三氧化二砷可以有效的用來治療急性前髓性細胞白血病病人而且對於人類實體瘤細胞也有造成細胞凋亡或細胞週期停滯的能力。抑癌基因p21WAF1/ CIP1 (p21)會抑制細胞週期的進行。我們之前的研究發現三氧化二砷可以透過兩條路徑來調控p21。其中之一為p21被活化的EGFR-Ras-Raf-ERK1/2路徑，另一條為p21被抑制的JNK路徑。我們也發現三氧化二砷會磷酸化c-Jun的N端並和TG-interacting factor (TGIF)作用，來抑制p21的活化。TGIF可以經由兩種機制來抑制轉錄，直接和啟動子上的homeodomain作用，或是透過吸引其他的轉錄因子例如c-Jun。因此，本實驗的目的是探討TGIF在三氧化二砷調控的p21啟動子以及在癌症中所扮演的角色。首先，我們證明了TGIF位於細胞核內；為了證明TGIF和c-Jun在完整的細胞中會有交互作用，我們利用螢光能量共振轉移(FRET)的技術。FRET是一種確認蛋白質在完整細胞中是否有交互作用的方法。我們建構了CFP-c-Jun和YFP-TGIF兩個融合蛋白質。透過FRET的方式我們觀察到人類子宮頸上皮癌A431細胞株裡CFP-c-Jun和YFP-TGIF有交互作用。利用第63、73胺基酸突變的融合蛋白質c-Jun(S63/73A)或不同片段的TGIF來探討交互作用的位置，結果證明了c-Jun N端的磷酸化和TGIF交互作用是必須的而且c-Jun是透過TGIF的N端來進行交互作用。另外也觀察到尿路上皮癌的病人檢體中，TGIF蛋白質有過度表現的現象。未來我們會進一步探討三氧化二砷對TGIF是否有轉譯後修飾的作用也會繼續觀察TGIF在尿路上皮癌細胞中過度表現的意義。

As2O3 has been effectively used to treat acute promyelocytic leukemia and can induce apoptosis or cell cycle arrest in human solid tumors. p21WAF1/ CIP1 (p21) is well known for its function as a cell cycle inhibitor and a tumor suppressor. In our previous results, As2O3 can regulate p21 expression through at least two opposing pathways. One is the EGFR-Ras-Raf-ERK1/2 pathway, which is involved in the As2O3-induced p21 activation. The other is the JNK pathway, which inhibits As2O3-induced p21 activation. We further found that N-terminal domains of c-Jun could be phosphorylated by As2O3-induced JNK activation, bound to TG-interacting factor, and then repressed p21 activation. TG-interacting factor (TGIF) is a transcriptional co-repressor or repressor and functions as a co-repressor through two mechanisms. TGIF directly binds the promoter via the homeodomain to repress the transcription of promoter, or serve as a repressing partner associated with other transcription factor like c-Jun. Therefore, the aim of this study is to study the role of TGIF in As2O3-regulated p21 promoter and in cancer cells. First, we demonstrated that TGIF localizes in nucleus. To confirm the interaction of TGIF with c-Jun in intact cells, we performed fluorescent resonance energy transfer (FRET) assay. FRET is a suitable methodology to confirm protein-protein interactions in intact cells. We constructed fusion proteins of CFP-c-Jun or YFP-TGIF, respectively. After over-expression of these two fusion proteins in human epidermoid carcinoma A431 cells, the protein-protein interaction was observed by FRET in fixed cells. The results showed the occurrence of intermolecular FRET between CFP-c-Jun and YFP-TGIF. We also used fusion proteins of point mutation c-Jun (S63/73A) or various truncations of TGIF protein to study the direct binding domains. The requirement of c-Jun N-terminal phosphorylation for the interaction and the N-terminal domain of TGIF as a binding site to c-Jun were also proved. Furthermore, over-expression of TGIF in urothelial cell carcinoma patient’s specimens was observed by immunohistochemistry. The post-translational modification of TGIF by As2O3 and its expression in urothelial cell carcinoma will be further elucidated in the future.

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