臺灣博碩士論文加值系統

膠達納黴素(Geldanamycin, GA)是熱休克蛋90(heat shock protein 90, HSP90)及葡萄糖調控蛋白94(glucose regulated protein94, GRP94)的抑制物且可能成為有效的抗癌藥物。過去我們實驗室發現以未達致死藥量的GA處理9L大鼠腦瘤細胞(rat brain tumor, RBT)會引發內質網逆境反應，導致未摺疊蛋白質反應(unfolded protein response, UPR)而誘發葡萄糖調控蛋白的大量表現。這些葡萄糖調控蛋白是在基因轉錄的層次受到調控，在葡萄糖調控蛋白的啟動子序列上存在有數個內質網逆境反應轉錄元件(ER stress response element, ERSE)，具有CCAATN9 CCACG序列高度保留性，其中N9是指含CG為主的9個鹼基，此轉錄元件在內質網逆境誘發葡萄糖調控蛋白的調控中扮演必要且是決定性的角色。
在此論文中我們以9L大鼠腦瘤細胞為材料，探討GA誘發9L大鼠腦瘤細胞中葡萄糖調控蛋白94表現所參與的訊息傳遞路徑。葡萄糖調控蛋白94是存在於內質網的伴護蛋白，我們發現GA引起的內質網逆境會在基因層次調控葡萄糖調控蛋白94而活化其大量表現。我們分析葡萄糖調控蛋白94的啟動子序列並針對轉錄元件設計不同長度片段的啟動子，轉殖進入報導基因載體中並進行分析，發現內質網逆境反應轉錄元件4及CRE-BP1/c-Jun 扮演重要的角色。此外，點突變及電泳遷移實驗也支持此一結果並指出其他的ERSE亦參與其中。在預處理抑制劑的實驗中，我們發現絲胺酸激酶抑制劑4-(2-氨乙基)苯磺酰氟( AEBSF)會抑制活化轉錄因子(activating transcription factor 6, ATF6)被酶切而抑制葡萄糖調控蛋白94被活化。總結而言，我們發現在GA所引起的內質網逆境中，葡萄糖調控蛋白94的活化主要是透過內質網逆境反應轉錄元件及活化轉錄因子6的參與。

Geldanamycin (GA), a benzoquinone ansamycin, is an inhibitor of heat shock protein 90 (HSP90)/ glucose-regulated protein 94 (GRP94) and has been implicated as a potent anti-cancer drug. In our previous study, we found that GA with sublethal dose provoked the ER stress in 9L rat brain tumor (RBT) cells and induced glucose-regulated proteins under unfold protein response (UPR) at transcriptional level. The promoter of grp genes contain multiple copies of the ER stress response element (ERSE), with a consensus of CCAAT(N9)CCACG, which is critical and necessary for transcription induction. Herein, we showed that GRP94, an ER resident chaperone, was induced under GA treatment in 9L rat brain tumor (RBT) cells and the mRNA level of grp94 was peaked at 8 h for about 9 fold. We analyzed the promoter sequence of grp94 according to rat genome resource and designed reporter vectors containing progressive-deleted promoters of grp94. In reporter gene assays, ERSE4 and CRE-BP1/c-Jun played the major roles in GA-induced grp94 expression. Moreover, with mutagenesis clones we further confirmed this result although other ERSEs still have involved in. By inhibitors screening, pretreatment of KT5720, BIM I, or Gö6983 partially decreased GA-induced GRP94 expression, suggesting the involvement of PKA and PKC. Pretreatment of AEBSF blocked proteolysis of ATF6 abolished GA-induced GRP94 expression. Taken together, we found that under GA induced ER stress in 9L cells, activation of GRP94 were mainly through ERSEs by the transcription factor ATF6.

中文摘要 …………………………………………………………… 1
ABSTRACT …………………………………………………………… 2
ABBREVIATIONS …………………………………………………… 3
INTRODUCTION ……………………………………………………… 5
EXPERIMENTAL PROCEDURES ……………………………………… 9
RESULTS …………………………………………………………… 14
DISCUSSION ……………………………………………………… 19
REFERENCES ……………………………………………………… 23
FIGURE LEGENDS ………………………………………………… 29
FIGURES …………………………………………………………… 32
TABLES …………………………………………………………… 40

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