臺灣博碩士論文加值系統

p53 蛋白質，有基因組守護員之稱，當細胞面臨壓力時， p53 致使細胞週期暫停和細胞凋亡等機制以維持基因的穩定性。p63 蛋白質為 p53 家族成員之一，對於上皮細胞的生長以及恆定扮演重要角色，其isoform ΔNp63α 蛋白質主要為維持上皮細胞的貼附能力。我們先前研究已發現 ΔNp63α 會與 CTEN 啟動子結合並調控其基因表現，參與細胞貼附的機制，以及隨著前列腺癌惡化程度提高，CTEN 與p63 的 mRNA 也會隨之下降。因此本論文假設 ΔNp63α 促進 CTEN 表現增加，能降低前列腺癌細胞惡化程度。本論文研究發現，在前列腺癌細胞中過量表現 ΔNp63α 蛋白質會促進CTEN mRNA及蛋白質表現量增加，然而增加的幅度並不大，我們認為除了外源表現的 ΔNp63α之外，在前列腺癌細胞中亦有其他的轉錄因子或 co-activator 共同參與調控 CTEN 的表現機制。另一方面，我們發現無論 p63 存在與否， p53 均抑制 CTEN promoter 轉錄活性，而在人類正常前列腺細胞株 RWPE-1 中，過量表現 p53 會造成 CTEN mRNA 及蛋白質表現量下降，我們以染色質免疫沉澱 (ChIP) 方式，證明了 p53 蛋白質會結合至 CTEN 啟動子上 -81~+25的 p53 預測結合位置上。此外，我們發現以 cisplatin 造成 DNA 損傷使細胞面臨生存壓力時，會促進 p53 表現量增加，並造成 CTEN mRNA 以及蛋白質表現量均下降之結果。本論文證實 p53 會結合在 ΔNp63α 亦辨識結合之 CTEN 啟動子位置上，抑制 CTEN 基因表現，並可能參與在細胞 DNA 損傷壓力的機制中。

p53, the guardian of genome, is involved in cell cycle arrest and apoptosis to maintain the stability of genome in cellular stress. p63, a member of the p53 transcriptional factor family, is a master of regulation of epithelial homeostasis and development. ΔNp63α, one of p63 isoforms, enhances the ability of cell adhesion. Our previous study revealed that ΔNp63α binds to putative p53 binding sites present in CTEN promoter and transcriptionally regulates CTEN expression involved in prostate cell adhesion. Furthermore, CTEN and p63 down-regulation correlates with prostate cancer progression from primary tumors to metastatic lesions. Therefore, we hypothesize that overexpression of ΔNp63α might enhance CTEN expression and hamper prostate cancer cell progression. In our study, overexpression of ΔNp63α in prostate cancer cells induced the mRNA and protein levels of CTEN. However the increases are slight. We assume that there might be other transcription factors or co-activators involved in the regulation of CTEN gene expression in prostate cancer cells in addition to exogenously expressed ΔNp63α. On the other hand, we found that p53 down-regulates CTEN promoter activity whether ΔNp63α exists or not. Also, overexpression of p53 decreased CTEN mRNA and protein in non-malignant prostate cell line, RWPE-1. By using the chromatin immunoprecipitation (ChIP) assay, we demonstrated that p53 binds to CTEN promoter within the region between -80 to +25. Moreover, by using cisplatin to induce DNA damage and celluar stress, we discovered that cisplatin exposure caused increased p53 and decreased the mRNA and protein levels of CTEN. In conclusion, our findings reveal that p53 binds to the CTEN promoter region which also recognized by ΔNp63α. p53 repressed the expression of CTEN in response to DNA damage stress.

縮寫表 iv
摘要 vi
Abstract vii
1 本論文之研究背景 1
1.1 p53家族成員簡介 1
1.2 p53 於細胞中所扮演之功能 1
1.3 p63 結構以及功能 2
1.4 p53 與 ΔNp63α 對相同目標基因的調控及對細胞功能之影響 4
1.5 CTEN 的結構 4
1.6 CTEN 調控細胞遷移能力 5
1.7 CTEN 的基因表現受到 ΔNp63α 的調控並與前列腺細胞貼附表現有關 6
1.8 本論文研究目的與重要性 7
2 材料與方法 8
2.1 Cell preparation 8
2.2 Plasmid preparation 9
2.3 Cell transfection 11
2.4 Dual luciferase assay 11
2.5 RNA analysis 12
2.6 Protein analysis 13
2.7 Immuno-precipitation (IP) 14
2.8 Chromatin Immuno-precipitation assay, ChIP 14
2.9 Radiation 16
2.10 Cisplatin treatment 16
3 研究結果 17
3.1 外源表現 ΔNp63α 於前列腺細胞中能促進 CTEN 表現 17
3.2 p53 蛋白質抑制 CTEN 啟動子活性 17
3.3 p53 抑制 CTEN mRNA 及蛋白質表現 18
3.4 p53 辨識結合於 CTEN 啟動子之序列 19
3.5 以放射線方式，誘導 p53 蛋白質表現觀察 CTEN 表現 20
3.6 以 cisplatin 誘導 p53 表現造成 CTEN mRNA 及蛋白質表現下降 21
4 討論與未來的研究方向 22
5 參考資料 27
6 圖與表 33

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