EMP2 (Epithelial membrane protein-2)，為GAS3/PMP22 family的成員，以tetraspan形式表現在細胞膜上之蛋白質，已知可調控細胞生長、分化、細胞凋亡、以及影響細胞與細胞外基質蛋白質的相互作用。EMP2基因在肺癌病人之正常肺組織的表現量較肺腫瘤組織高約2~162倍。從MTT assay可知，當CL1-0細胞EMP2過度表現時，並不影響生長速率，而CL1-5細胞的生長速度有下降的趨勢。經由流式細胞儀分析細胞週期的分佈，發現控制組與實驗組並沒有太大差異。EMP2表現量增加時，在細胞正常貼附的狀況下(foci formation)，不影響CL1-0細胞群落形成的能力，但CL1-5的細胞群落卻有變小的現象；而在細胞不貼附的狀態下(anchorage independent)，EMP2過度表現可造成CL1-0以及CL1-5細胞形成細胞群落的能力下降。在沒有血清的狀況下，過度表現EMP2會引起CL1-0細胞的死亡。活體外試驗(in vitro)我們發現大量表現EMP2會降低CL1-5細胞的侵犯能力，更重要的是，將過度表現EMP2的CL1-5細胞經皮下注射至裸鼠中，沒有發現裸鼠有癌細胞轉移現象。由本研究結果顯示過度表現EMP2可以減緩細胞的生長，促進細胞在無血清狀態下的死亡，抑制腫瘤的侵犯，再加上先前在肺癌病人的組織中，偵測出EMP2在正常組織有較高的表現量，這些發現暗示著EMP2可能經過其抑癌基因的功能而影響肺癌細胞腫瘤形成的能力。

Epithelial membrane protein-2 (EMP2), a member of GAS3/PMP22 family, is a tetraspan protein and has been implicated in the modulation of cell growth, differentiation, cell apoptosis, and cell-extracellular matrix protein interactions. The mRNA level of EMP2 in normal lung was 2- to 162- fold higher than that in lung cancer. Overexpression of EMP2 in CL1-0 cells did not alter cell growth rate examined by MTT assay, but decrease the growth rate of CL1-5 cells compared with the control cells. There were no obvious differences in cell cycle distribution between parental cells and cells with overexpressed EMP2 examined by flow cytometry. Similar to MTS assay, increasing the expression of EMP2 decreased foci formation in CL1-5 cells, but no effect on foci formation in CL1-0 cells. However, overexpression of EMP2 decreased anchorage independent growth in both CL1-0 and CL1-5 cells. EMP2 induced CL1-0 cells death when serum was depleted from the culture medium. Elevation of EMP2 expression in CL1-5 cells reduced invasive ability in vitro. More importantly, when EMP2 overexpressed CL1-5 cells were injected subcutaneously into nude mice, none of the animals developed lung metastasis. In this study, overexpressed EMP2 restricted the cell growth, promoted cell death on serum deprivation and inhibited tumor invasion. All these findings together with the differential expression pattern of EMP2 in lung cancer patients suggest that EMP2 may influence lung tumorigenicity through a functional tumor suppressor phenotype.

壹、 中文摘要 1
貳、 英文摘要 2
參、 縮寫表 3
肆、 前言
一、 肺癌流行病學 4
二、 鑑定參與肺癌形成的基因 4
三、 人類EMP2基因 6
伍、 材料與儀器
一、 酵素 8
二、 商業套組 8
三、 試藥 8
四、 質體 9
五、 細胞株 9
六、 裸鼠品系 9
七、 儀器 9
陸、 實驗方法
一、 質體DNA的萃取 ( Plasmid extract ) 11
二、 建立穩定表現EMP2的細胞株 13
三、 肺癌細胞株培養 17
四、 篩選抗G418的Stable clone 19
五、 細胞染色體DNA的萃取 20
六、 細胞RNA的萃取 20
七、 利用去氧核糖核酸酶除去total RNA裡的染色體DNA 21
八、 反轉錄反應 ( Reverse Transcription；RT ) 22
九、 定量PCR(Real time PCR) 22
十、 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) 分析法 22
十一、 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) 分析法 23
十二、 計數細胞數目 (counting cell numbers) 23
十三、 流式細胞分析 (Flow cytometry) 24
