肝細胞生長因子 (HGF) 是一個多功能的促細胞分裂劑，透過與c-Met受體的結合引起訊號傳遞活化細胞內的相關調控蛋白，進而刺激細胞增生、提高細胞活動力以及影響血管新生與形態發生。HGF與c-Met的過度表達是腫瘤惡性轉化與轉移的重要特徵。而抑制HGF與其受體c-Met結合進而阻斷所引發的訊息傳遞路徑，或許可以為癌症治療提供另一項有力途徑。因此，本研究以HGF的kringle domain為單位設計了四個重組基因片段 (NK1、NK2、NK3和NK4)，將這些片段分別組裝至真核細胞的表達載體，轉染進人類乳癌細胞株MDA MB 435s，並利用含有抗生素G418的選擇性培養基篩選獲得穩定轉染細胞株，進而探討重組蛋白rNKs在乳癌細胞中的表達與影響。活體外細胞增生實驗結果顯示，重組蛋白rNKs的表達顯著抑制了細胞生長。而由傷痕癒合實驗結果得知，相較於對照組的未轉染正常細胞與GFP空載體轉染細胞，NK轉染細胞的移行能力明顯被抑制。本研究結果指出，重組蛋白rNKs對人類乳癌細胞的增生與移行具有抑制效果，值得進一步進行活體內實驗探討其對於乳癌治療之潛力。

Hepatocyte growth factor (HGF) is a multifunctional mitogen, stimulating cell proliferation, motility, angiogenesis and morphogenesis via activating its receptor, c-Met tyrosine kinase. Overexpression of HGF and c-Met has been shown as a characteristic of cancer transformation and metastasis. Inhibition of HGF/c-Met signaling may abrogate the malignant and metastatic states of cancer cells and offer a useful therapeutic approach for treating cancers. Thus with the aim of creating inhibitors to HGF-cMet signaling, we constructed plasmids containing truncated N-terminus of HGF, NK1, NK2, NK3 and NK4, respectively, and transfected into MDA MB 435S cells by electroporation. After selection with antibiotics, stable transfectants were obtained. Proliferation assay showed that the truncated NKs significantly inhibited the growth of the cells. Moreover, wound healing assay showed that migration of the NK-transfected cells were also significantly inhibited in comparison with GFP-transfected and nontransfected cells. These results therefore suggest that truncated NKs may be good inhibitors to HGF/c-Met signaling in the proliferation and migration of human breast cancer cells in vitro. In the future, the in vivo animal model is needed to be carried out to further clarify the clinical values of truncated NKs for application to cancer therapy.

Abstract •••••••••••••••••••••••••••• 1
摘要••••••••••••••••••••••••••••• 2
Abbreviations•••••••••••••••••••••••••• 3
Chapter 1 Introduction•••••••••••••••••••••• 4
Chapter 2 Material and Methods••••••••••••••••• 11
2.1 The sources of hHGF cDNA, vectors, cell lines and reagents•••••• 11
2.2 Cloning of truncated HGFs, NK1, NK2, NK3 and NK4•••••••• 12
2.2.1 Preparation of template DNA fragment for PCR••••••••••12
2.2.2 Amplification of truncated HGF genes by PCR••••••••••12
2.2.3 TA cloning•••••••••••••••••••••••• 14
2.2.4 Purification of plasmids••••••••••••••••••• 15
2.2.5 Identification of pGEMT/NK1~4 plasmids••••••••••• 15
2.2.6 Sub-cloning of truncated HGF genes to the expression vector•••• 16
2.3 Establishment and confirmation of stable transfectants••••••••16
2.3.1 Cell and culture condition•••••••••••••••••• 16
2.3.2 Transfection and selection of stable tranfectants••••••••••17
2.3.3 Isolation of genomic DNA and PCR•••••••••••••• 18
2.3.4 Western blotting•••••••••••••••••••••• 19
2.5 In vitro cell assays of stable transfectants•••••••••••••20
2.5.1 Cells and culture conditions••••••••••••••••••20
2.5.2 In vitro cell proliferation assay•••••••••••••••• 20
2.5.3 In vitro wound healing assay••••••••••••••••• 21
2.6 Statistical analysis•••••••••••••••••••••• 21
Chapter 3 Results•••••••••••••••••••••••• 22
3.1 Cloning of truncated HGF genes, NK1, NK2, NK3 and NK4••••• 22
3.2 Confirmation of stable transfectants expressing truncated HGFs•••• 22
3.3 In vitro cell proliferation assay••••••••••••••••• 24
3.4 In vitro wound healing assay••••••••••••••••• 25
Chapter 4 Discussion•••••••••••••••••••••• 28
References•••••••••••••••••••••••••• 36

Beviglia, L., Matsumoto, K., Lin, C. S., Ziober, B. L., and Kramer, R. H. (1997). Expression of the c-Met/HGF receptor in human breast carcinoma: correlation with tumor progression. Int J Cancer 74(3): 301-9.
