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研究生:賴享玫
研究生(外文):Lai, Xiang ME
論文名稱:組蛋白去乙醯化酶媒介細胞週期G2/M停滯:去乙醯化酶與MAPK-ERK訊息路徑交互作用關係
論文名稱(外文):The Mechanisms of HDAC Inhibitor-mediated G2/M Arrest:The Crosstalk between HDAC and MAPK-ERK pathway
指導教授:孫光煥孫光煥引用關係查岱龍查岱龍引用關係郭耀文郭耀文引用關係
指導教授(外文):Sun, Guang HuanCha, Tai LungGuo, Yaw Wen
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:64
中文關鍵詞:組蛋白去乙醯化酶細胞週期
外文關鍵詞:Histone deacetylasecell cycle
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  組蛋白乙醯化酶(HDAC)負責組蛋白和非組蛋白去乙醯化、調節基因轉錄、穩定蛋白質功能,在許多癌症類型中觀察到HDACs異常表達和活性增加,因此,組蛋白去乙醯化酶抑制劑(HDACi)已被引入臨床研究和臨床試驗,來探討其抗癌活性,來自天然來源或合成化合物的HDACi皆在開發中,LBH589屬於hydroxmate類HDACi並擁有強大的抗癌活性,現在進入II/III期臨床試驗,其中包括血癌和腫瘤。
  在先前的研究中指出,HDACi會導致癌細胞生長抑制、分化和凋亡,其中已知HDACi活化了p21基因促進G1期停滯,在近年來,也少數發現HDACi會誘導一些癌細胞停滯在G2/M期。然而,細胞停滯在G2/M期的分子機制仍不清楚。在本實驗室先前的研究中,我們發現LBH589可誘導多種腫瘤細胞(腎細胞癌和前列腺癌細胞)停滯在G2/M期細胞週期,我們指出HDACi導致G2/M期停滯是透過抑制HDAC3和HDAC6造成Aurora A和Aurora B蛋白質降解,但是,在某些細胞如LNCaP、293T經由HDACi處理可以看到誘發早期M期停滯,但卻不是藉由降解Aurora kinase,我們猜測可能有兩種截然不同的途徑─aurora kinase dependent and independent pathways─負責HDACi誘發腫瘤細胞停滯在G2/M期。在此研究結果指出,HDACi處理LNCaP、293T誘發早期M期停滯並激活ERK活性,另外,ERK下游的磷酸酶CDC25C增加其磷酸化的信號,在同一時間我們也觀察到其受質CDC2抑制磷酸根依然存在,經過LBH589處理後癌細胞株停滯在M期早期是由於ERK1/2一直被活化且CDC2不活化。根據以往的研究,我們知道HDACs能與蛋白磷酸酶(protein phosphatases)相互作用以反映不同的生物性刺激。有趣的是,protein phosphatases涉及ERK1/2 cascade pathway或參與調節CDC25C,因此我們認為,在細胞週期中,一連串事件發生的時間先後取決於HDACs /protein phosphatases控制。
Histone deacetylases (HDAC) are responsible for deacetylating histones and non-histone proteins, regulating gene transcription, protein function and stability. Aberrant expression and increased activity of HDACs are observed in many cancer types. Therefore histone deacetylase inhibitors (HDACi) have been introduced into preclinical studies and clinical trials to investigate their anticancer activity. There are varieties of HDACi derived from natural sources or synthetic compounds under development. LBH589 belonged to hydroxmate group HDACi posesses potent anti-cancer activity and now enters into phase II/III clinical trials with hematologic and solid cancers.
