臺灣博碩士論文加值系統

S100A2是屬於S100鈣離子結合蛋白質家族中的一員，它能藉分子間的雙硫鍵自我形成同型二聚體結合蛋白。在我們先前研究已證實細胞核內S100A2的表現量降低，可提供早期口腔癌病人高危險復發的診斷標記。此外，之前的研究也證實在體外及體內試驗中，S100A2具有抑制腫瘤的效果。為了找出S100A2抑制腫瘤的分子機轉，我們利用並列親和層析法(Tandem Affinity Purification)、二維凝膠電泳(2D-GE)及MOLTI-TOF質譜儀，找尋可以與S100A2作用的新穎蛋白質。首先建立高度穩定表達S100A2的細胞株，從蛋白質混合液中利用並列親和層析法，純化出S100A2蛋白質複合體。其中Enolase 1重複被發現於並列親和層析法純化出的S100A2蛋白質複合體中。我們更進一步使用短載性轉染方式、免疫共沈澱法及免疫螢光顯微鏡觀察，再次證明S100A2與Enolase 1之間的結合。值得注意的是，在正常人類口腔角質細胞株(NOK)、口腔癌前細胞株、口腔癌細胞株及高度穩定表達S100A2的表現載體細胞株中，我們發現S100A2與Enolase 1之間的蛋白質表現是呈現反向相關的情況。我們也發現若高度表達Enolase 1的表現，則會促進口腔癌細胞的生長速率、移動能力、侵襲能力及集落形成能力；若抑制Enolase 1的表現，則會抑制口腔癌細胞的生長速率、移動能力、侵襲能力及集落形成能力，因此推測Enolase 1可能是透過拮抗S100A2扮演促癌基因的角色。此外，利用免疫螢光顯微鏡觀察，我們發現S100A2可以增加Enolase 1在細胞核內的分佈。綜合以上發現，我們還需要更進一步研究S100A2與Enolase 1結合所扮演的功能，及去釐清在口腔癌細胞生長機轉中Enolase 1所扮演的角色。

S100A2 is a member of S100 calcium-binding protein family and can form homodimer by intermolecular disulfide bonds. Our previous studies demonstrated that the loss of nuclear S100A2 may serve as an independent prognostic marker for early-stage oral cancer patients at high risk of recurrence. Moreover, S100A2 manifested an anti-tumor effect in vitro and in vivo. To find out the underlying mechanism responsible for S100A2-meidated anti-tumor effect, we used tandem affinity purification (TAP), 2D-GE and MOLTI-TOF mass spectrometry to fingerprint the interacting partners of S100A2. Following establishing stable clones expressing S100A2 in TAP expression vector, total protein lysate was harvested for subsequent precipitation of S100A2-associated protein complexes. Among the putative S100A2-interacting proteins identified from TAP, Enolase 1 was further confirmed by immunoprecipitation and immunofluorescence microscopy. Intriguingly, the expression of Enolase 1 and S100A2 was inversely related in not only S100A2-stable clones but also oral cancer specimens. Knocking down the expression of Enolase 1 slowed down the growth, migration, invasion and colony formation of oral cancer growth while overexpression of Enolase 1 enhanced the cell the growth, migration, invasion and colony formation in the presence of S100A2, suggesting an oncogenic role of Enolase 1. By using immunofluorescence microscopy, we found S100A2 could enhance nuclear distribution of Enolase 1. The exact function of S100A2 interacting with Enolase 1 and the role of Enolase 1 in oral cancer growth warrant more studies.

