臺灣博碩士論文加值系統

砷是一種有致癌性的類金屬物質，長期暴露下容易引發血管病變、癌症甚或死亡。Aurora-A激�’b許多癌症細胞和臨床檢體中的表現量均顯著增加。我們先前的研究中發現烏腳病地區膀胱癌患者的腫瘤組織中Aurora-A激�’章L度表現的機率明顯高於一般地區，顯示砷暴露可能與Aurora-A激�﹞j量表現有關。本篇研究的重點為探討表觀遺傳調控 (epigenetic regulation) 中之DNA甲基化是否參與砷所誘發的Aurora-A過度表現。Bisulfite sequencing PCR及methylation-specific PCR的結果顯示膀胱癌、皮膚癌細胞株及臨床皮膚癌檢體中Aurora-A啟動子內CpG小島上的28個CpG雙核�˙臚云滬M嘧啶都沒有被甲基修飾之情形，將細胞培養在含1 μM砷的培養液4周也不會對Aurora-A啟動子之甲基化有任何影響。總而言之，砷引起的Aurora-A過度表現並非經由Aurora-A啟動子內CpG小島之去甲基化來活化基因轉錄。
Ras是一廣為人知的致癌基因。藉由二維電泳比較經誘發H-Ras12V過度表現和未經誘發的7-4細胞其蛋白質體的差異我們選定出二十個有兩倍表現差異的蛋白，經LC-MS/MS蛋白質鑑定其中之一為phosphoglycerate mutase I (PGAM1)，且此蛋白表現量可能與Ras活性相關。本研究進一步建立了可以透過誘發來製造具有生物素標記之H-Ras12V重組蛋白的大腸桿菌，利用此系統可更專一地搜尋和H-Ras有直接互動的新穎蛋白，藉此深入探討這些蛋白在Ras相關癌症中所扮演之角色。

Arsenic is an oncogenic metalloid. Long-term exposure to arsenic may lead to vascular diseases, cancers and death. Aurora-A is a mitotic kinase which is overexpressed in numerous cancer cell lines and clinical specimens. We previously unraveled that the incidence of Aurora-A protein overexpression in the bladder cancer patients of black-foot disease endemic areas is significantly higher than in other areas, suggesting that arsenic exposuremay induce Aurora-A overexpression. In the present study we elucidate whether the DNA methylation of CpG islands within Aurora-A promoter is involved in arsenic-induced Aurora-A overexpression. The cytosines of the 28 CpG dinucleotides on CpG islands within Aurora-A promoter are unmethylated in bladder and skin cancer cell lines as well as clinical skin cancer specimens determined by bisulfite sequencing PCR (BSP) and methylation-specific PCR (MSP). All together, long-term (4 weeks) exposure to arsenic (1 μM) of E7 cells doesn’t affect the methylation status of Aurora-A promoter. Arsenic-induced Aurora-A overexpression is not through demethylation of CpG islands within Aurora-A promoter.
Ras is a well-known oncogene. Comparing the protein profile between cells with and without H-Ras12V overexpression total of 20 H-Ras-related proteins with approximate 2-fold expression difference were identified. By LC-MS/MS protein identification, one of them is phosphoglycerate mutase I (PGAM1), which may correlate with Ras activity. Furthermore, an E. coli strain that expresses inducible biotin-tagged H-Ras12V recombinant protein was established. This system will be used for the identification of novel Ras-interacted proteins, which will shed lights on the therapy against Ras-related cancers.

中文摘要 I
Abstract II
誌謝 III
Contents IV
Figure List VI
Appendix List VIII
Introduction
I. Overview of Aurora gene family 1
II. Role of Aurora-A in normal and cancer cells 2
III. Aurora-A may act as a mediator in arsenic-associated tumors 3
IV. DNA methylation in normal and tumor cells 5
V. Role of DNA hypomethylation in arsenic-induced Aurora-A overexpression 6
VI. Overview of Ras 7
VII. Ras research in new direction 7
VIII. Strategies to screen for novel Ras-related proteins 10
Materials and Methods
I. Culture condition 11
II. Western blotting 11
III. Reverse-transcription PCR 12
IV. In vitro DNA methylation 14
V. Cell cycle synchronization 14
VI. Bisulfite-sequencing PCR 15
VII. Methylation-specific PCR 16
VIII. Promoter assay 18
IX. Two-dimensional electrophoresis 18
X. Cloning 21
Results
I. Long-term and low-dosage inorganic arsenic exposure induces Aurora-A protein expression 23
II. DNA methylation plays a role in Aurora-A expression at mRNA and protein levels 23
III. No DNA methylation is detected by bisulfite sequencing PCR in three regions within Aurora-A promoter 24
IV. Differential Aurora-A expression during mitosis is not dynamically regulated by DNA methylation 25
V. CpG islands within Aurora-A promoter are unmethylated confirmed by methylation-specific PCR 26
VI. 5-Aza-dC treatment induces Aurora-A mRNA and protein expression through promoting the transcription of Aurora-A 27
VII. An IPTG-inducible H-Ras protein expression system is used to screen for novel Ras-related proteins 28
VIII. Construction of a plasmid bearing a H-ras12V gene with biotin-tag (AviTag) at N-terminus 29
Discussion 31
References 39
Background 80

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