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研究生:鄭雅玲
研究生(外文):Ya-Ling Cheng
論文名稱:肝醣合成激酶-3β在三氧化二砷誘導角質細胞內p21WAF1/CIP1表現所扮演的角色
論文名稱(外文):Role of glycogen synthase kinase-3β in ATO-induced p21 WAF1/CIP1 expression in keratinocyte
指導教授:黃暉升
指導教授(外文):Huei-Sheng Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學檢驗生物技術學系碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:70
中文關鍵詞:肝醣合成激酶-3β
外文關鍵詞:glycogen synthase kinase-3βATOAs2O3p21WAF1/CIP1ERKAKTc-Jun
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  • 被引用被引用:0
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三氧化二砷(As2O3, ATO)是一種無臭無味的白色霜狀粉末,俗稱砒霜。砷化物存在於整個大自然界中,包括在飲用水、土壤、空氣中。由於它具有強烈毒性,因此長期曝露在含有砷化物的環境之下則有能會造成一些疾病,如:熟為人知的烏腳病、皮膚病,甚至是皮膚癌等等。儘管如此,ATO用在治療上已經有2000多年的歷史,尤其在急性前骨髓性白血病(APL)上有亮眼的療效。近年來更有文獻指出ATO也可用於治療牛皮癬(psoriasis)等皮膚疾病以及一些惡性腫瘤,像是大腸癌、乳癌等。根據研究報導ATO是藉由誘導p21蛋白質(細胞週期蛋白依賴性激酶抑制劑, cyclin dependent kinase inhibitor)進而導致細胞週期停擺(cell cycle arrest)進而凋亡(apoptosis)。在實驗室過去研究中發現利用ATO刺激人類上皮癌細胞A431 (human epidermal carcinoma)會藉由兩條互相拮抗的訊息傳遞路徑來調控p21的表達。一條是經由JNK (c-Jun N-terminal kinase磷酸化c-Jun 的N端Ser-63/73的位置然後吸引TGIF/HDAC1進而抑制p21表現。相反的,ATO會藉由持續活化Ras/Raf/ERK pathway增加c-Fos的表現進一步和c-Jun結合去吸引p300而促使p21的表現。由此可知,在整個ATO刺激過程中,c-Jun似乎扮演一個adaptor藉由吸引不同的因子來調控p21的表達。就我們所知,c-Jun是一個轉錄因子,透過C端上的DNA binding domain和下游基因結合進而調控目標基因(target gene)的表現。而c-Jun的C端有磷酸化位點可藉由肝醣合成激酶-3β (GSK-3β)加以磷酸化而導致c-Jun和target gene結合能力及其蛋白質的穩定度下降。另一方面,GSK-3β在一般狀態或是UV照射之下也會促使p21蛋白質降解。因此,我們的假設是ATO所誘導的p21表現會藉由抑制GSK-3β的活性來促使c-Jun的C端去磷酸化進而增加p21啟動子的活性,以及增加p21蛋白質的穩定性。首先,利用DNA affinity precipitation assay,我們發現c-Jun的C端去磷酸化確實會增加與p21啟動子之間的結合並增加p21的表現。而ATO及GSK-3β inhibitor, lithium chloride (LiCl),均會增加GSK-3β的N端Ser-9的磷酸化及誘導p21表現,呈現時間與劑量的關係。更進一步我們發現此GSK-3βSer9 的磷酸化是透過MEK/ERK這條路徑而不是PI3K/Akt 路徑。但是,藉由LY294002抑制Akt pathway卻會增強ATO誘導p21表現。除此,ATO和LiCl也會增加p21蛋白質的半衰期。總括來說,我們認為在角質細胞中,ATO可以藉由抑制GSK-3β的活性而使得c-Jun的C端呈現去磷酸化的狀態以增加p21啟動子的活性及其蛋白質的穩定以誘導p21的表現。
Arsenic trioxide (ATO) has been effectively used to treat a variety of ailments, such as acute promyelocytic leukemia, solid tumors, and some skin diseases, via induction of cell cycle arrest or apoptosis. Our previous studies show that ATO-induced JNK pathway can phosphorylate N-terminus (Ser-63/73) of c-Jun to recruit TGIF/HDAC1 to suppress p21WAF1/CIP1 (p21) expression, but the induced sustained Ras/Raf/ERK pathway can enhance c-Fos expression to dimerize with c-Jun to recruit p300 to increase p21 expression. That is, c-Jun might act as an adaptor in response to ATO to regulate p21 expression. It has been reported that glycogen synthase kinase-3β (GSK-3β) can phosphorylate the C-terminus (Ser-243) of c-Jun to decrease its protein stability and DNA binding ability. In addition, GSK-3β can also increase the degradation of p21 in resting condition or UV irradiation. Therefore, we hypothesize that ATO-induced p21 expression might be through both the inhibition of GSK-3β to dephosphorylate the C-terminus (Ser-243) of c-Jun to induce p21 promoter activity, and the increase of p21 protein stability. Using DNA affinity precipitation assay, ATO could dephosphorylate the C-terminus (Ser-243) of c-Jun to enhance its binding to p21 promoter, and result in p21 expression. Then, we found that the phosphoarylation of GSK-3βSer9 and p21 expression could be increased by ATO or LiCl (GSK-3β inhibitor) treatment in time- and dose-dependent manners, respectively. Furthermore, the ATO-induced phosphorylation of GSK-3βSer9 was through MEK/ERK pathway, but not PI3K/Akt pathway. However, blockage of the PI3K/Akt pathway could enhance ATO-induced p21 expression. Besides, the stability of p21 protein was prolonged by ATO or LiCl treatment. Taken together, we suggest that inhibition of GSK-3β by ATO can dephosphorylate the C-terminus (Ser-243) of c-Jun to enhance p21 promoter activity and its protein stability to increase p21 expression in keratinocyte.
Introduction 1
Hypothesis and Specific aims 13
Materials and Methods 14
Results 32
Conclusion 36
Discussion 37
References 41
Figures 51
Appendix 60
Author profile 70
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