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研究生:魏迪卿
研究生(外文):Wei,Dicing
論文名稱:探討TOPK/PBK所誘導之細胞移行機制及其在癌症治療上所扮演之潛在角色
論文名稱(外文):Mechanistic investigations of TOPK/PBK-induced cell migration and the potential role in cancer therapy.
指導教授:賴金美
指導教授(外文):Lai,JinMei
口試委員:周秀慧黃奇英
口試委員(外文):Chou,ShiuhueyHuang,Chi-Ying F
口試日期:2011-07-26
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:59
中文關鍵詞:TOPKAKT細胞移行肺癌癌症治療
外文關鍵詞:TOPKAKTmigrationlung cancer therapy
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TOPK/PBK (T-LAK cell-originated protein kinase/ PDZ-binding kinase)為一個在台灣女性肺腺癌組織及轉移性腫瘤中過度表現的Ser/Thr蛋白質激酶基因。先前實驗室已發現TOPK可以經由影響PTEN穩定度來調控PI3K/AKT dependent pathway 以促進細胞的移行及侵入能力。本論文進而分析TOPK之PDZ binding domain對其促進細胞移行及侵入能力的影響,此外,亦在細胞內降低TOPK表現量以評估TOPK於EGF所誘導之AKT磷酸化中所扮演之抑制角色。同時我驗證了TOPK和H2AX在M phase時期之結合能力,顯示當TOPK被活化時,H2AX可能可做為其之受質。進一步瞭解TOPK是否藉著與H2AX結合影響到DNA修復機制時,結果顯示默化TOPK表現明顯降低cisplatin所誘導之H2AX磷酸化並增加細胞死亡情形,接著,我亦在免疫螢光實驗中觀察到默化TOPK表現明顯降低cisplatin所誘導之H2AX磷酸化,此外,TOPK與cisplatin所誘導之H2AX磷酸化位在相同的subcellular localization,意謂著TOPK與DNA damage所誘發之H2AX磷酸化有功能上之關聯性。整體而言,上述結果顯示TOPK會影響EGF所誘導之AKT活化及肺癌細胞對化療藥物cisplatin之感受性,說明TOPK可做為肺癌的分子標幟(molecular target)之潛力。
TOPK/PBK (T-LAK cell-originated protein kinase/ PDZ-binding kinase) is a Ser/Thr protein kinase gene that is over-expressed in female lung adenocarcinoma tissues and in metastatic tumors. TOPK can promote cell migration and invasion via modulation of PI3K/PTEN/AKT dependent pathway. This thesis further identified the role of PDZ binding domain of TOPK in its involvement in cell migration/invasion. In addition, TOPK-depleted cells were used to demonstrate its role on reducing EGF induced AKT-Ser473 phosphorylation. Meanwhile, I confirmed the interaction of TOPK and H2AX at M phase, indicating that H2AX may serve as a substrate of TOPK when TOPK is activated. To test whether TOPK could affect the DNA repair mechanism by binding with H2AX, the results showed that cisplatin-induced H2AX phosphorylation was decreased and followed cell death was increased in TOPK- depleted cells. Next, I used immunofluorescence to show that cisplatin-induced H2AX phosphorylation was decreased in TOPK-depleted cells. Moreover, TOPK and cisplatin-induced H2AX phosphorylation had the same subcellular localization, suggesting that TOPK in DNA damage induced H2AX phosphorylation is functionally linked. Overall, our results demonstrate the role of TOPK in affecting EGF induced AKT activation and in responding to cisplatin treatment, which may have implications for TOPK as a molecular target for lung cancer.
中文摘要 ............................................................................................................1
Abstract ...........................................................................................................2
縮寫表 .............................................................................................................3
壹、 序論 ......................................................................................................4
一、 肺癌的發生率、死亡率及其分類 ................................................................................4
二、 基因與癌症 .................................................................................................7
三、 肺癌與基因變異相關性 .........................................................................................8
四、 肺癌的基因體研究..............................................................................................9
五、 TOPK/PBK基因的發現及其功能研究...............................................................................11
六、 研究動機與目標 ..............................................................................................15
貳、 實驗材料 ...................................................................................................17
一、 細胞株及大腸桿菌株..........................................................................................17
二、 化學藥品及材料套組劑 .......................................................................................17
三、 抗體及親和性膠體 ............................................................................................19
參、 實驗方法 ...................................................................................................21
一、 勝任細胞的製備 ..............................................................................................21
二、 轉形作用 ...................................................................................................22
三、 細胞繼代培養 ...............................................................................................22
四、 細胞轉染....................................................................................................22
五、 蛋白質濃度測定 ..............................................................................................23
六、 正十二烷硫酸鈉-聚苯烯鹽胺膠體電泳及西方墨點法...................................................................23
七、 細胞移行、侵入能力分析.......................................................................................23
八、 免疫沉澱....................................................................................................24
九、 免疫螢光....................................................................................................24
十、 細胞活性測試................................................................................................25
肆、 結果 ......................................................................................................27
...一、 探討PDZ binding domain在TOPK誘導之細胞移行及侵入之角色.........................................................27
二、 TOPK可誘發PTEN independent之細胞移行 ........................................................................28
三、 評估TOPK是否可抑制EGF所誘導之AKT磷酸化........................................................................28
四、 利用免疫沉澱分析證明H2AX在細胞中(ex vivo)可與TOPK結合..........................................................30
五、 評估TOPK可否影響化療藥物cisplatin誘導之H2AX活化...............................................................31
六、 評估TOPK可否影響化療藥物cisplatin引發之細胞死亡 ...............................................................32
伍、 討論 ......................................................................................................34
一、 TOPK調控PTEN蛋白質穩定性之機制 ...............................................................................34
二、 TOPK在EGFR/PI3K/AKT signaling活化所扮演的角色...............................................................35
三、 TOPK在DNA受損時所扮演的角色 ................................................................................36
四、 TOPK對於p21表現的影響 .......................................................................................37
陸、 圖表 ......................................................................................................38
Fig 1. PDZ domain of TOPK may play a role in regulating PI3K/AKT signaling and
cell migration/invasion. .................................................................................39
Fig 2. TOPK can conduct PI3K/AKT independent cell migration. ...................................................40
Fig 3. Test the condition of EGF-induced AKT phosphorylation in A549 cells.......................................41
Fig 4. Knockdown of TOPK decreased EGF-induced AKT phosphorylation in
A549-Luc shTOPK stable clone. ............................................................................42
Fig 5. Knockdown of TOPK decreased EGF-induced AKT phosphorylation in A549
cells (siRNA). ...........................................................................................43
Fig 6. TOPK interact with H2AX at least during M phase...........................................................44
Fig 7. Examine the time period of cisplatin-induced H2AX phosphorylation in A549 cells.........................45
Fig 8. Knockdown of TOPK decreases cisplatin-induced H2AX phosphorylation in
TOPK shRNA stably transfected cells......................................................................46
Fig 9. Knockdown of TOPK decreases cisplatin-induced H2AX phosphorylation in
TOPK siRNA transfected cells.............................................................................47
Fig 10.Knockdown of TOPK decreases cisplatin-induced H2AX phosphorylation in
TOPK shRNA stably transfected cells. .....................................................................48
Fig 11.TOPK and H2AX may co-localize in the nucleus is response to cisplatin treatment..........................49
Fig 12.Knockdown TOPK decreases cisplatin-induced cell death....................................................50
柒、 附錄......................................................................................................52
S1. TOPK expression is up-regulated in lung adenocarcinoma.
S2. Protein identified as candidate TOPK interaction proteins.
捌、 參考文獻 ...................................................................................................54

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