Chibby (或PIGEA-14)為細胞核內致癌基因Beta-catenin 的拮抗因子，然而Chibby在癌腫瘤形成過程中所扮演的角色，與Chibby其生物功能是否必須進入細胞核內後才能執行，皆尚未被探討與證實。 在本研究中，我們將Chibby cDNA轉殖至帶有與綠螢光蛋白或Flag標記融合之的哺乳類細胞表達載體中，再利用細胞轉染與免疫螢光染色觀察外生性Chibby蛋白質在細胞內所在的位置。 在轉染後的子宮頸癌HeLa細胞中，我們發現Chibby蛋白可以在存在於細胞核與細胞質中或細胞質中某些特定胞器(如:高基式體或內質網)，但約有50%以上的細胞，其Chibby蛋白會被送到細胞核內。 而在子宮頸癌細胞株內Chibby基因的過度表現不僅可有效抑制Beta–catenin的轉錄活性，並誘發細胞程式死亡(apoptosis)，也可以抑制癌細胞的增生、移動能力與細胞群落產生能力，經由此實驗結果更加提高Chibby具有腫瘤抑制功能的可能性。另一方面，我們將Chibby基因序列中所預測之核定位序列(nuclear localization sequence; NLS)的位置(Chibby第123-126胺基酸)突變， 再將突變後的基因送入細胞內表現，發現Chibby核定位序列突變蛋白無法進入細胞核內，只能存在於細胞質中，也因此證實Chibby基因的核定位序列位於第123-126個胺基酸上。有趣的是，我們發現Chibby核定位序列突變基因在子宮頸癌細胞中，仍然可以誘發細胞程式死亡、抑制Beta–catenin轉錄活性與抑制腫瘤細胞增生、移動並細胞群落形成能力。 經由基因表達分析實驗，我們還發現Chibby核定位序列突變基因可以藉著在細胞質中與Beta–catenin結合，並阻止Beta–catenin進入細胞核內，來達到抑制Beta–catenin轉錄活性的目的。 综合以上的結果，Chibby基因可以在細胞質與細胞核中，作為 Beta–catenin的拮抗因子，與行使其腫瘤抑制的功能。

ABSTRACT
Chibby (or PIGEA-14) is a novel antagonist of the Beta-catenin pathway in nucleus. However, the tumor-suppressing function of Chibby and the importance of nuclear targeting to the cellular functions of Chibby have not been validated. By fusion of Chibby cDNA with green fluorescent protein (GFP) or Flag-tag, it was found that exogenous Chibby expression was detected in the nucleus as well as cytoplasm of transfected HeLa cells, but with a preferential nuclear localization (more than 50% cells with nuclear Chibby expression). Chibby overexpression significantly abrogated the cellular Beta–catenin activities and induced apoptosis in HeLa cells. Moreover, Chibby gene delivery attenuated the proliferation, migration, and anchorage-independent growth of HeLa cells, supporting the tumor suppressor function of Chibby. Mutation or deletion of the predicted nuclear localization sequence (NLS), at residues 123-126, significantly promoted the cytoplasmic localization of Chibby, indicating residues 123-126 is the NLS domain of Chibby. Interestingly, ecotopic expression of Chibby NLS mutants remained capable of inducing apoptosis and inhibiting Beta–catenin activities in HeLa cells. Besides, overexpression Chibby NLS mutants effectively attenuated the viability, motility and colonies formation of HeLa cells. Expression analysis revealed that Chibby NLS mutants retained Beta-catenin in the cytoplasm and prevented its nuclear entry, thereby inhibiting the Beta-catenin transcriptional activities. In summary, Chibby shuttles between nucleus and cytoplasm, and possesses the functions of tumor suppressor and Beta-catenin antagonist.

Index
Abstract in Chinese----------------------------------------------I
Abstract in English---------------------------------------------III
Contents
Introduction---------------------------------------------------------1
Material and methods---------------------------------------------9
Results---------------------------------------------------------18
Discussion------------------------------------------------------25
References----------------------------------------------------------29
Figures and legends-------------------------------------------------36

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