十四、 細胞侵犯試驗 (Modified Boyden chamber) 25
十五、 群落形成試驗 (Colony formation) 25
十六、 軟培養基非附著性生長試驗 (Achorage independent assay) 26
十七、 細胞凋亡試驗 (Apoptosis assay) 26
十八、 活體腫瘤形成試驗 (Tumorigenicity assay) 27
十九、 統計分析 27
柒、 結果
一、 利用西方點墨法偵測穩定表現EMP2的細胞株 28
二、 利用RT-real time PCR定量細胞株中EMP2 mRNA過度表現的效果 29
三、 利用MTT assay觀察過度表現EMP2對CL1-0細胞生長的影響 30
四、 利用MTS assay觀察過度表現EMP2對CL1-5細胞生長的影響 31
五、 以流式細胞儀分析過度表現EMP2對細胞週期的影響 31
六、 利用細胞群落形成試驗(foci colony formation assay)觀察EMP2 overexpression對細胞群落形成的影響 32
七、 利用細胞不貼附試驗(anchorage independent assay)觀察EMP2過度表現對細胞群落形成的影響 33
八、 過度表現EMP2基因在無血清之壓力下對細胞凋亡的影響 35
九、 CL1-5細胞中EMP2基因過度表現對細胞侵犯能力的影響 35
十、 CL1-5細胞中EMP2基因過度表現對腫瘤形成的影響 36
捌、 討論
一、 N21與EMP2之間的關係 38
二、 過度表現的效果 39
三、 人類EMP2基因於癌症的角色 39
四、 抑癌基因的探討 40
五、 人類EMP2基因於生理功能的角色 44
玖、 圖目錄
圖一、 利用西方點墨法偵測穩定表現EMP2的CL1-0細胞株 46
圖二、 利用西方點墨法偵測穩定表現EMP2的CL1-5細胞株 47
圖三、 轉殖EMP2之CL1-0細胞株中，EMP2 mRNA過度表現的情形 48
圖四、 轉殖EMP2之CL1-5細胞株中，EMP2 mRNA過度表現的情形 49
圖五、 利用MTT assay 觀察EMP2 過度表現對CL1-0細胞生長的影響 50
圖六、 利用MTS assay 觀察EMP2 過度表現對CL1-5細胞生長的影響 51
圖七、 經流式細胞儀分析過度表現EMP2對CL1-0細胞週期的影響 52

圖八、 經流式細胞儀分析過度表現EMP2對CL1-5細胞週期的影響 53
圖九、 利用細胞群落形成試驗(foci colony formation assay)觀察EMP2過度表現對CL1-0細胞群落形成的影響 54
圖十、 利用細胞群落形成試驗(foci colony formation assay)觀察EMP2過度表現對CL1-5細胞群落形成的影響 55
圖十一、 利用細胞不貼附試驗(anchorage independent assay)
觀察EMP2 過度表現對CL1-0細胞群落形成的影響 56
圖十二、 利用細胞不貼附試驗(anchorage independent assay)觀察EMP2 過度表現對CL1-5細胞群落形成的影響 57
圖十三、 在細胞不貼附試驗(anchorage independent assay)，CL1-5細胞中EMP2過度表現之細胞群落大於250 μm所佔之比例 58
圖十四、 經流式細胞儀分析過度表現EMP2在無血清的壓力下對CL1-0細胞週期的影響 59
圖十五、 經流式細胞儀分析過度表現EMP2在無血清的壓力下對CL1-5細胞週期的影響 60
圖十六、 CL1-5細胞中EMP2基因過度表現對細胞侵犯能力
的影響 61
圖十七、 CL1-5細胞中EMP2基因過度表現對腫瘤形成的影響 62
圖十八、 CL1-5細胞株所誘發的腫瘤形態 63
圖十九、 裸鼠腫瘤的生成率 64
拾、 表目錄
表一、 引子序列 65
表二、 過度表現EMP2的細胞株相對於18s rRNA表現量的相關性 66
拾壹、 附圖
附圖一、 EMP2過度表現質體的構築 68
拾貳、 參考文獻 69

Adams, J.M. and Cory, S. (1998) The Bcl-2 protein family: arbiters of cell survival. Science, 281, 1322-1326.
Ben-Porath, I., Kozak, C.A. and Benvenisty, N. (1998) Chromosomal mapping of Tmp (Emp1), Xmp (Emp2), and Ymp (Emp3), genes encoding membrane proteins related to Pmp22. Genomics, 49, 443-447.
Berditchevski, F. (2001) Complexes of tetraspanins with integrins: more than meets the eye. J Cell Sci, 114, 4143-4151.
Chu, Y.W., Yang, P.C., Yang, S.C., Shyu, Y.C., Hendrix, M.J., Wu, R. and Wu, C.W. (1997) Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol, 17, 353-360.
Dedhar, S. (2000) Cell-substrate interactions and signaling through ILK. Curr Opin Cell Biol, 12, 250-256.
Dong, J.T., Lamb, P.W., Rinker-Schaeffer, C.W., Vukanovic, J., Ichikawa, T., Isaacs, J.T. and Barrett, J.C. (1995) KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science, 268, 884-886.
Doree, M. and Galas, S. (1994) The cyclin-dependent protein kinases and the control of cell division. Faseb J, 8, 1114-1121.