目錄
項目 頁數
中文摘要 Ⅵ
英文摘要 Ⅷ
致謝 IX
圖目錄 Ⅳ
英文縮寫對照表 XI
壹､緒論 1
一､什麼是口腔癌 2
二､口腔癌的發生率 3
三､口腔癌的致病因素 4
四､S100家族成員的介紹 5
五､S100A2的介紹及研究近況 13
六､醣類代謝與癌症的介紹 14
七､Enolase 1的介紹及研究近況 16
八､研究動機、目的與策略 17
貳､材料與方法 19
一、 正常口腔上皮細胞 (NOK) 之分離 20
二、 口腔癌細胞來源及資料 20
三、 細胞之培養 (Cell culture) 21
四、 建構pNTAP-S100A2重組質體 (Establishment of pNTAP-S100A2) 22
五、 建立S100A2穩定轉染細胞株 (Establishment of
S100A2 stable transfected cell line) 24
六、 並列親和性層析法 (Tandem affinity purification) 25
七、 膠體染色分析 (Coomassie blue stain assay) 27
八、 西方墨點法 (Western blot) 29
九、 二維電泳與質譜分析 (2D-GE and Mass Spectrometry) 31
十、 免疫沈澱法 (Immunoprecipitation) 36
十一、 免疫螢光染色法 (Immunofluorescence) 38
十二、 免疫組織染色法 (Immunohistochemistry) 40
十三、 建構pcDNA-Enolase 1重組質體 (Establishment of
pcDNA-Enolase 1) 42
十四、 建立Enolase 1穩定轉染細胞株 (Establishment of
Enolase 1 stable transfected cell line) 44
十五、 建立Enolase 1 knocking-down 穩定轉染細胞株 (Establishment
of Enolase 1 knocking-down stable
transfected cell line) 45
十六、 細胞生長速率 (Cell growth rate) 46
十七、 侵襲能力測試 (Invasion ability assay) 46
十八、 傷口癒合實驗 (Wound healing assay) 47
十九、 集落培養分析 (Colony Formation Assay) 48
參､結果 49
一、 尋找與S100A2結合的新穎蛋白質-Enolase 1 50
二、 建立穩定表現Enolase 1及Enolase 1 knocking-down
細胞株 51
三、 觀察S100A2與Enolase 1之間表現量的差異 52
四、 穩定表現Enolase 1及Enolase 1 knocking-down細胞株
之生長速率 (Growth rate) 52
五、 穩定表現Enolase 1及Enolase 1 knocking-down細胞株
之移動速率 (Migration rate) 53
六、 穩定表現Enolase 1及Enolase 1 knocking-down細胞株
之侵襲能力 (Invasion ability) 54
七、 穩定表現Enolase 1及Enolase 1 knocking-down細胞
株之形成集落能力 (Colony forming assay) 55
肆､討論 56
伍､參考文獻 61
陸､圖與表 68

1.Owen, G., J. Berry, and P. Bicknell, Hamartoma of the tongue. J Laryngol Otol, 1993. 107(4): p. 363-7.
2.Silverman, S., Jr., Precancerous lesions and oral cancer in the elderly. Clin Geriatr Med, 1992. 8(3): p. 529-41.
3.Zitsch, R.P., 3rd, Carcinoma of the lip. Otolaryngol Clin North Am, 1993. 26(2): p. 265-77.
4.Silverman, S., Jr., M. Gorsky, and F. Lozada, Oral leukoplakia and malignant transformation. A follow-up study of 257 patients. Cancer, 1984. 53(3): p. 563-8.
5.Isobe, T. and T. Okuyama, The amino-acid sequence of S-100 protein (PAP I-b protein) and its relation to the calcium-binding proteins. Eur J Biochem, 1978. 89(2): p. 379-88.
6.Isobe, T. and T. Okuyama, The amino-acid sequence of the alpha subunit in bovine brain S-100a protein. Eur J Biochem, 1981. 116(1): p. 79-86.
7.Fritz G., H.C., 3D structures of the calcium and zinc binding S100 proteins, in: Messerschmidt A, Bode W, Cygler W (Eds.),. Handbook of Metalloproteins, Wiley, Chichester, 2004: p. 529-540.
8.Marenholz, I., C.W. Heizmann, and G. Fritz, S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature). Biochem Biophys Res Commun, 2004. 322(4): p. 1111-22.
9.Santamaria-Kisiel, L., A.C. Rintala-Dempsey, and G.S. Shaw, Calcium-dependent and -independent interactions of the S100 protein family. Biochem J, 2006. 396(2): p. 201-14.
10.Schafer, B.W. and C.W. Heizmann, The S100 family of EF-hand calcium-binding proteins: functions and pathology. Trends Biochem Sci, 1996. 21(4): p. 134-40.
11.Ridinger, K., et al., S100A13. Biochemical characterization and subcellular localization in different cell lines. J Biol Chem, 2000. 275(12): p. 8686-94.
12.Donato, R., Intracellular and extracellular roles of S100 proteins. Microsc Res Tech, 2003. 60(6): p. 540-51.