In previous studies, HDACi treatment resulted in growth arrest, differentiation, and apoptosis of cancer cells. Some studies demonstrate that HDACi treatment resulted in elevation of p21 contributing to G1 cell-cycle arrest. Although less common, HDACi also induced G2/M cell-cycle arrest of cancer cells. However, the molecular mechanism involved in G2/M arrest remains unclear. In our previous study, we found that LBH589 can induce G2/M cell-cycle arrest in various cancer cell lines including renal cancer cells and prostate cancer cells. We also demonstrated that HDACi treatment resulted in G2/M arrest through degradation of aurora kinases A and B by inhibition of HDAC3 and HDAC6. However, HDACi-mediated early M phase arrest can be observed in some cell lines such as LNCaP, 293T without downregulation of aurora kinase levels. We hypothesize that there may be two distinct aurora kinase dependent and independent pathways responsible for HDACi-mediated G2/M cell-cycle arrest in cancer cells, respectively. Our results showed that HDACi treatment resulted in activating ERK activity in aurora-independent but not in aurora-dependent cancer cells with early M phase arrest. Although ERK downstream phosphatase, CDC25C, is increased its phosphorylated signal, we observed that CDC2 remain inhibitor signal at the same time. After LBH589 treatment, ERK1/2 Activity sustainability and CDC2 inactivation result in arresting at prophase. According to previous studies, we knew that HDACs interact with protein phosphatases to reflect different biological stimulation. Interestingly, protein phosphates involve ERK1/2 cascade pathway or regulate CDC25C. We consider that a series of cell cycle temporal consequences occurring depend on HDACs/protein phosphates complexes control.
謝誌............................................................................................................I
目錄……………………………………………………………………...II
圖目錄…………………………………………………………………...V
ABSTRACT……………………………………………………………VII
摘要.......................................................................................................VIII
第一章  緒言………………………………………………………....…1
 第一節 組蛋白去乙醯化酶抑制劑……………………………....…1
  壹 組蛋白去乙醯化抑制劑對於癌症治療研究………............…1
  貳 組蛋白去乙醯化酶抑制劑分類及功能…………………....…2
 參 組蛋白去乙醯化酶分類及其功能………………………....…3
 第二節 組蛋白去乙醯化酶與細胞週期停滯…………………....…4
第三節 組蛋白去乙醯化酶LBH589………………….....……....…5
第四節 G2/M Transition…………………………………………......6
壹 細胞週期G2/M……………………………………....……......6
貳 MAPK-ERK Pathway…………………..…………....……......6
第五節 研究動機………………………………………………....…7
第二章  材料與方法……………………………………………………9
 第一節 細胞培養……………………………………………………9
 第二節 CIP Assay…………………………………………………..14
 第三節 免疫螢光染色......................................................................15
 第四節 Diamidino-2-phenylindole (DAPI) 細胞核染色………….16
 第五節 Propidium iodide (PI) 染色測定細胞週期…………...16
 第六節 藥物毒殺存活率........................…………………………..17
 第七節 ROS Detection Assay……………………………………...18
 第八節 免疫分析(Immunoassay)……….……………………...19
 第九節 轉染作用(Transfection)………………………………..24
第三章  結果.........……………………………………………...……..25
 第一節 組蛋白去乙醯化酶抑制劑(LBH589)造成細胞株停滯
在細胞分裂期..…………………………………………....25
  壹 LBH589造成細胞生長抑制.………………………………...25
  貳 LBH589使細胞停滯在分裂期…….………………………...25
第二節 組蛋白去乙醯化酶抑制劑(LBH589)誘發ERK 1/2激
酶活化.. ……………………………………………....…...26
壹 ERK 1/2活化……………………………………………....…26
貳 抑制ERK 1/2活化能減少細胞停滯在分裂期…………...…26
第三節 ERK 1/2激酶活化影響磷酸水解酶CDC25C磷酸態改變..27
壹 ERK 1/2活化與CDC25C表現情形之關係…………....…....27
貳 CDC2抑制訊號維持不變………………………………....…28
第四節 ERK 1/2激酶非以傳統RAS-RAF-MAPK路徑活化....…28
第四章  討論…………………………………………………………..30
 第一節 293T及LNCaP對於LBH589反應差異…………….…..30
 第二節 細胞週期停滯在分裂期早期之因素……………………..30
 第三節 CDC25C磷酸化修飾………………………………....…...31
 第四節 水平式調控MEK 1/2-ERK 1/2訊息傳遞…………....…...32
第五章  結論…………………………………………………………..33
第六章  參考文獻……………………………………………………..34
附圖 pCMV5- Y185F-ERK2 plasmid……………….……………..38
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