中文摘要
細胞在正常生理下，如果受到熱、重金屬、病毒、藥物等外來因子的刺激後 ，細胞會因因應這些刺激而產生熱休克蛋白(Heat Shock proteins)。而一般熱休克蛋白可因分子量分成四群: 1.熱休克蛋白110(HSP110)，2.熱休克蛋白90(HSP90) ，3.熱休克蛋白70(HSP70) ，4.熱休克蛋白70KD以下的熱休克蛋白。熱休克蛋白含有保護子(Chaperone)的功能，可以幫助新合成或受破壞之蛋白質的折疊，使其具有應有正確的結構和功能，當細胞處於逆境環境下，將會誘發細胞中熱休克蛋白表現增加。之前的研究發現，膠達納黴素 (geldanamycin, GA)處理下會造成細胞之中蛋白質的折疊不正常，引發細胞之逆境反應而誘導熱休克蛋白的合成增加，特別是熱休克蛋白70s(HSP70s)。

在此論文中我們以H460人類非小型肺癌細胞為材料，探討GA誘發H460人類非小型肺癌細胞中HSP70表現所參與的訊息傳遞路徑。在我們的研究中發現，經過GA處理的H460人類非小型肺癌細胞，其細胞內HSP70蛋白質的表現會隨著GA的濃度以及處理時間的改變而改變，並且利用北方點墨(Northern Blot)實驗發現，GA是影響HSP70的轉錄過程。由於人類熱休克蛋白70含有許多isoforms存在，利用2-D及北方點墨(Northern Blot)的分析讓我們知道GA其所誘發熱休克蛋白70s主要為HSP70A(HSP70-1)熱休克蛋白，另外我們是第一個發現GA所誘導的HSP70D可能會透過PKC等Pathway去誘發。在kinas inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7)完全抑制HSP70A蛋白質的表現，而HA1043則無明顯的抑制作用，同時，H-7，H-8及HA1043對GA所誘發mRNA的增加也有相同的抑制現象。因此，可以知道在H460人類非小型肺癌細胞，GA所誘發HSP70A表現增加的訊息傳遞路徑中，細胞內的激鋂(Kinase)扮演重要的角色。在另一方面，利用electrophoretic mobility shift assay (EMSA)實驗，可以清楚的了解細胞核中轉錄蛋白和promoter的結合情形。因此經由EMSA的實驗，我們得到在GA處理狀況下轉錄蛋白會和熱休克轉錄序列(Heat shock element)有結合增加的現象。同時轉錄蛋白和HSE結合增加的現象也會被H-7，H-8所抑制，因此，我們更清楚的了解，HSE轉錄序列在GA所誘發HSP70表現增加的訊息傳遞過程裡扮演重要的角色。

ABSTRACT
Geldanamycin (GA) specifically binds to HSP90 and disrupts the interaction of HSP90 and its target proteins. The binding will lead to substrate protein dissociation from HSP90 and may affect their structures and functions. Herein, we showed that exposure of non-small lung cancer H460 cells to 0.5 mM GA leads to enhancement of the synthesis of the 70 kDa heat shock proteins (HSP70s). The induction of HSP70s by GA is concentration- and time-dependent and this process coincides to the accumulation of its mRNA. By using the specific probes for hsp70-1 and hsp70b, we found that HSP70-1 is abundantly involved in GA-induced HSP70s. Furthermore importantly, we demonstrated for the first time that the PKC pathway is significant in this process since it is inhibited by H-7 and H-8 but not affected by HA1043. Similar results can be found in the protein and mRNA level. These data lead us to conclude that PKC pathway plays a major role in the GA-induced HSP70-1 expression in H460 cells. By using the electrophoretic mobility shift assay (EMSA), we showed that proximal Heat shock element (HSE1) was responsible for the inducible expression of HSP70. We found that the nuclear extracts prepared from GA-treated cells exhibited a significant increase in binding activity toward Heat shock element. Moreover, this increase in binding activity toward the Heat shock element is reduced by H-7 and H-8 but not affected by HA1004. We concluded that the PKC pathway is the major pathway involved in GA-induced expression of HSP70-1 in H460 cells.

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