13.Lin, J., et al., Inhibition of p53 transcriptional activity by the S100B calcium-binding protein. J Biol Chem, 2001. 276(37): p. 35037-41.
14.Grigorian, M., et al., Tumor suppressor p53 protein is a new target for the metastasis-associated Mts1/S100A4 protein: functional consequences of their interaction. J Biol Chem, 2001. 276(25): p. 22699-708.
15.Kriajevska, M., et al., Metastasis-associated Mts1 (S100A4) protein modulates protein kinase C phosphorylation of the heavy chain of nonmuscle myosin. J Biol Chem, 1998. 273(16): p. 9852-6.
16.Eberhard, D.A., et al., Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclear export signal and by p11 binding. J Cell Sci, 2001. 114(Pt 17): p. 3155-66.
17.Heierhorst, J., et al., Ca2+/S100 regulation of giant protein kinases. Nature, 1996. 380(6575): p. 636-9.
18.Millward, T.A., et al., Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins. Embo J, 1998. 17(20): p. 5913-22.
19.Rambotti, M.G., et al., S100B and S100A1 proteins in bovine retina:their calcium-dependent stimulation of a membrane-bound guanylate cyclase activity as investigated by ultracytochemistry. Neuroscience, 1999. 92(3): p. 1089-101.
20.Wu, T., et al., P11, a unique member of the S100 family of calcium-binding proteins, interacts with and inhibits the activity of the 85-kDa cytosolic phospholipase A2. J Biol Chem, 1997. 272(27): p. 17145-53.
21.Scotto, C., et al., Calcium and S100B regulation of p53-dependent cell growth arrest and apoptosis. Mol Cell Biol, 1998. 18(7): p. 4272-81.
22.Zimmer, D.B., et al., S100A1 regulates neurite organization, tubulin levels, and proliferation in PC12 cells. J Biol Chem, 1998. 273(8): p. 4705-11.
23.Lee, S.W., et al., Down-regulation of a member of the S100 gene family in mammary carcinoma cells and reexpression by azadeoxycytidine treatment. Proc Natl Acad Sci U S A, 1992. 89(6): p. 2504-8.
24.Sakaguchi, M., et al., Relationship between contact inhibition and intranuclear S100C of normal human fibroblasts. J Cell Biol, 2000. 149(6): p. 1193-206.
25.Most, P., et al., S100A1: a regulator of myocardial contractility. Proc Natl Acad Sci U S A, 2001. 98(24): p. 13889-94.
26.Donato, R., S-100 proteins. Cell Calcium, 1986. 7(3): p. 123-45.
27.Donato, R., S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles. Int J Biochem Cell Biol, 2001. 33(7): p. 637-68.
28.Komada, T., et al., Novel specific chemtactic receptor for S100L protein on guinea pig eosinophils. Biochem Biophys Res Commun, 1996. 220(3): p. 871-4.
29.Nagy, N., et al., S100A2, a putative tumor suppressor gene, regulates in vitro squamous cell carcinoma migration. Lab Invest, 2001. 81(4): p. 599-612.
30.Hofmann, M.A., et al., RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell, 1999. 97(7): p. 889-901.
31.Zimmer, D.B. and J.G. Dubuisson, Identification of an S100 target protein: glycogen phosphorylase. Cell Calcium, 1993. 14(4): p. 323-32.
32.Ruse, M., et al., S100A7, S100A10, and S100A11 are transglutaminase substrates. Biochemistry, 2001. 40(10): p. 3167-73.
33.Li, Z.H., et al., Mts1 regulates the assembly of nonmuscle myosin-IIA. Biochemistry, 2003. 42(48): p. 14258-66.
34.Ehlermann, P., et al., Right ventricular upregulation of the Ca(2+) binding protein S100A1 in chronic pulmonary hypertension. Biochim Biophys Acta, 2000. 1500(2): p. 249-55.
35.Remppis, A., et al., Altered expression of the Ca(2+)-binding protein S100A1 in human cardiomyopathy. Biochim Biophys Acta, 1996. 1313(3): p. 253-7.
36.Li, Y., et al., S100 beta increases levels of beta-amyloid precursor protein and its encoding mRNA in rat neuronal cultures. J Neurochem, 1998. 71(4): p. 1421-8.
