Investigation on the Mechanism(s) Mediating Bcl10 Protein Translocation Between Nucleus and Cytoplasm

胃相關黏膜組織淋巴瘤的發生與Helicobacter pylori的感染相當有關，所以在抗生素的治療下約有50%至70%的病患會痊癒；但仍有相當比例的病患無法以抗生素治療來治癒。而Bcl10蛋白質在核內的不正常表現被認為是這些無法以抗生素治療的病患相當重要的指標之一。以免疫組織染色觀察Bcl10蛋白質主要表現在正常B細胞的細胞質中，而其弁鄍D要是參與T細胞與B細胞受體調控NF-kB活化的路徑。所以在這篇論文裡我們所想要探討的問題是Bcl10是藉由何種機制的調控而進入細胞核。 第一部分，我們分析了包括T細胞淋巴瘤、Burkitt’s淋巴瘤、肝癌、鼻咽癌、子宮頸癌上皮、肺癌、以及乳癌等細胞株內Bcl10的表現及分佈。以北方及西方墨點分析皆發現在這些細胞株內的確有Bcl10蛋白質的表現。在這些細胞株內，Bcl10蛋白質分佈在細胞核與細胞質內，且這樣的細胞內分佈似乎與NF-kB活性的高低無關。 接下來，我們在Bcl10基因後面接上綠螢光蛋白基因(GFP)以便直接在顯微鏡下觀察Bcl10在細胞內的分佈；同時我們也製備Bcl10L41R來研究CARD domain對於調控Bcl10蛋白質在細胞內分佈的影響。Bcl10蛋白質和Bcl10GFP融合蛋白質在細胞內可以活化NF-kB，而Bcl10L41R蛋白質則失去活化NF-kB的能力，這樣的結果與其他的研究報告相符。大量表現Bcl10蛋白質和Bcl10GFP融合蛋白質，會在細胞質內形成絲狀構造；但CARD domain突變的Bcl10L41R和Bcl10L41RGFP融合蛋白質則均勻分佈在細胞核、質內。所以CARD domain對Bcl10活化NF-kB和形成絲狀構造相當重要。另外一方面，我們也在SDS-PAGE的實驗中發現Bcl10蛋白質的位置較Bcl10L41R蛋白質高；而以鹼性去磷酸酵素處理後Bcl10位置高於Bcl10L41R的這種現象就消失，因此Bcl10蛋白質在細胞內會被磷酸化。除此之外，我們也利用放射性磷酸鹽標定實驗證實Bcl10蛋白質的確會在細胞內會被磷酸化，而且CARD domain在Bcl10蛋白質的磷酸化過程中也扮演了相當重要的角色。 Bcl10GFP融合蛋白質在細胞內形成的絲狀構造並不會受到外界的刺激（如TPA、Sodium butyrate、UV、ionizing irradiation、TNF-ㄐ^而改變；但是HBx蛋白質卻可以藉由降低Bcl10GFP融合蛋白質的表現，使Bcl10GFP融合蛋白質在細胞內呈現均勻的分佈。但HBx蛋白質降低Bcl10GFP融合蛋白質表現的機制需進一步的研究探討。此外，我們也成左瑪鼢犍X可誘導穩定表現Bcl10GFP融合蛋白質的Hep G2細胞株。與之前在短暫細胞轉染的實驗中不同，Bcl10GFP融合蛋白質在細胞內並不會形成絲狀構造，而是呈現均勻的細胞內分佈。Bcl10GFP融合蛋白質在誘導細胞株與短暫細胞轉染間顯著不同的表現，我們將會在內文進行探討。Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is highly associated with the infection of Helicobacter pylori. Approximately 50 to 70 % of patients with MALT lymphoma can be cured with anti-H. pylori antibiotic treatment. Aberrant nuclear expression of Bcl10 protein in MALT lymphoma cells was demonstrated to be one of the markers in patients showing no response to the antibiotic therapy. Bcl10 protein was shown to be an essential signaling molecule in TCR- (T cell receptor) or BCR- (B cell receptor) mediated NF-kB activation. Immunohistochemical analysis revealed that Bcl10 protein was mainly distributed in the cytoplasm of normal B cells. We would like to elucidate the molecular mechanism(s) mediating the aberrant nuclear translocation of Bcl10 protein in this study. Firstly, expression level and pattern of distribution of Bcl10 protein were examined in several mammalian cell lines, including T cell lymphoma, Burkitt’s lymphoma, hepatoma, nasopharyngeal carcinoma, cervical carcinoma, lung carcinoma and breast carcinoma. Northern blot and western blot analysis indicated that Bcl10 protein was ubiquitously expressed in all cell lines tested. Bcl10 protein was found to be distributed in both cytoplasm and nucleus irrespective of the NF-kB activity in these cell lines. Secondly, for direct visualization of Bcl10 protein under fluorescence microscope, expression vector with Bcl10 gene fused with green fluorescence protein (GFP) gene was generated. Meanwhile, mutant Bcl10L41R was