β-catenin 在前列腺癌細胞株PC3 內影響
轉錄因子Tcf4 蛋白穩定性之新功能的探討

β-catenin 在維持細胞正常生長上扮演兩種重要功能。一種為與 E-cadherin 以及α-catenin 結合作為細胞貼附複合體的次單元之ㄧ，另一種則為參與β-catenin /Tcf 訊息調控路徑，作為轉錄因子Tcf/Lef family 共同啟動子（coactivator）。β-catenin /Tcf 訊息調控路徑在胚胎發育､幹細胞新生､細胞癌化的過程當中皆扮演著十分重要的角色。許多癌症中，包括前列腺癌､皮膚癌､大腸癌以及肝癌上我們都可以發現，β-catenin 本身或是控制β-catenin 水解與否的分子，皆有突變的情形。所有的證據均顯示β-catenin 參與癌症的形成但其分子機制卻還不明。在我們對於前列腺癌的研究當中，為了解β-catenin 如何影響Tcf4 訊息傳遞路徑並參與前列腺癌形成，我們選擇含有Tcf4 訊息之前列腺癌細胞株 PC3，並篩選轉殖入含有flag-tag 的野生型以及持續活化突變型β-catenin(T41A, S45A)的PC3 當做模式細胞以研究β-catenin 如何影響Tcf4 訊息傳遞路徑。我們的結果顯示， β-catenin 會增加核內Tcf4 蛋白質含量。而更進一步的證據顯示，此一現象產生之原因為β-catenin 增強Tcf4 蛋白質穩定性。另外，隨著核內Tcf4 蛋白質含量增加，Tcf4 所主導之轉錄活性也隨之增加。為了解β-catenin 是否影響Tcf4 結合到Tcf4 專一DNA 序列，我們抽取出轉殖入不同型β-catenin PC3 的核內蛋白，利用electrophoretic 3 mobility shift assay 試圖了解β-catenin 是否影響Tcf4 結合於其專一的DNA 序列，結果我們發現在PC3 上無法看見specific-binding。同樣在對照組或轉植入持續表現活化型β-catenin 之293 細胞株中我們也無法看見specific-binding。有趣的是，當我們利用anti-flag antibody-conjugated agarose 將β-catenin 純化出來後，依照相同步驟跑electrophoretic mobility shift assay 卻可以在之前未純化的 non-specific binding 相同位置發現specific β-catenin-Tcf4-DNA complex。根據此一結果我們推測，這條specific β-catenin-Tcf4-DNA complex 可能隱藏在non-specific binding 的下方。另外，過度表現 β-catenin 卻無法看見super-shift band 則暗示著我們β-catenin 對於 Tcf4 結合到其專一DNA sequence 上為必需。最後，我們想更進一步了解持續活化突變型β-catenin 是否影響細胞在沒有血清情況下之存活率，我們的結果顯示，有持續活化突變型β-catenin 的PC3 其存活率是比對照組來的高出許多。

關鍵字

前列腺癌,β-catenin,Tcf4,PC3

並列摘要

Two functions of β-catenin have been proposed to influence cell behavior. One is the component of cell adhesion complex as a connecting molecule between E-cadherin and α-catenin, and the other is the coactivator of Tcf/Lef family of transcription factors .The β-catenin /Tcf pathway plays a critical role in embryo development , stem cell renew, and also contributes to carcinogenesis. Mutations in the components controlling β-catenin toward proteasomal degradation including APC and axin, or β-catenin itself have been identified in several cancer types including prostate, skin, colon and liver. Therefore, the mutated β-catenin might be a key point to cause tumor formation. To determine the function of β-catenin participate in Tcf-4 signaling in prostate cancer, we used PC3 cell that contains Tcf-4 transcription factor with over-expression of wild type and mutated form of flag-tag β-catenin as model. This mutated form is β-Catenin (T41A, S45A). By using this model, we have identified a new role of β-catenin by increasing the amount of Tcf4 in the nuclear. Further evidence indicated that this increasing is due to β-catenin enhancing stability of Tcf4. Significantly, this increasing amount of Tcf4 was consistent with Tcf4-driving transcription activity. To know β-catenin effect on the DNA binding activity of Tcf4, we performed electrophoretic mobility shift assay by using the nuclear extracts from the PC3 cells transfected with the vector control, wild type or mutated form of β-catenin. We found no specific-binding among these nuclear extracts. This phenomenon was also demonstrated by using the nuclear extracts from the 293 cells transfected with the control vector or mutated form of β-catenin. Furthermore, we concentrated β-catenin with anti-flag antibody-conjugated agarose and performed the same assay. Interestingly, the specific β-catenin-Tcf4-DNA complex appeared in the same position as the non-specific binding by using the crude nuclear extracts. It suggested that the specific β-catenin-Tcf4-DNA complex was hidden in the non-specific binding band in the assay using the crude nuclear extract. With over-expression of β-catenin, no super-shift band was observed. This implied that β-catenin is essential for Tcf4 to interact with its specific DNA sequence. In 5 addition, we examined whether mutated form of β-catenin can enhance the cell survival under serum-free condition. The results indicated that the PC3 cells overexpressing mutated form of β-catenin has higher survival rate than that of the control cells under serum-free condition.