腫瘤易感基因TSG101具有調控基因表現、囊泡運輸及細胞生長與分化等多項生物功能，然而對於調控TSG101基因功能之訊號傳遞路徑並不清楚。根據我們的研究成果發現，不論是在人類正常表皮或是人工培養之類表皮組織中，TSG101之表現強度與分化狀態成正相關；同時我們也發現，在鈣誘導角質細胞分化之過程中，若抑制TSG101在細胞內之表現量，則會同步抑制分化作用之繼續進行，顯示TSG101在分化過程當中扮演著非常重要之角色。另外，藉由下列之實驗結果亦證實了調控TSG101表現之訊號傳遞路徑與 PKC有關；第一、PKC活化劑TPA可正向調控TSG101及keratin 10之表現，第二、同時添加 TPA及PKC抑制劑GF 109203X，能有效阻斷 TPA誘導之TSG101及keratin 10表現。已有研究報導指出TSG101啟動子區域具有Sp1結合位點，我們的分析結果顯示鈣與TPA會刺激Sp1之磷酸化及其轉錄活性，同時也會促進TSG101啟動子之活性，但此作用並不會發生在Sp1結合位點有突變之TSG101啟動子。而GF 109203X阻斷鈣與TPA之作用，同樣也可以阻斷TSG101之啟動子活性，顯示其為PKC依賴性步驟；綜合上述結果可推論，PKC-Sp1之訊號傳遞可藉由促進TSG101之表現而啟動角質細胞之早期分化程序。抑制小鼠細胞內tsg101之功能表現會導致小鼠產生腫瘤，說明了tsg101具有腫瘤抑制劑之功能。然而很多針對人體腫瘤組織之分析，或是細胞特異性tsg101基因剔除小鼠之實驗卻得到不一致之結果。本研究之另一結果發現人類頭頸部鱗狀上皮癌組織之TSG101蛋白質表現情形與p16INK4a及acetylated-histone H4之表現呈負相關(N=98, p<0.001)。因此，進一步使用人類喉癌上皮細胞HEp-2調整改變細胞內TSG101表現量，實驗結果發現TSG101會逆向調控p16INK4a在細胞內之mRNA及蛋白質表現情形。藉由染色質免疫沉澱方法(ChIP)也證實了，TSG101會降低p16INK4a 啟動子區染色質上結合之acetylated-histone H4量，而抑制p16INK4a 啟動子活性。此外，共軛顯微鏡觀察及免疫共沉澱分析之結果皆顯示，TSG101會與HDAC1及SUMO-1在細胞核內形成複合體，而TSG101會呈劑量效應地促進細胞中HDAC1受SUMO修飾之作用，進而提昇其酵素活性；綜合以上實驗結果可知，我們的實驗設計首先證明了TSG101可以促進HDAC1之SUMO-1修飾作用，進而逆向調控p16INK4a之表現，此機制可能為造成頭頸部腫瘤鱗狀上皮癌發展過程中，p16INK4a表現量下降之另一重要原因，並為TSG101參與頭頸部腫瘤進展時，p16INK4a基因表現之epigenetic調控提供有力證據。

Tumor Susceptibility Gene 101, TSG101, exhibits multiple biological functions including the regulation of gene transcription, vesicular trafficking, cellular growth and differentiation. However, the signals involve in the regulation of TSG101 gene functions are unclear. In this present study, we observed congruous TSG101 up-regulation and the differentiation status of keratinocyte in both human foreskin tissue and reconstructed organotypic skin culture. In addition, we found an essential and downstream role of TSG101 in calcium-induced early keratinocyte differentiation since TSG101 siRNA inhibits this process. Our results also indicate a PKC-dependent mechanism is involved based on the following findings. First, a PKC agonist, TPA up-regulates TSG101 and keratin 10 under low calcium condition. Second, co-treatment of keratinocytes with GF 109203X, a PKC inhibitor, blocks TPA-induced TSG101 and keratin 10 up-regulation. Previous report indicates TSG101 gene exhibits a TATA-less and Sp1-containing promoter. Our analysis further shows that both calcium and TPA stimulate phosphorylation of Sp1 and the corresponding TSG101 wild type promoter activity, but not the activity of Sp1 site mutant TSG101 promoter. The co-treatment with GF 109203X blocks the above effects of calcium and TPA, implying that this is a PKC signaling-dependent process. Taken together, these data suggest a PKC-Sp1 signaling is involved in early differentiation switch of keratinocyte through up-regulation of TSG101. Functional inactivation experiment indicates that tsg101 is a tumor suppressor in mouse model. However, many studies using human tumor specimens or conditional knockout mouse give discrepant and contradictive results. Therefore, the role of TSG101 in human cancer remains illusive. Here we demonstrate an inverse correlation between TSG101 and p16INK4a or acetylated- histone H4 protein expression profiles in human head and neck squamous cell carcinomas (HNSCC) (N=98, p<0.001). Using conditioned human HEp2 cells, we confirm that TSG101 negatively modulates p16INK4a expression. Chromatin immunoprecipitation and the subsequent PCR analysis reveal that TSG101 dose-dependently decreases the amount of acetylated histone H4-associated chromatin on p16INK4a promoter. In addition, TSG101 interacts and colocalizes with HDAC1 and SUMO-1 in the nucleus. Furthermore, TSG101 confers a dose-dependent effect on promoting HDAC1 SUMOylation, hence its activity. Taken together, our data demonstrate for the first time that TSG101 can promote SUMO-1 modification of HDAC1, which impacts on down-regulation of p16INK4a gene expression, providing evidence whereby TSG101 might participate in the epigenetic silencing of p16INK4a during the development of HNSCC.

中文總摘要------------------------------------------------ 1
英文總摘要------------------------------------------------ 3
縮寫文字對照表-------------------------------------------- 5
第一章 總緒論
一、腫瘤易感基因TSG101與人類癌症之相關性-------- 8

二、TSG101基因位置與蛋白質功能結構-------------- 8

三、TSG101與細胞週期之關係--------------------- 14
四、角質細胞分化過程中TSG101與p21 waf1/cip1之交互作用--- 18
五、研究方向與目的----------------------------- 20

第二章 角質細胞分化過程中TSG101之功能角色
前言--------------- --------------------------- 23
實驗材料與方法--------------------------------- 25
實驗結果--------------------------------------- 33
結果討論--------------------------------------- 37
圖表------------------------------------------- 40
第三章 TSG101在p16INK4a啟動子染色質重塑之角色探討
前言------------------------------------------- 53
實驗材料與方法--------------------------------- 55
實驗結果--------------------------------------- 67
結果討論--------------------------------------- 72
圖表------------------------------------------- 76
綜合討論------------------------------------------------- 94
參考文獻------------------------------------------------- 96

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