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研究生:曾雅詩
研究生(外文):Ya-Shih Tseng
論文名稱:5-fluorouracil以及Aurora-A對Ras致癌基因轉形細胞之作用和機轉
論文名稱(外文):The effects and the related mechanisms of 5-fluorouracil as well as Aurora-A on Ras oncogene transformed cells
指導教授:劉校生
指導教授(外文):Hsiao-Sheng Liu
學位類別:博士
校院名稱:國立成功大學
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:2005
畢業學年度:93
語文別:英文
論文頁數:139
中文關鍵詞:Aurora-A5-FURas 致癌基因
外文關鍵詞:5-FURasAurora-A
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  在許多人類癌症細胞中都發現有Ras致癌基因的突變,包括胰臟癌、大腸直腸癌、肺癌、乳癌及膀胱癌。雖然過去已經發展出許多針對Ras致癌基因相關癌症的新式治療方法,5-flurorouracil (5-FU) 仍然是一個廣汎使用的化療藥物。在許多人類癌症細胞中也都發現有致癌基因Aurora-A過量表現的情形,包括乳癌、卵巢癌、大腸直腸癌、攝護腺癌、胰臟癌、膀胱癌、肝癌及腦癌。這份研究主要致力於研5-FU對帶有Ras致癌基因突變之轉形細胞之作用機轉曁探討在膀胱癌中Aurora-A過量表現和Ras致癌基因突變發生之關聯以及Aurora-A過量表現對Ras致癌基因突變轉形細胞之影響。我們的研究結果顯示帶有Ras致癌基因突變之轉形細胞對於5-FU比較敏感,此乃起因於Ras致癌基因突變之轉形細胞在處理5-FU後會活化caspase-3,改變粒線體膜電位,並產生DNA片斷使細胞走向凋亡之路。進一步的研究顯示Bcl-2、Raf-1及 PI3K在此過程中扮演重要關鍵的角色。帶有Ras致癌基因突變之轉形細胞在處理5-FU後,會有細胞週期異常的現象,此乃肇因於cyclin E、p21Cip1/Waf-1及磷酸化Cdc 2 的異常表現。另一方面,在Aurora-A過量表現對於帶有Ras致癌基因突變之轉形細胞的作用與影響的研究方面,我們發現在37位膀胱癌病人中,有59.4% 的病人具有Aurora-A過量表現;在Aurora-A過量表現的病人中,有31.8% 的病人具有Ras致癌蛋白質第12位置突變,有45.5% 的病人是來自烏腳病盛行地區。對膀胱細胞株,E7,處理不同濃度之砷,會使得Aurora-A 蛋白質的表現隨著砷濃度的增加而增加,這意味著在膀胱癌中,砷可能可以促進Aurora-A 蛋白質的表現。共同表現高量Aurora-A及RasV12致癌蛋白質之細胞與只表現RasV12致癌蛋白質之細胞比較,可明顯的增加focus 的形成及在BALB/c老鼠中之腫瘤形成。Aurora-A 會促進Ras下游的訊息傳遞分子,包括MEK/ERK,AKT以及RalA的活化。進一步的研究顯示共同表現高量Aurora-A及RasV12致癌蛋白質所增強之focus形成乃導因於MEK/ERK磷酸化的增加;而且我們也發現Aurora-A本身並不會活化ERK路徑,Aurora-A需要有活化態之Ras 存在下才可促進ERK路徑之活化。綜合以上結果,帶有Ras致癌基因突變之轉形細胞對於抗癌藥5-FU較敏感,而表現高量Aurora-A會促進帶有Ras致癌基因突變之轉形細胞之致癌性,瞭解這些機轉可對人類治療Ras相關之癌症的方向上有所貢獻。
  About 30% of human cancers, including pancreas adenocarcinoma, colon cancer, lung cancer, breast carcinoma, and bladder carcinoma, harbor mutations of ras oncogene. Despite that several novel therapeutic approaches have been designed to treat Ras-related cancers, 5-fluorouracil (5-FU) is still a widely used agent. Overexpression of Aurora-A are also detected in diverse human cancer cell lines and cancers, including breast, ovarian, colon, prostate, pancreas, bladder, medulloblastoma, hepatocellular carcinoma, and gliomas. This study intends to reveal the effectand the mechanism of 5-FU on Ha-rasV12 transformants as well as the relation between overexpression of Aurora-A and Ras codon-12 mutation in bladder cancers and the effects of these two oncogenes in cellular transformation. Our results demonstrated that cells harboring Ha-rasV12 were susceptible to 5-FU treatment through Ras-Raf-1 related apoptosis process, which was demonstrated by caspase-3 activation, loss of mitochondria membrane potential and DNA fragmentation. Further study showed that Bcl-2 as well as PI3K pathways could block 5-FU-induced apoptosis under Ha-ras-overexpressed condition. In addition, under Ras overexpressed condition, 5-FU treatment caused cell cycle disruption is possible through aberrant expression of cyclin E and p21Cip1/Waf-1 as well as phosphorylation of Cdc 2. We also found that 59.4% of bladder tumor tissues showed overexpression of Aurora-A; and 31.8% of patients with Aurora-A overexpression also had Ha-ras codon 12 mutation, indicating a relation between these two components. In addition, 45.5% of patients with Aurora-A overexpression were from BDF endemic area, suggesting the environmental factor arsenic may be involved. Cell line study further demonstrated that arsenic treatment of the immortalized bladder cells, E7, increased the expression level of Aurora-A in a dose dependent manner, supporting out speculation that arsenic may be responsible for Aurora-A overexpression in bladder cancers. Coexpression WT-Aurora-A and RasV12 in the cells significantly increased the focus formation as well as tumor size in BABL/c mice compared with expression RasV12 only in the cells. Further analysis demonstrated that WT-Aurora-A enhanced the focus formation through Ras-related MEK/ERK pathway. All together, the cells overexpressing Ha-rasV12 becomes sensitive to 5-FU. In contrast, the same cells overexpressing Aurora-A becomes more tumorigenic. Our findings will provide new aspects for design tumor therapy and understanding tumor progress of Ras-related cancers.
Abstract (Chinese) ----------------------------------------------------------------------------- I
Abstract (English) ------------------------------------------------------------------------------ III
Acknowledgments------------------------------------------------------------------------------- V
Contents------------------------------------------------------------------------------------------ i
Abbreviations------------------------------------------------------------------------------------ v
Index of Tables----------------------------------------------------------------------------------- vii
Index of Figures--------------------------------------------------------------------------------- viii
Index of Appendix------------------------------------------------------------------------------- x