generated to examine the role of CARD (caspase recruitment domain) domain in Bcl10-mediated effects. Bcl10 as well as Bcl10GFP fusion protein were shown to activate NF-kB activity in a reporter assay. Bcl10L41R mutant failed to activate NF-kB as reported by others. Overexpression of Bcl10 and Bcl10GFP fusion protein resulted in the formation of filamentous structure in the cytoplasm. Bcl10 mutants, Bcl10L41R and Bcl10L41RGFP, showed homogenous distribution in the cytoplasm and nucleus. These results indicated that intact CARD domain was essential for cytoplasmic filaments formation and NF-kB activation. Interestingly, in SDS polyacrylamide gel, wild type Bcl10 protein acted as a slower-migrating molecule as compared to mutant Bcl10L41R. Alkaline phosphatase treatment of wild type Bcl10 protein abrogated the slower-migrating phenomenon, implying that Bcl10 might be a phosphorylated protein. In-vivo phosphate labeling experiment confirmed that Bcl10 was modified by phosphorylation intracellularly and CARD domain was essential in the process. While a lot of stress-inducing agents (such as TPA, sodium butyrate, UV, ionizing irradiation. TNF-a) failed to affect the cytoplasmic filamentous structure of ectopically-expressed Bcl10GFP protein, hepatitis B virus X protein (HBx) reduced the expression level of Bcl10GFP, resulting in a homogenous distribution of Bcl10GFP protein inside the cells. The molecular mechanism by which HBx reduced the expression level of Bcl10GFP awaits further investigation. Also, we have successfully cloned a Hep G2 cell line with inducible expression of Bcl10GFP fusion protein. Different from the cytoplasmic filamentous structure as seen in the transient transfection experiment, Bcl10GFP showed homogenous distribution in the nucleus and cytoplasm in this selected clone. The significance of these findings will be discussed in the text.英文摘要…………………………………………………………………I 中文摘要………………………………………………………………III 目錄……………………………………………………………………IV 壹、 序論…………………………………………………………1 貳、 本論文研究目的……………………………………………11 參、 材料與方法…………………………………………………12 一、 質體的構築…………………………………………………12 二、 細菌轉形 (Transformation)……………………………12 三、 小量質體製備 (Mini-plasmid preparation)…………12 四、 大量質體製備 (Maxi-plasmid preparation)………… 13 五、 細胞培養 (Cell culture)………………………………14 六、 冷凍細胞 (Frozen cells)………………………………15 七、 解凍細胞 (Cell reconstitution)…………………… 15 八、 細胞轉染 (Transfection) …………………………… 15 九、 SDS-PAGE………………………………………………… 15 十、 西方墨點分析 (Western blot analysis)…………… 16 十一、 北方墨點分析(Northern blot analysis)………………17 十二、 Cytospin………………………………………………… 19 十三、 Bcl10免疫螢光染色與Hoechst染色………………………19 十四、 GFP螢光觀察與Hoechst染色………………………………19 十五、 免疫沉澱與去磷酸酵素作用…………………………… 20 十六、 放射性磷酸鹽標定……………………………………… 20 十七、 冷光酵素測定( Luciferase assay) ………………… 21 肆、 結果…………………………………………… …………22 伍、 討論……………………………………………………… 27 陸、 結果圖表………………………………………………… 32 柒、 附錄圖表………………………………………………… 46 捌、 參考文獻………………………………………………… 6