Ｔ細胞第四因子(Tcf4)與細胞死亡相關蛋白(Daxx)結合作用之探討

Abstract

近年來的研究報導指出，在人類大腸癌細胞株細胞中，因為抑癌基因APC的突變或是乙型卡特寧本身的突變，導致細胞核內的人類T細胞因子與乙型卡特寧蛋白複合物活性的持續活化，因而造成人類T細胞因子所調節的下游基因失控是造成大腸上皮細胞的轉型以及息肉生成的主要原因。事實上，人類T細胞因子與乙型卡特寧蛋白複合物對轉譯活性的調節，不管是在胚胎發育或是癌症生成的過程中都存在著相當的意義。在嘗試探討T細胞因子在癌症生成過程中所可能扮演的角色之前，必須先釐清T細胞因子的調控網絡如何運作。雖然T細胞因子可以透過HMG直接與去氧核醣核酸結合，然而這樣的結合在缺乏與乙型卡特寧結合形成蛋白複合體時，T細胞因子並不足以獨立啟動其下游基因的轉譯，除此之外，更多的實驗結果顯示乙型卡特寧不只有如上所述與T細胞因子形成蛋白質複合體以啟動下游基因的轉譯活性，同時也可以將T細胞因子對下游基因的調控功能，由原本的轉譯抑制轉換為轉譯活化，鑑於對相同下游基因的不同作用，科學家們深信必定存在有一嚴謹的開關，得以嚴密調控並且決定細胞發育生長過程中不同的命運。 在T細胞因子轉譯活性的調控中，可能存在與T細胞因子結合的介質蛋白以及其彼此之間交互作用的機轉激發我們相當的研究興趣。由於在人類大腸上皮細胞中存在大量的T細胞第四因子蛋白，因此實驗設計以T細胞第四因子為餌，透過酵母菌雜交的方式順利分離出可能與T細胞第四因子結合的候選蛋白-人類細胞死亡相關蛋白：Daxx。 Daxx蛋白最早被發現是在細胞質中扮演Fas/JNK相關訊息傳遞的媒介蛋白，不過近年來更多的證據指出，其實Daxx蛋白主要是存在細胞核中，扮演基因轉譯活性的調節者。藉由在人類293T細胞中的共同免疫沈澱法以及在酵母菌Y190菌種中的雜交法，證實了Daxx蛋白與T細胞第四因子蛋白之間的結合作用，並且定出T細胞第四因子與Daxx蛋白的結合區間。在細胞核內，Daxx蛋白藉由減低T細胞第四因子與去氧核醣核酸結合的能力而達到抑制T細胞第四因子轉譯活性。因此，在人類大腸癌細胞中大量表現Daxx蛋白時，T細胞第四因子下游基因的調控也跟著改變，包括有cyclinD1和Hath-1基因，同時造成人類大腸癌細胞生長週期G1時期的停頓。除此之外，在人類大腸腺癌病人的檢體中，比較該病人癌化的與正常的大腸配對組織，Daxx蛋白的表現則有明顯減少的現象。綜合以上的發現推論Daxx蛋白可能透過與T細胞第四因子的結合，繼而調控人類細胞的生長週期，以達到細胞增生與細胞分化的生理目的。 另外，當共同表現Daxx蛋白時，可以透過SDS-PAGE的分離，觀察到另一個較小型態的T細胞第四因子蛋白的存在，並且隨著Daxx蛋白的表現劑量或時間有專一的相關聯性。T細胞第四因子的轉譯活性也可以被SUMO-1以及PIAS3加強，不過這樣的轉譯加強效果仍然可以被Daxx蛋白所抑制。以上的研究發現與推論，除了可以進一步勾勒出T細胞第四因子與Daxx蛋白結合時對人類生理功能的影響，同時也可以提供創新的思考模式，達到癌症治療的目的。

Recently, it has been reported that the nuclei of colon carcinoma cell lines contain constitutively active Tcf4 / b-catenin complexes as a direct consequence of either loss of function of the tumor suppressor gene APC or gain of function mutations in b-catenin itself. This is believed to result in the uncontrolled transcription of TCF target genes, leading to transformation of colon epithelial cells and initiation of polyp formation. Regulation of the transcriptional activity of b-catenin / Tcf4 has important implications for embryonic development as well as for carcinogenesis in the intestinal epithelium. Prior to discuss the potential role for LEF / TCF transcription factors in cancer, it is important to outline the mechanism by which they have been proposed to operate. Although LEF / TCF transcription factors bind directly to DNA through their HMG domains, they are incapable of independently activating gene transcription. On the other hand, most experimental data support the view that TCF is a repressor when Wnt does not convert it into an activator. TCF can repress target genes as well as activate those same target genes in cells instructed to change developmental fate, there are several mechanisms by which this switch is achieved. We are interested in the possibility of the bridging protein that interacts with TCF during the regulation of transcriptional activity. Because of its predominant expression in the human colonic epithelium, the full-length Tcf4 is used as bait in yeast two-hybridization, and Daxx is isolated. Daxx, a human cell death associated protein, has been reported to mediate the Fas / JNK-dependent signals in the cytoplasm. However, several lines of evidence have suggested that Daxx is mainly located in the nucleus and functions as a transcriptional regulator. Co-immunoprecipitation in HEK-293T cells and yeast two-hybrid screen in Y190 cells are performed to identify the interaction between Tcf4 and Daxx, and co-immunoprecipitation is also used to map the binding regions of Tcf4. In the nucleus, Daxx reduces DNA binding activity of Tcf4 and represses Tcf4 transcriptional activity. Overexpression of Daxx alteres expression of genes downstream of Tcf4, including cyclin D1 and Hath-1, and induces G1 phase arrest in colon cancer cells. Besides, a reduction in Daxx protein expression is observed in colon adenocarcinoma tissue when compared with normal colon tissue. These findings suggest a possible physiological function of Daxx, via interaction with Tcf4, to regulate cell cycle progression, and hence cell proliferation and differentiation. Furthermore, a small form of Tcf4 is observed specifically in SDS-PAGE when Daxx is co-expressed in a dose- and time-dependent manner. Moreover, transcriptional activity of Tcf4 is enhanced by SUMO-1 and PIAS3, but this transactivation still can be repressed by Daxx. Taken together, these findings not only outline the functional link between Tcf4 and Daxx, but also provide a new idea to develop novel cancer therapies.