Evans, W.H. and Martin, P.E. (2002) Gap junctions: structure and function (Review). Mol Membr Biol, 19, 121-136.
Fabbretti, E., Edomi, P., Brancolini, C. and Schneider, C. (1995) Apoptotic phenotype induced by overexpression of wild-type gas3/PMP22: its relation to the demyelinating peripheral neuropathy CMT1A. Genes Dev, 9, 1846-1856.
Fernandez, A., Udagawa, T., Schwesinger, C., Beecken, W., Achilles-Gerte, E., McDonnell, T. and D''Amato, R. (2001) Angiogenic potential of prostate carcinoma cells overexpressing bcl-2. J Natl Cancer Inst, 93, 208-213.
Forbes, A., Wadehra, M., Mareninov, S., Morales, S., Shimazaki, K., Gordon, L.K. and Braun, J. (2007) The tetraspan protein EMP2 regulates expression of caveolin-1. J Biol Chem.
Fu, S., Rogowsky, P., Nover, L. and Scanlon, M.J. (2006) The maize heat shock factor-binding protein paralogs EMP2 and HSBP2 interact non-redundantly with specific heat shock factors. Planta, 224, 42-52.
Galbiati, F., Razani, B. and Lisanti, M.P. (2001a) Emerging themes in lipid rafts and caveolae. Cell, 106, 403-411.
Galbiati, F., Volonte, D., Liu, J., Capozza, F., Frank, P.G., Zhu, L., Pestell, R.G. and Lisanti, M.P. (2001b) Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism. Mol Biol Cell, 12, 2229-2244.
Giehl, K. (2005) Oncogenic Ras in tumour progression and metastasis. Biol Chem, 386, 193-205.
Gupta, S. and Knowlton, A.A. (2005) HSP60, Bax, apoptosis and the heart. J Cell Mol Med, 9, 51-58.
Jankowski, S.A., Mitchell, D.S., Smith, S.H., Trent, J.M. and Meltzer, P.S. (1994) SAS, a gene amplified in human sarcomas, encodes a new member of the transmembrane 4 superfamily of proteins. Oncogene, 9, 1205-1211.
Jetten, A.M. and Suter, U. (2000) The peripheral myelin protein 22 and epithelial membrane protein family. Prog Nucleic Acid Res Mol Biol, 64, 97-129.
Kedar, D., Baker, C.H., Killion, J.J., Dinney, C.P. and Fidler, I.J. (2002) Blockade of the epidermal growth factor receptor signaling inhibits angiogenesis leading to regression of human renal cell carcinoma growing orthotopically in nude mice. Clin Cancer Res, 8, 3592-3600.
Lavoie, J.N., L''Allemain, G., Brunet, A., Muller, R. and Pouyssegur, J. (1996) Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem, 271, 20608-20616.
Leitinger, B. and Hogg, N. (2002) The involvement of lipid rafts in the regulation of integrin function. J Cell Sci, 115, 963-972.
Liehr, T., Kuhlenbaumer, G., Wulf, P., Taylor, V., Suter, U., Van Broeckhoven, C., Lupski, J.R., Claussen, U. and Rautenstrauss, B. (1999) Regional localization of the human epithelial membrane protein genes 1, 2, and 3 (EMP1, EMP2, EMP3) to 12p12.3, 16p13.2, and 19q13.3. Genomics, 58, 106-108.
Liu, H.S., Chen, C.Y., Lee, C.H. and Chou, Y.I. (1998) Selective activation of oncogenic Ha-ras-induced apoptosis in NIH/3T3 cells. Br J Cancer, 77, 1777-1786.
Lobsiger, C.S., Magyar, J.P., Taylor, V., Wulf, P., Welcher, A.A., Program, A.E. and Suter, U. (1996) Identification and characterization of a cDNA and the structural gene encoding the mouse epithelial membrane protein-1. Genomics, 36, 379-387.
London, E. and Brown, D.A. (2000) Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts). Biochim Biophys Acta, 1508, 182-195.
Lukas, J., Bartkova, J. and Bartek, J. (1996) Convergence of mitogenic signalling cascades from diverse classes of receptors at the cyclin D-cyclin-dependent kinase-pRb-controlled G1 checkpoint. Mol Cell Biol, 16, 6917-6925.
Maecker, H.T., Todd, S.C. and Levy, S. (1997) The tetraspanin superfamily: molecular facilitators. Faseb J, 11, 428-442.
Melkonian, K.A., Ostermeyer, A.G., Chen, J.Z., Roth, M.G. and Brown, D.A. (1999) Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J Biol Chem, 274, 3910-3917.
Musgrove, E.A. (2006) Cyclins: roles in mitogenic signaling and oncogenic transformation. Growth Factors, 24, 13-19.
Rak, J., Yu, J.L., Kerbel, R.S. and Coomber, B.L. (2002) What do oncogenic mutations have to do with angiogenesis/vascular dependence of tumors? Cancer Res, 62, 1931-1934.
