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系統識別號 U0026-0812200914341823
論文名稱(中文) 含雙色胺酸功能區氧化還原酶在Akt/mTOR訊息傳遞中之調控性角色
論文名稱(英文) Regulatory role of WW domain-containing oxidoreductase in Akt/mTOR signaling
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 96
學期 2
出版年 97
研究生(中文) 黃資婷
研究生(英文) Tzu-Ting Huang
學號 s4695408
學位類別 碩士
語文別 中文
論文頁數 71頁
口試委員 指導教授-林以行
指導教授-徐麗君
口試委員-張南山
口試委員-詹明修
中文關鍵字 含雙色胺酸功能區氧化還原酶 
英文關鍵字 AKT  mTOR  WW domain-containing oxidoreductase 
學科別分類
中文摘要 WWOX基因可以表現出含雙色胺酸功能區氧化還原酶 (WW domain-containing oxidoreductase;又稱為WWOX、FOR或WOX1) 這個腫瘤抑制性蛋白質 (tumor suppressor)。WOX1的高度表現可以引起多種癌細胞進行細胞凋亡 (apoptosis)。本實驗室先前在鱗狀上皮細胞癌 (squamous cell carcinoma; SCC) 的研究中證明,SCC-15細胞以抗癌藥物methotrexate (MTX) 處理之後,細胞中WOX1蛋白質的表現量會增加,而且使得細胞更容易進行凋亡。有趣的是,在MTX刺激後的SCC-15細胞中也觀察到細胞自噬現象 (autophagy) 的減少。細胞自噬是一種細胞內的降解 (degradation) 系統,可以藉此產生小分子來供給細胞在營養缺乏 (starvation) 時使用,這個機制被認為有助於細胞存活。Mammalian target of rapamycin (mTOR) 進行磷酸化以後可以抑制細胞自噬的現象,而在MTX刺激後的SCC-15細胞中,mTOR的磷酸化情形增加,根據這個線索,我們推測WOX1可能會調控mTOR的訊息傳遞。我們的實驗結果顯示,在高度表現WOX1的SCC-15細胞中,mTOR下游的受質p70 S6 kinase (p70 S6K)及eIF4E binding protein 1 (4E-BP1) 的磷酸化程度增加,而WOX1導致p70 S6K蛋白質磷酸化程度增加的現象會受到mTOR抑制劑rapamycin的作用而減弱。為了更進一步探討WOX1的調控機制,我們也檢測上游的激酶Akt。高度表現WOX1的SCC-15細胞中也發現Akt在Ser473位置的磷酸化情形增加。當細胞處於營養缺乏的狀態、或是使用PI3 kinase抑制劑wortmannin處理後,WOX1所引起的Akt與p70 S6k磷酸化的程度都會降低。令人興奮的是,WOX1會減少protein phosphatase 2A catalytic subunit (PP2Ac) 蛋白質表現量及其甲基化 (methylation)的情形。我們也用Glutathione S-transferase pull-down及免疫沈澱 (coimmunoprecipitation) 實驗證實了WOX1和PP2Ac在SCC-15細胞中有結合的作用。綜合以上結果,我們認為在SCC-15細胞中,WOX1可能是藉由對PP2Ac蛋白質表現與甲基化的負調控,來活化Akt/mTOR的訊息傳遞路徑。
英文摘要 The WWOX gene encodes a candidate tumor suppressor WW domain–containing oxidoreductase (designated as WWOX, FOR or WOX1). Overexpression of WOX1 induces apoptosis in several types of cancer cells. Previous study in our laboratory has demonstrated that methotrexate (MTX), an anticancer drug, increases WOX1 expression in squamous cell carcinoma (SCC)-15 cells and sensitizes these cells to apoptosis. Interestingly, MTX treatment inhibits autophagy, a bulk degradation process for survival during starvation, in SCC-15 cells. Phosphorylation of autophagy-inhibitory mammalian target of rapamycin (mTOR) is upregulated in MTX-treated SCC-15 cells. In light of these findings, we hypothesize that WOX1 regulates mTOR signalling. Our results showed that ectopic overexpression of WOX1 increased phosphorylation of mTOR downstream substrates p70 S6 Kinase (p70 S6K) and eIF4E binding protein 1 (4E-BP1) in SCC-15 cells. WOX1-induced phosphorylation of p70 S6K was downregulated by the treatment of rapamycin, an mTOR inhibitor. To further explore the regulatory mechanism of WOX1, the upstream kinase Akt was examined. Our data showed that WOX1 induced phosphorylation of Akt at Ser473 in SCC-15 cells. WOX1-induced phosphorylation of Akt and p70 S6K was suppressed by starvation or a PI3 kinase inhibitor wortmannin. Most strikingly, we determined that WOX1 downregulated the protein expression and methylation of protein phosphatase 2A catalytic subunit (PP2Ac). Glutathione S-transferase pull-down assay and coimmunoprecipitation demonstrated the interaction of WOX1 with PP2Ac in SCC-15 cells. Together, our results suggest that WOX1 modulates Akt/mTOR signaling pathway by downregulating the protein expression and methylation of PP2Ac in SCC-15 cells.
論文目次 中文摘要 I
英文摘要 II
致謝 III
總目錄 IV
圖目錄 VI
緒論
含雙色胺酸功能區氧化還原脢 1
結構 1
磷酸化調控 2
相結合的蛋白質 2
細胞內分佈 3
細胞中的功能與角色 3
本實驗室針對WOX1在鱗狀上皮細胞癌的研究 6
AKT/mTOR訊息傳遞路徑 7
實驗目的 9
材料與方法
A. 材料 10
A-1 細胞株 10
A-2 試劑藥品 10
A-3 抗體 11
A-4 耗材 12
A-5 儀器 13
B. 方法 14
B-1 製備細胞培養液 14
B-2 細胞繼代培養 (附著型細胞) 15
B-3 細胞數目測定 15
B-4 大腸桿菌之轉型 (Transformation) 16
B-5 製備質體DNA 17
B-6 細胞轉型 (Transfection)—電穿孔法(Electroporation) 18
B-7 萃取細胞蛋白質 19
B-8 蛋白質濃度定量 20
B-9 聚丙烯醯胺膠體電泳 (SDS-PAGE) 20
B-10 西方墨點法 (Western Blot) 22
B-11 GST (glutathione S-transferase) 融合蛋白質萃取 25
B-12 GST pull-down assay 26
B-13 共同免疫沈澱 (Co-immunoprecipitation) 28
B-14 細胞免疫螢光染色 (Immunofluorescence staining) 28
實驗結果
1. WOX1使p70 S6K磷酸化程度增加 30
2. WOX1增加p70 S6K蛋白質磷酸化程度的現象會受到mTOR
抑制劑rapamycin的影響而減低 30
3. WOX1促使p70 S6K及Akt磷酸化增加的現象在營養缺乏的狀態
、以及PI3 kinase抑制劑wortmannin處理之下會被抑制 31
4. 在WOX1高度表現的細胞當中protein phosphatase 2A
catalytic subunit (PP2Ac) 的蛋白質表現程度有降低的現
象 32
5. WOX1與PP2A之交互作用 34
討論
WOX1對於Akt/mTOR訊息傳遞路徑的影響 36
細胞凋亡與mTOR/p70 S6K的活化 39
WOX1和PP2A的關係 40
參考文獻 42
圖附錄 57
自述 71
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