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研究生:莊靜萍
研究生(外文):Ching-Ping Chung
論文名稱:HDAC抑制劑對膀胱癌細胞株之抗DNA損傷基因之影響
論文名稱(外文):The effect of HDAC inhibitor on DNA damage resistant genes in U1 (late stage), U4 (early stage) and B23 down-regulated U1 bladder cancer cells
指導教授:翁一鳴
指導教授(外文):Benjamin Yat Ming Yung
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:63
中文關鍵詞:核仁磷酸蛋白
外文關鍵詞:Nucleophosmin/B23
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核仁磷酸蛋白(B23)是細胞核內具有多功能的磷酸蛋白,它在腫瘤細胞及增生細胞的含量比在正常休止期的細胞高出許多,顯示B23具有促進細胞增生及致癌基因的特性。過去我們實驗室的結果觀察到在膀胱癌晚期的細胞,例如MGH-U1細胞中B23含量高於早期細胞(MGH-U4)並且對於會造成DAN損傷的抗癌藥物具有較高的抗藥性,因此我們想要了解癌症細胞對於化學藥物的抗藥性是否和B23相關。另外過去研究中也發現B23會與AP-2α形成一個具有轉錄調控功能的複合物,我們選擇具有對抗DAN損傷的基因做探討,發現這些基因的啟動子上可能具有AP-2α結合的位置,並且發現在MGH-U1細胞中,這些基因的表現量都多於早期膀胱癌細胞(MGH-U4)。
另外我們使用ㄧ種抗癌藥物是histone deacetylase的抑制劑,當我們將這ㄧ類的抗癌藥物作用於膀胱癌細胞並且觀察抗DAN損傷的基因的表達,發現只有部份基因受到藥物的影響,進一步去探討基因轉錄機制,B23會與AP-2α共同結合在基因啟動子上的AP-2α結合位置,並且histone deacetylase的抑制劑會減少此種結合的表現。這些證據顯示出B23可以藉由其他的轉錄因子結合來參與基因轉錄層面的調控。
Nucleophosmin/B23 (NPM/B23) was a multifunctional nucleolar phosphoprotein. In the analysis of clinical cancer tissues, cancers of later stages or recurrent cancers seem to have more NPM/B23 protein. We supposed that MGH-U1 bladder cancer cells (late stage) are more resistant to chemotherapeutic drugs than MGH-U4 (early stage) cells. In other study, twenty genes were found to be up-regulated in cell lines that are resistant to several chemotherapeutic drugs. In this study, a number of genes (9 of 22) were highly expressed in MGH-U1 cells compared with MGH-U4 cells. Previous study has shown that NPM/B23 is involved in regulation of heat shock 60kd protein 1 through interaction with AP-2α as well as binding to the AP-2α sites in the promoter of heat shock 60kd protein 1. We wanted to know whether these genes were regulated by NPM/B23 during anti-cancer drug treatment or not.
Another anti-cancer agent is histone deacetylase inhibitor (HDACis), such as suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). The action of HDAC inhibitors on gene expression is selective and restricted to a relatively small number of the chemotherapeutic resistant genes. Moreover, the binding of the NPM/B23-AP-2α complex to the AP-2α binding site of Hsp60 promoter are decreased by TSA treatment. The evidence suggests that NPM/B23 can involve in the transcriptional regulation by associating with transcription factor AP-2α during HDAC inhibitor treatment.
Chapter I Introduction………..……………………..…………………..1
Nucleophosmin (B23)………………………………….…………...1
Bladder cancer….…………………….………………………………3
Doxorubicin (Dox) and Trichostatin-A (TSA)………………………5
Activator protein 2 (AP-2)………………………………………...6
Objectives of this study………………………………………..….8
Chapter II Materials and Methods…………………………………....9
Cell culture…………….…………………..…….…………..….….9
Western blotting………………………………………………..…..9
Cell transfection……….…...…………………….…….………..…10
Luciferase and β-galactosidase activity assays………………..…..11
RNA isolation and Reverse transcription-PCR (RT-PCR)…..…...11
Identification of transcription factor binding sites……………..…12
Chromatin immunoprecipitation (ChIP) assay…………………....12
Statistical analysis...…………………………………………….…14
Chapter III Results……………………………………………….…....15
Treatment of doxorubicin (Dox) increased NMP/B23 and c-Myc protein level in MGH-U1 cells…………………….……………….15
The NPM/B23 promoter activity was up-regulated by treatment of Dox………..…………..................................................................…15
Dox increased c-Myc recruitment to B23 promoter………………..16
RNA expression of DNA damage resistant genes in MGH-U1 and MGH-U4 cells……………………………………………………...16
Effect of HDAC inhibitors in the DNA damage resistant genes expression in MGH-U1 cells………………………………………18
In vivo binding of B23 and AP-2α to the Heat shock 60kd protein 1 promoter……………………………………………………………19
Effect of DNA damage drugs on B23 down-regulated MGH-U1 cell growth……………………………………………………………...20
RNA expression of the DNA damage resistant genes in B23 down-regulated MGH-U1 cells…………………………………….20
RNA expression of AP-2α in B23 down-regulated MGH-U1 cells..21
Chapter IV Discussion………………………………………...……….22
Chapter V References………………………………………………….25
Chapter VI Figures……………………………………..……………..34
Chapter VII Appendix………………………..………….……………44
Chapter VIII Table.................................................................................48
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