37.Sheng, J.G., R.E. Mrak, and W.S. Griffin, S100 beta protein expression in Alzheimer disease: potential role in the pathogenesis of neuritic plaques. J Neurosci Res, 1994. 39(4): p. 398-404.
38.Van Eldik, L.J. and W.S. Griffin, S100 beta expression in Alzheimer's disease: relation to neuropathology in brain regions. Biochim Biophys Acta, 1994. 1223(3): p. 398-403.
39.Ryckman, C., et al., Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion. J Immunol, 2003. 170(6): p. 3233-42.
40.Grigorian, M., et al., Effect of mts1 (S100A4) expression on the progression of human breast cancer cells. Int J Cancer, 1996. 67(6): p. 831-41.
41.Hsieh, H.L., et al., Expression analysis of S100 proteins and RAGE in human tumors using tissue microarrays. Biochem Biophys Res Commun, 2003. 307(2): p. 375-81.
42.Guerreiro Da Silva, I.D., et al., S100P calcium-binding protein overexpression is associated with immortalization of human breast epithelial cells in vitro and early stages of breast cancer development in vivo. Int J Oncol, 2000. 16(2): p. 231-40.
43.Wicki, R., et al., Repression of the candidate tumor suppressor gene S100A2 in breast cancer is mediated by site-specific hypermethylation. Cell Calcium, 1997. 22(4): p. 243-54.
44.Engelkamp, D., et al., Six S100 genes are clustered on human chromosome 1q21: identification of two genes coding for the two previously unreported calcium-binding proteins S100D and S100E. Proc Natl Acad Sci U S A, 1993. 90(14): p. 6547-51.
45.Ilg, E.C., B.W. Schafer, and C.W. Heizmann, Expression pattern of S100 calcium-binding proteins in human tumors. Int J Cancer, 1996. 68(3): p. 325-32.
46.Glenney, J.R., Jr., M.S. Kindy, and L. Zokas, Isolation of a new member of the S100 protein family: amino acid sequence, tissue, and subcellular distribution. J Cell Biol, 1989. 108(2): p. 569-78.
47.Lee, S.W., C. Tomasetto, and R. Sager, Positive selection of candidate tumor-suppressor genes by subtractive hybridization. Proc Natl Acad Sci U S A, 1991. 88(7): p. 2825-9.
48.Pedrocchi, M., et al., Expression of Ca(2+)-binding proteins of the S100 family in malignant human breast-cancer cell lines and biopsy samples. Int J Cancer, 1994. 57(5): p. 684-90.
49.Matsubara, D., et al., Differential expression of S100A2 and S100A4 in lung adenocarcinomas: clinicopathological significance, relationship to p53 and identification of their target genes. Cancer Sci, 2005. 96(12): p. 844-57.
50.Lauriola, L., et al., Prognostic significance of the Ca(2+) binding protein S100A2 in laryngeal squamous-cell carcinoma. Int J Cancer, 2000. 89(4): p. 345-9.
51.Nagy, N., et al., The Ca2+-binding S100A2 protein is differentially expressed in epithelial tissue of glandular or squamous origin. Histol Histopathol, 2002. 17(1): p. 123-30.
52.Heighway, J., et al., Expression profiling of primary non-small cell lung cancer for target identification. Oncogene, 2002. 21(50): p. 7749-63.
53.El-Rifai, W., et al., Gastric cancers overexpress S100A calcium-binding proteins. Cancer Res, 2002. 62(23): p. 6823-6.
54.Kyriazanos, I.D., et al., Expression and prognostic significance of S100A2 protein in squamous cell carcinoma of the esophagus. Oncol Rep, 2002. 9(3): p. 503-10.
55.Santin, A.D., et al., Gene expression profiles in primary ovarian serous papillary tumors and normal ovarian epithelium: identification of candidate molecular markers for ovarian cancer diagnosis and therapy. Int J Cancer, 2004. 112(1): p. 14-25.
56.Xia, L., et al., CaN19 expression in benign and malignant hyperplasias of the skin and oral mucosa: evidence for a role in regenerative differentiation. Cancer Res, 1997. 57(14): p. 3055-62.
57.Tan, M., et al., Transcriptional activation of the human S100A2 promoter by wild-type p53. FEBS Lett, 1999. 445(2-3): p. 265-8.
