the heat shock protein 90 (HSP90) molecular chaperone for folding, is markedly decreased by geldanamycin (GA) and by radicicol (RA), and the mechanisms of these down-regulations are well-studied. However, the upstream mechanism for GA and RA to induce heat shock protein-70 (HSP70) expression through disrupting a chaperone complex of the inactive transcription factor, heat shock factor-1 (HSF1), subsequently activating and binding to the heat shock elements in the promoter region of hsp70 is largely unknown. This study is to investigate the differential effects of GA and RA on HSP70 synthesis for short-term treated with human non-small lung cancer H460 cells (H460 cells). Although both GA and RA induced the expression of HSP70 in H460 cells, only GA caused the induction of HSP70 synthesis within 5 min while RA takes for 2 h. RT-PCR and immunoblotting analysis showed that the GA-induced prompt HSP70 expression associated with the mRNA accumulation and protein hyperphosphorylation of HSF1. The potent protein kinase C (PKC) inhibitor H-7 dramatically suppresses HSP70 expression and HSF1 hyperphosphorylation in GA and RA-treated cells, suggesting that PKC is at least one of the common mediator for both drugs. Furthermore, GA produces a prompt intracellular calcium ([Ca2+]i) spike while RA did not show the obvious raise in [Ca2+]i. To test the importance of calcium in these pathways, cells were cultured with calcium free medium, and these results in suppression of HSP70 induced synthesis by GA and RA. This indicates that calcium is required in both pathways. I hypothesize that the prompt induction of calcium might be responsible for the fast induction of HSP70 in GA, if so, the ecotopic addition of calcium might force the otherwise slow-inductive RA to elicit a prompt HSP70 induction just as that GA does. As cells were treated with CaCl2, RA acquired the prompt induction ability like GA as I expected. Taken together, GA possesses the prompt induction ability to increase HSP70 expression through prompt induction of calcium and activation of PKC and HSF1. The fact that RA can mimic the prompt induction ability of GA after exogenous high calcium is provided, suggesting that common signaling pathways are shared by these HSP90 binding drugs, GA and RA, and that the differential calcium induction in the beginning might contribute to the differential HSP70 induction patterns between GA and RA.
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