Birchmeier, C., Birchmeier, W., Gherardi, E., and Vande Woude, G. F. (2003). Met, metastasis, motility and more. Nat Rev Mol Cell Biol 4(12): 915-25.
Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M., Kmiecik, T. E., Vande Woude, G. F., and Aaronson, S. A. (1991). Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251(4995): 802-4.
Brinkmann, V., Foroutan, H., Sachs, M., Weidner, K. M., and Birchmeier, W. (1995). Hepatocyte growth factor/scatter factor induces a variety of tissue-specific morphogenic programs in epithelial cells. J Cell Biol 131(6 Pt 1): 1573-86.
Bussolino, F., Di Renzo, M. F., Ziche, M., Bocchietto, E., Olivero, M., Naldini, L., Gaudino, G., Tamagnone, L., Coffer, A., and Comoglio, P. M. (1992). Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 119(3): 629-41.
Chan, A. M., Rubin, J. S., Bottaro, D. P., Hirschfield, D. W., Chedid, M., and Aaronson, S. A. (1991). Identification of a competitive HGF antagonist encoded by an alternative transcript. Science 254(5036): 1382-5.
Cioce, V., Csaky, K. G., Chan, A. M., Bottaro, D. P., Taylor, W. G., Jensen, R., Aaronson, S. A., and Rubin, J. S. (1996). Hepatocyte growth factor (HGF)/NK1 is a naturally occurring HGF/scatter factor variant with partial agonist/antagonist activity. J Biol Chem 271(22): 13110-5.
Corso, S., Comoglio, P. M., and Giordano, S. (2005). Cancer therapy: can the challenge be MET? Trends Mol Med 11(6): 284-92.
Danilkovitch-Miagkova, A., and Zbar, B. (2002). Dysregulation of Met receptor tyrosine kinase activity in invasive tumors. J Clin Invest 109(7): 863-7.
Di Renzo, M. F., Olivero, M., Giacomini, A., Porte, H., Chastre, E., Mirossay, L., Nordlinger, B., Bretti, S., Bottardi, S., and Giordano, S. (1995). Overexpression and amplification of the met/HGF receptor gene during the progression of colorectal cancer. Clin Cancer Res 1(2): 147-54.
Di Renzo, M. F., Olivero, M., Katsaros, D., Crepaldi, T., Gaglia, P., Zola, P., Sismondi, P., and Comoglio, P. M. (1994). Overexpression of the Met/HGF receptor in ovarian cancer. Int J Cancer 58(5): 658-62.
Fan, S., Ma, Y. X., Gao, M., Yuan, R. Q., Meng, Q., Goldberg, I. D., and Rosen, E. M. (2001). The multisubstrate adapter Gab1 regulates hepatocyte growth factor (scatter factor)-c-Met signaling for cell survival and DNA repair. Mol Cell Biol 21(15): 4968-84.
Ferracini, R., Di Renzo, M. F., Scotlandi, K., Baldini, N., Olivero, M., Lollini, P., Cremona, O., Campanacci, M., and Comoglio, P. M. (1995). The Met/HGF receptor is over-expressed in human osteosarcomas and is activated by either a paracrine or an autocrine circuit. Oncogene 10(4): 739-49.
Garcia-Guzman, M., Dolfi, F., Zeh, K., and Vuori, K. (1999). Met-induced JNK activation is mediated by the adapter protein Crk and correlates with the Gab1 - Crk signaling complex formation. Oncogene 18(54): 7775-86.
Gherardi, E., Sandin, S., Petoukhov, M. V., Finch, J., Youles, M. E., Ofverstedt, L. G., Miguel, R. N., Blundell, T. L., Vande Woude, G. F., Skoglund, U., and Svergun, D. I. (2006). Structural basis of hepatocyte growth factor/scatter factor and MET signalling. Proc Natl Acad Sci U S A 103(11): 4046-51.
Giordano, S., Di Renzo, M. F., Narsimhan, R. P., Cooper, C. S., Rosa, C., and Comoglio, P. M. (1989). Biosynthesis of the protein encoded by the c-met proto-oncogene. Oncogene 4(11): 1383-8.
Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Miyazaki, H., Hashimoto, S., and Daikuhara, Y. (1988). Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. J Clin Invest 81(2): 414-9.
Guerin, C., Luddy, C., Abounader, R., Lal, B., and Laterra, J. (2000). Glioma inhibition by HGF/NK2, an antagonist of scatter factor/hepatocyte growth factor. Biochem Biophys Res Commun 273(1): 287-93.
Hara, H., Nakae, Y., Sogabe, T., Ihara, I., Ueno, S., Sakai, H., Inoue, H., Shimizu, S., Nakamura, T., and Shimizu, N. (1993). Structural study of the N-linked oligosaccharides of hepatocyte growth factor by two-dimensional sugar mapping. J Biochem 114(1): 76-82.
Hartmann, G., Naldini, L., Weidner, K. M., Sachs, M., Vigna, E., Comoglio, P. M., and Birchmeier, W. (1992). A functional domain in the heavy chain of scatter factor/hepatocyte growth factor binds the c-Met receptor and induces cell dissociation but not mitogenesis. Proc Natl Acad Sci U S A 89(23): 11574-8.
Hirao, S., Yamada, Y., Koyama, F., Fujimoto, H., Takahama, Y., Ueno, M., Kamada, K., Mizuno, T., Maemondo, M., Nukiwa, T., Matsumoto, K., Nakamura, T., and Nakajima, Y. (2002). Tumor suppression effect using NK4, a molecule acting as an antagonist of HGF, on human gastric carcinomas. Cancer Gene Ther 9(8): 700-7.
Hirono, S. (1988). [Characteristics and partial purification of human hepatocyte growth factor in plasma from patients with fulminant hepatic failure]. Fukuoka Igaku Zasshi 79(11): 839-50.
Jeffers, M., Rong, S., and Vande Woude, G. F. (1996). Enhanced tumorigenicity and invasion-metastasis by hepatocyte growth factor/scatter factor-met signalling in human cells concomitant with induction of the urokinase proteolysis network. Mol Cell Biol 16(3): 1115-25.
Kubota, T., Fujiwara, H., Amaike, H., Takashima, K., Inada, S., Atsuji, K., Yoshimura, M., Matsumoto, K., Nakamura, T., and Yamagishi, H. (2004). Reduced HGF expression in subcutaneous CT26 tumor genetically modified to secrete NK4 and its possible relation with antitumor effects. Cancer Sci 95(4): 321-7.
Lamorte, L., Kamikura, D. M., and Park, M. (2000). A switch from p130Cas/Crk to Gab1/Crk signaling correlates with anchorage independent growth and JNK activation in cells transformed by the Met receptor oncoprotein. Oncogene 19(52): 5973-81.
Lee, H. S., Huang, G. T., Sheu, J. C., Chiou, L. L., Horng, M. C., Lai, M. Y., Chen, D. S., and Lee, S. C. (1995). Lack of critical domains in the beta-chain of hepatocyte growth factor. Biochem Biophys Res Commun 210(3): 1017-24.
Lokker, N. A., and Godowski, P. J. (1993). Generation and characterization of a competitive antagonist of human hepatocyte growth factor, HGF/NK1. J Biol Chem 268(23): 17145-50.
Lokker, N. A., Mark, M. R., Luis, E. A., Bennett, G. L., Robbins, K. A., Baker, J. B., and Godowski, P. J. (1992). Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding. EMBO J 11(7): 2503-10.
Ma, P. C., Maulik, G., Christensen, J., and Salgia, R. (2003). c-Met: structure, functions and potential for therapeutic inhibition. Cancer Metastasis Rev 22(4): 309-25.
Matsumoto, K., and Nakamura, T. (2003). NK4 (HGF-antagonist/angiogenesis inhibitor) in cancer biology and therapeutics. Cancer Sci 94(4): 321-7.
Matsumoto, K., and Nakamura, T. (2005). Mechanisms and significance of bifunctional NK4 in cancer treatment. Biochem Biophys Res Commun 333(2): 316-27.
Miyazawa, K., Kitamura, A., Naka, D., and Kitamura, N. (1991). An alternatively processed mRNA generated from human hepatocyte growth factor gene. Eur J Biochem 197(1): 15-22.
Montesano, R., Soriano, J. V., Malinda, K. M., Ponce, M. L., Bafico, A., Kleinman, H. K., Bottaro, D. P., and Aaronson, S. A. (1998). Differential effects of hepatocyte growth factor isoforms on epithelial and endothelial tubulogenesis. Cell Growth Differ 9(5): 355-65.
