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研究生:李莉甄
論文名稱:膠達那黴素和瑞迪士可黴素於人類非小型肺癌細胞中透過過磷酸化熱休克調控因子1而誘發熱休克蛋白70表現之研究
論文名稱(外文):Comparative Studies of Geldanamycin- and Radicicol-Induced Heat Shock Protein 70 Expression Through Hyperphosphorylating Heat Shock Factor 1 in Human Non-Small Lung Cancer H460 Cells
指導教授:黎耀基黎耀基引用關係傅化文傅化文引用關係
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:47
中文關鍵詞:膠達那黴素瑞迪士可黴素熱休克調控因子1熱休克蛋白70蛋白質激酶C鈣離子
外文關鍵詞:geldanamycinradicicolHSF1HSP70PKCCa2+
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在膠達那黴素(geldanamycin)與瑞迪士可黴素(radicicol)作用下,生物細胞內許多依賴熱休克蛋白90(heat shock protein 90)維持構型的調節蛋白與訊號傳遞蛋白的活性會明顯降低,對此種負調節的機制研究很多;然而,對此兩種黴素如何透過活化熱休克調控因子1(heat shock factor1)與熱休克蛋白70(heat shock protein 70)基因上游的啟動子(promoter)做結合,導致熱休克蛋白70之表現的正調節的機制,卻瞭解不多。本文探討這兩種藥物,在短時間處理人類非小型肺癌細胞H460下,對於熱休克蛋白70誘使上的差異。我發現雖然膠達那黴素與瑞迪士可黴素在人類非小型肺癌細胞H460內都能誘使熱休克蛋白70的表現,膠達那黴素能夠刺激細胞在僅5分鐘,並在沒有培養基的情況下恢復六小時,就達到熱休克蛋白70的最大誘使量;瑞迪士可黴素則要刺激細胞直到二小時並在沒有培養基的情況下恢復六小時,才達到熱休克蛋白70的最大誘使量。藉由反轉錄-聚合酶連鎖反應(RT-PCR)與免疫墨點(immunoblotting)分析法證明,與熱休克調控因子1的核醣核酸累積量(mRNA)和過磷酸化(hyperphosphorylation)增加的時間點有關。在細胞前處理H-7做為蛋白質激酶C(PKC)抑制劑時,這兩種藥物所引發的熱休克調控因子過磷酸化增加的情形,都會被明顯的抑制下來,暗示蛋白質激酶C是這兩種藥物的共同中介物。此外,我們也發現膠達那黴素會在處理細胞的瞬間增加細胞內鈣離子的濃度,但是瑞迪士可黴素卻沒有這種鈣離子瞬間效應。為了瞭解鈣離子在這兩種藥物所誘使熱休克蛋白70表現的重要性,細胞被培養在無鈣培養基,造成這兩種藥物所引發的熱休克蛋白70表現,都會被明顯的抑制下來;暗示鈣離子是這兩種藥物的必須中介物。我推論若鈣離子瞬間效應,造成膠達那黴素能夠刺激細胞在僅5分鐘引發的熱休克蛋白70最大表現,強迫外加的鈣離子,應可促使原本須長時間的瑞迪士可黴素,在短時間引發的熱休克蛋白70大量表現。當細胞被培養在CaCl2培養基,造成瑞迪士可黴素穫得如膠達那黴素的瞬間誘發力,證實我的推論。綜合上述實驗結果,顯示膠達那黴素有瞬間誘發力,透過鈣離子、蛋白質激酶C、熱休克調控因子,以正調節熱休克蛋白70表現。從瑞迪士可黴素可穫得如膠達那黴素瞬間誘發力,證明共同訊號傳遞中介物之存在;其誘發所須時間之不同,可能差別於鈣離子對細胞的起動步驟。
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.
中文要………………………………………………………………………1
Abstract……………………………………………………………………3
Introduction………………………………………………………………5
Materials and methods…………………………………………………10
Results……………………………………………………………………14
Discussion……………………………………………………………---19
Footnotes…………………………………………………………………23
Reference…………………………………………………………………24
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