Abstract
This thesis started by using geldanamycin (GA) to treat rat brain tumor (RBT) 9L cells. By observing cell stress response, we initiated the study of the differentially inductive mechanisms on heat shock protein 90 isoforms. Geldanamycin (GA) is an ansamycin-derivative benzoquinone compound, which was originally isolated as a natural product with anti-fungal activity. GA could inhibit the essential ATPase activity of HSP90 and 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. But reports had shown that GA treatment could also induce heat-shock response; especially including HSP70, GRP78 and GRP94 in RBT 9L cells. In this study, we found only a 1/10 dose of GA (i.e., 0.5 贡M) could induce HSP90, compared to 5 贡M GA to induce those heat shock proteins like HSP70, GRP78 and GRP94. Furthermore, HSP90 exists two highly consistent isoforms, HSP90α and HSP90β, in mammalian cells. We further separated HSP90 isoforms by decrease pH (to 8.0) and percentage (9%) of PAGE gel; the results showed apparently differential induction of HSP90 isoforms through GA treatment (HSP90α > HSP90β). On the other hand, the consistencies were confirmed from RNA to protein level by real time qPCR analysis, Western Blotting and de novo synthetic analysis. Our results demonstrated that gene level regulation controlled the differential induction of HSP90 isoforms. According to the promoter sequences of hsp90α and hsp90β, we evaluated the different importance of some transcriptional elements by Electrophoresis Mobility Shift Assay (EMSA). Interestingly, differential induction of HSP90α and β is related to the differential binding activities to HSEs in hsp90α and hsp90β promoters. In addition, we also observed the binding strengths on HSEs might imply how HSP90α is more inducible isoforms than HSP90β. On the other hand, the results of binding activities on basic transcription elements showed that GC-box (sp-1 site) involved in inductive level of HSP90α汹and HSP90β the binding on TATA-box and CRE were respectively related to the induction of HSP90α and HSP90β. In conclusion, treatment with GA facilitate HSF1 binding to the distinct HSE sites on the promoters of hsp90α and hsp90β and further induce HSP90s. It agreed with the observations of differential induction on mRNA and protein level in 9L cells under treatment with GA.