中文摘要
肺癌是癌症死亡率排名之首，約有85%的患者屬於非小型細胞肺癌，同時只有約13%的肺癌病人可達到五年以上的存活率。GCIP 是一個Grap2 and Cyclin-D Interacting Protein，此蛋白在結構上屬於helix-loop-helix leucine zipper protein的一種。文獻上報導此蛋白在高分化組織上有較高表現量，同時也發現到在乳癌、肝癌、大腸癌上GCIP的表現量都較低，原因由於此蛋白質會去抑制CyclinD1蛋白的表現量，進而減緩細胞增生速率以及細胞週期的進行。因此我們在低表現GCIP的高侵犯性肺癌細胞中建立過度表現GCIP的穩定細胞株，同時觀察到GCIP會使細胞增生速率下降並且使細胞停留在G1時期，並且使得部分細胞走向老化的型態，因此為了更加確定GCIP的角色，先前我們利用mammalian two hybrid偵測到GCIP會透過HLH domain和Id1具有交互作用，而Id1 全名為inhibitor of differentiation or DNA binding，同樣屬於 helix-loop-helix protein。更進一步我利用免疫沉澱法以及核質分離實驗證實此兩蛋白具有交互作用不論是外送又或者是在穩定表現GCIP的肺癌細胞株當中。更重要的是這兩個蛋白質交互作用的位置位於細胞核當中。而先前文獻當中提及Id1在大部分惡性癌症中表現量較高，Id1又以肺癌當中表現量高於其它癌症，由於98%肺癌病人患者中發現BMP2蛋白大量表現，此蛋白可活化Id1基因，進而使得蛋白表現量上升。同時超表現Id1也會使得腫瘤細胞更具有侵犯能力且癒後較差，原因之一便是透過活化MAPK下游訊息傳遞路徑，因此透過過度表現GCIP藉由交互作用的方式可以抑制Id1所活化和腫瘤有相關的路徑，我們更進一步在細胞層級的研究上利用過度表現GCIP觀察到有和內生性的Id1交互作用，進而影響到Id1所活化的下游MAPK之訊息路徑，透過此路徑影響Id1所造成細胞轉移的功能。另外在動物體內的活體分析發現到過度表現GCIP的老鼠腫瘤大小較對照組小，且具有顯著差異(P = 0.0023)。在此研究中我首度證實GCIP可以透過和Id1的交互作用進而影響Id1所活化和細胞轉移相關的訊息路徑，因此可以透過超表現GCIP的方法來達到降低Id1所造成的癌症惡化情形，藉此可以運用在治療癌症的新標的。

Abstract
Lung cancer is the leading cause of cancer-related deaths, and approximately 85% of lung cancer cases are classified as non-small-cell lung cancer (NSCLC). Currently, prognosis for advanced and metastatic NSCLC is relatively poor in the medical field, and the 5-year survival rates are less than 13%. GCIP, the D-type cyclin–interacting protein 1, is a human homologue of the MAID protein that reduces cell growth and cell cycle inhibition. It is a helix-loop-helix leucine zipper protein that is mainly expressed in terminally differentiated tissues and was observed to have lower expression levels in human breast, prostate, and colon tumor tissues. Our previous study revealed that GCIP through HLH domain is able to interact with Id1 (inhibitor of differentiation or DNA binding protein 1) in HEK293T cells by mammalian two hybrid. Since overexpression of Id1 is associated with an aggressive phenotype and poor clinical outcome in lung cancer and ectopic Id1 expression has activated cell migration and invasion in NSCLC have been reported, it is interesting to investigate whether the GCIP-Id1 interaction occurs in lung cancer and whether GCIP exhibits effects counteracting those of Id1 in NSCLC. In this study, we demonstrated that GCIP is able to interact with Id1 in 293T cells cotransfected with c-Myc-GCIP and Flag-Id1 by immunoprecipitation assays. To examine whether GCIP functions as a tumor suppressor in lung cancer, H1299 stably overexpressing GCIP cells, G6 and G9, were established. Cellular fractions and immunoprecipitation assays revealed that Id1-GCIP complex was present mainly in the nucleus of G6 cells. Confocal microscope analysis also confirmed that Id1 and GCIP were co-localized in stable cells. Stable cells exhibited a significant reduction in cell proliferation and colony formation ability compared to its mock-transfected, indicating that GCIP expression level was negatively associated with cell proliferation and tumor cell colony formation ability. Since stable cells has senescence-like morphology with an enlarged and flattened shape, and endogenous SA-βgal activity was higher in stable cells, we propose that overexpression of GCIP resulted in cell-cycle arrest and induced cellular senescence in stable cells. In addition, our data also revealed that activated Id1-induced MAPK pathway was suppressed by GCIP produced in stable cells. Furthermore, in vivo tumorigenesis analyses revealed that tumor sizes from GCIP overexpression cells were significantly smaller than those from control cells (P = 0.0023). Here, for the first time, we propose that activated Id-mediated signaling pathways involved in tumor cell migration, invasion, and metastasis could be suppressed by GCIP overexpression.

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蔡宗霖學長(2005)探討GCIP與ID蛋白質之間的交互作用及其生理影響：57
顏郁蓁學姊(2008)探討GCIP蛋白在乳癌中抑制腫瘤生成所扮演的角色：69