58.Jost, C.A., M.C. Marin, and W.G. Kaelin, Jr., p73 is a simian [correction of human] p53-related protein that can induce apoptosis. Nature, 1997. 389(6647): p. 191-4.
59.Kaghad, M., et al., Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell, 1997. 90(4): p. 809-19.
60.Lapi, E., et al., S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation. Oncogene, 2006. 25(26): p. 3628-37.
61.O., L.K.a.M., Über die enzymatische Umwandlung von Phosphoglyzerinsäure in Brenztraubensäure und Phosphorsäure (Enzymatic transformation of phosphoglyceric acid into pyruvic and phosphoric acid). Biochem. Z., 1934. 273: p. 60–72.
62.Phelps, J.C.a.M.E., Positron emission tomography scanning: current and future applications. Annu. Rev. Med, 2002. 53: p. 89-112.
63.Xu, R.H., et al., Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer Res, 2005. 65(2): p. 613-21.
64.Giallongo, A., et al., Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase. Proc Natl Acad Sci U S A, 1986. 83(18): p. 6741-5.
65.Moscato, S., et al., Surface expression of a glycolytic enzyme, alpha-enolase, recognized by autoantibodies in connective tissue disorders. Eur J Immunol, 2000. 30(12): p. 3575-84.
66.Aaronson, R.M., et al., Non-neuronal enolase is an endothelial hypoxic stress protein. J Biol Chem, 1995. 270(46): p. 27752-7.
67.Subramanian, A. and D.M. Miller, Structural analysis of alpha-enolase. Mapping the functional domains involved in down-regulation of the c-myc protooncogene. J Biol Chem, 2000. 275(8): p. 5958-65.
68.Wang, W., et al., Identification of alpha-enolase as a nuclear DNA-binding protein in the zona fasciculata but not the zona reticularis of the human adrenal cortex. J Endocrinol, 2005. 184(1): p. 85-94.
69.Ito, S., et al., Differential expression of the human alpha-enolase gene in oral epithelium and squamous cell carcinoma. Cancer Sci, 2007. 98(4): p. 499-505.
70.Feo, S., et al., ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1). FEBS Lett, 2000. 473(1): p. 47-52.
71.Altenberg, B. and K.O. Greulich, Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes. Genomics, 2004. 84(6): p. 1014-20.
72.Pancholi, V., Multifunctional alpha-enolase: its role in diseases. Cell Mol Life Sci, 2001. 58(7): p. 902-20.
73.Wu, W., et al., Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry. Clin Exp Metastasis, 2002. 19(4): p. 319-26.
74.Takashima, M., et al., Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma: association with tumor progression as determined by proteomic analysis. Proteomics, 2005. 5(6): p. 1686-92.
75.Tsai, S.T., et al., S100A2, a potential marker for early recurrence in early-stage oral cancer. Oral Oncol, 2005. 41(4): p. 349-57.
76.Tsai, W.C., et al., Cyclooxygenase-2 is involved in S100A2-mediated tumor suppression in squamous cell carcinoma. Mol Cancer Res, 2006. 4(8): p. 539-47.
77.Oda, D. and E. Watson, Human oral epithelial cell culture I. Improved conditions for reproducible culture in serum-free medium. In Vitro Cell Dev Biol, 1990. 26(6): p. 589-95.
78.Lin, S.C., et al., Establishment of OC3 oral carcinoma cell line and identification of NF-kappa B activation responses to areca nut extract. J Oral Pathol Med, 2004. 33(2): p. 79-86.
79.Werner, U. and T. Kissel, In-vitro cell culture models of the nasal epithelium: a comparative histochemical investigation of their suitability for drug transport studies. Pharm Res, 1996. 13(7): p. 978-88.
80.Kamata, N., et al., Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture. Cancer Res, 1986. 46(4 Pt 1): p. 1648-53.
81.Albini, A., et al., A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res, 1987. 47(12): p. 3239-45.
82.Hirama, M., et al., Osteopontin overproduced by tumor cells acts as a potent angiogenic factor contributing to tumor growth. Cancer Lett, 2003. 198(1): p. 107-17.
83.Gimona, M., et al., Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins. J Cell Sci, 1997. 110 ( Pt 5): p. 611-21.