Sawada, S., Yoshimoto, M., Odintsova, E., Hotchin, N.A. and Berditchevski, F. (2003) The tetraspanin CD151 functions as a negative regulator in the adhesion-dependent activation of Ras. J Biol Chem, 278, 26323-26326.
Shaw, R.J. and Cantley, L.C. (2006) Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature, 441, 424-430.
Sherr, C.J. (1996) Cancer cell cycles. Science, 274, 1672-1677.
Sherr, C.J. (2000) The Pezcoller lecture: cancer cell cycles revisited. Cancer Res, 60, 3689-3695.
Sinenko, S.A. and Mathey-Prevot, B. (2004) Increased expression of Drosophila tetraspanin, Tsp68C, suppresses the abnormal proliferation of ytr-deficient and Ras/Raf-activated hemocytes. Oncogene, 23, 9120-9128.
Sleiman, R.J., Catchpoole, D.R. and Stewart, B.W. (1998) Drug-induced death of leukaemic cells after G2/M arrest: higher order DNA fragmentation as an indicator of mechanism. Br J Cancer, 77, 40-50.
Taylor, V. and Suter, U. (1996) Epithelial membrane protein-2 and epithelial membrane protein-3: two novel members of the peripheral myelin protein 22 gene family. Gene, 175, 115-120.
Taylor, V., Welcher, A.A., Program, A.E. and Suter, U. (1995) Epithelial membrane protein-1, peripheral myelin protein 22, and lens membrane protein 20 define a novel gene family. J Biol Chem, 270, 28824-28833.
Udagawa, T., Fernandez, A., Achilles, E.G., Folkman, J. and D''Amato, R.J. (2002) Persistence of microscopic human cancers in mice: alterations in the angiogenic balance accompanies loss of tumor dormancy. Faseb J, 16, 1361-1370.
Wadehra, M., Dayal, M., Mainigi, M., Ord, T., Iyer, R., Braun, J. and Williams, C.J. (2006a) Knockdown of the tetraspan protein epithelial membrane protein-2 inhibits implantation in the mouse. Dev Biol, 292, 430-441.
Wadehra, M., Forbes, A., Pushkarna, N., Goodglick, L., Gordon, L.K., Williams, C.J. and Braun, J. (2005) Epithelial membrane protein-2 regulates surface expression of alphavbeta3 integrin in the endometrium. Dev Biol, 287, 336-345.
Wadehra, M., Goodglick, L. and Braun, J. (2004) The tetraspan protein EMP2 modulates the surface expression of caveolins and glycosylphosphatidyl inositol-linked proteins. Mol Biol Cell, 15, 2073-2083.
Wadehra, M., Iyer, R., Goodglick, L. and Braun, J. (2002) The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion. J Biol Chem, 277, 41094-41100.
Wadehra, M., Natarajan, S., Seligson, D.B., Williams, C.J., Hummer, A.J., Hedvat, C., Braun, J. and Soslow, R.A. (2006b) Expression of epithelial membrane protein-2 is associated with endometrial adenocarcinoma of unfavorable outcome. Cancer, 107, 90-98.
Wadehra, M., Su, H., Gordon, L.K., Goodglick, L. and Braun, J. (2003a) The tetraspan protein EMP2 increases surface expression of class I major histocompatibility complex proteins and susceptibility to CTL-mediated cell death. Clin Immunol, 107, 129-136.
Wadehra, M., Sulur, G.G., Braun, J., Gordon, L.K. and Goodglick, L. (2003b) Epithelial membrane protein-2 is expressed in discrete anatomical regions of the eye. Exp Mol Pathol, 74, 106-112.
Wang, C.X., Wadehra, M., Fisk, B.C., Goodglick, L. and Braun, J. (2001) Epithelial membrane protein 2, a 4-transmembrane protein that suppresses B-cell lymphoma tumorigenicity. Blood, 97, 3890-3895.
Weber, J.D., Raben, D.M., Phillips, P.J. and Baldassare, J.J. (1997) Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase. Biochem J, 326 ( Pt 1), 61-68.
Wilson, H.L., Wilson, S.A., Surprenant, A. and North, R.A. (2002) Epithelial membrane proteins induce membrane blebbing and interact with the P2X7 receptor C terminus. J Biol Chem, 277, 34017-34023.
Wu, Z.Z., Chien, C.M., Yang, S.H., Lin, Y.H., Hu, X.W., Lu, Y.J., Wu, M.J. and Lin, S.R. (2006) Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells. Mol Cell Biochem, 292, 99-105.
Yang, J.J., Kang, J.S. and Krauss, R.S. (1998) Ras signals to the cell cycle machinery via multiple pathways to induce anchorage-independent growth. Mol Cell Biol, 18, 2586-2595.