Background
I. Ras
The Ras family----------------------------------------------------------------------------- 1
Posttranslational modification of Ras proteins---------------------------------------- 2
Activation of Ras proteins--------------------------------------------------------------- 3
Signal pathways of Ras------------------------------------------------------------------- 3
II. The strategies used in treatment with Ras-related cancers
For inhibition of Ras-related signaling------------------------------------------------- 6
For induction of cell death--------------------------------------------------------------- 7
III. The program cell death (PCD) pathways
Type I PCD (Apoptotic pathways------------------------------------------------------- 8
Type II PCD (Autophagy) pathways---------------------------------------------------- 9
IV. Aurora-A
The Aurora-A kinases--------------------------------------------------------------------- 10
Aurora-A and mitosis--------------------------------------------------------------------- 11
Aurora-A and tumorigenesis------------------------------------------------------------- 12
V. The blackfoot disease (BFD) endemic area and cancers-------------------------- 13
VI. Specific aims of this study------------------------------------------------------------- 13

Materials and Methods
Plasmids------------------------------------------------------------------------------------ 15
Cells and cell culture---------------------------------------------------------------------- 15
Primers and probes------------------------------------------------------------------------ 16
Antibodies---------------------------------------------------------------------------------- 16
Tumor Tissues ----------------------------------------------------------------------------- 17
RNA preparation--------------------------------------------------------------------------- 18
cDNA preparation------------------------------------------------------------------------- 18
Genomic DNA preparation--------------------------------------------------------------- 18
Quantitative real-time PCR-------------------------------------------------------------- 18
Ha-ras codon-12 SNP--------------------------------------------------------------------- 19
Ki-ras codon-12/13 SNP----------------------------------------------------------------- 19
MTT assay---------------------------------------------------------------------------------- 20
Flow cytometry---------------------------------------------------------------------------- 20
DNA fragmentation assay---------------------------------------------------------------- 21
Caspase-3activity assay------------------------------------------------------------------ 22
Nuclear extraction------------------------------------------------------------------------- 22
Northern blotting-------------------------------------------------------------------------- 22
Western blotting--------------------------------------------------------------------------- 22
Ras and RalA pull-down assay---------------------------------------------------------- 23
Immunoprecipitation---------------------------------------------------------------------- 23
Transfection-------------------------------------------------------------------------------- 24
Establishment of stable clones---------------------------------------------------------- 24
Growth curve assay----------------------------------------------------------------------- 24
Focus formation--------------------------------------------------------------------------- 25
Luciferase activity and ��-galactosidase assay----------------------------------------- 25
Immunohistochemistry (IHC) stain---------------------------------------------------- 25
Arsenic treatment------------------------------------------------------------------------- 26
Statistic analysis--------------------------------------------------------------------------- 26

Part I: The effects of 5-fluorouracil on Ha-rasV12 transformants
Abstract---------------------------------------------------------------------------------- 28
Introduction ----------------------------------------------------------------------------- 29
Results----------------------------------------------------------------------------------- 31
Discussion------------------------------------------------------------------------------ 36

Part II: The relation between Aurora-A and Ha-ras
Abstract--------------------------------------------------------------------------------- 40
Introduction---------------------------------------------------------------------------- 42
Section I: The association between Aurora-A overexpression and Ha-ras
codon 12 mutation in bladder cancer.
Results---------------------------------------------------------------------------------- 46
Discussion----------------------------------------------------------------------------- 49
Section II: WT-Aurora-A overexpression enhances the tumorigenesis of
Ha-rasV12 transformants through MEK/ERK pathway.
Results---------------------------------------------------------------------------------- 52
Discussion----------------------------------------------------------------------------- 59

Summary---------------------------------------------------------------------------------------- 62
References--------------------------------------------------------------------------------------- 63
Tables--------------------------------------------------------------------------------------------- 85
Figures------------------------------------------------------------------------------------------- 92
Appendix---------------------------------------------------------------------------------------- 119
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