Naka, D., Ishii, T., Yoshiyama, Y., Miyazawa, K., Hara, H., Hishida, T., and Kidamura, N. (1992). Activation of hepatocyte growth factor by proteolytic conversion of a single chain form to a heterodimer. J Biol Chem 267(28): 20114-9.
Nakamura, T., Nawa, K., and Ichihara, A. (1984). Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats. Biochem Biophys Res Commun 122(3): 1450-9.
Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K., and Shimizu, S. (1989). Molecular cloning and expression of human hepatocyte growth factor. Nature 342(6248): 440-3.
Nakayama, H. (1988). Existence and clinical significance of human hepatocyte growth factor in sera and plasma from patients with fulminant hepatic failure. Fukuoka Igaku Zasshi 79(7): 537-47.
Peruzzi, B., and Bottaro, D. P. (2006). Targeting the c-Met signaling pathway in cancer. Clin Cancer Res 12(12): 3657-60.
Potempa, S., and Ridley, A. J. (1998). Activation of both MAP kinase and phosphatidylinositide 3-kinase by Ras is required for hepatocyte growth factor/scatter factor-induced adherens junction disassembly. Mol Biol Cell 9(8): 2185-200.
Ridley, A. J., Comoglio, P. M., and Hall, A. (1995). Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells. Mol Cell Biol 15(2): 1110-22.
Rosen, E. M., Knesel, J., and Goldberg, I. D. (1991). Scatter factor and its relationship to hepatocyte growth factor and met. Cell Growth Differ 2(11): 603-7.
Royal, I., Lamarche-Vane, N., Lamorte, L., Kaibuchi, K., and Park, M. (2000). Activation of cdc42, rac, PAK, and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation. Mol Biol Cell 11(5): 1709-25.
Russell, W. E., McGowan, J. A., and Bucher, N. L. (1984). Partial characterization of a hepatocyte growth factor from rat platelets. J Cell Physiol 119(2): 183-92.
Saga, Y., Mizukami, H., Suzuki, M., Urabe, M., Kume, A., Nakamura, T., Sato, I., and Ozawa, K. (2001). Expression of HGF/NK4 in ovarian cancer cells suppresses intraperitoneal dissemination and extends host survival. Gene Ther 8(19): 1450-5.
Sakkab, D., Lewitzky, M., Posern, G., Schaeper, U., Sachs, M., Birchmeier, W., and Feller, S. M. (2000). Signaling of hepatocyte growth factor/scatter factor (HGF) to the small GTPase Rap1 via the large docking protein Gab1 and the adapter protein CRKL. J Biol Chem 275(15): 10772-8.
Schwall, R. H., Chang, L. Y., Godowski, P. J., Kahn, D. W., Hillan, K. J., Bauer, K. D., and Zioncheck, T. F. (1996). Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2. J Cell Biol 133(3): 709-18.
Silvagno, F., Follenzi, A., Arese, M., Prat, M., Giraudo, E., Gaudino, G., Camussi, G., Comoglio, P. M., and Bussolino, F. (1995). In vivo activation of met tyrosine kinase by heterodimeric hepatocyte growth factor molecule promotes angiogenesis. Arterioscler Thromb Vasc Biol 15(11): 1857-65.
Sonnenberg, E., Meyer, D., Weidner, K. M., and Birchmeier, C. (1993). Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development. J Cell Biol 123(1): 223-35.
Stahl, S. J., Wingfield, P. T., Kaufman, J. D., Pannell, L. K., Cioce, V., Sakata, H., Taylor, W. G., Rubin, J. S., and Bottaro, D. P. (1997). Functional and biophysical characterization of recombinant human hepatocyte growth factor isoforms produced in Escherichia coli. Biochem J 326 ( Pt 3): 763-72.
Stamos, J., Lazarus, R. A., Yao, X., Kirchhofer, D., and Wiesmann, C. (2004). Crystal structure of the HGF beta-chain in complex with the Sema domain of the Met receptor. EMBO J 23(12): 2325-35.
Stoker, M., Gherardi, E., Perryman, M., and Gray, J. (1987). Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature 327(6119): 239-42.
Tamura, M., Gu, J., Matsumoto, K., Aota, S., Parsons, R., and Yamada, K. M. (1998). Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science 280(5369): 1614-7.
Wang, X., Le, P., Liang, C., Chan, J., Kiewlich, D., Miller, T., Harris, D., Sun, L., Rice, A., Vasile, S., Blake, R. A., Howlett, A. R., Patel, N., McMahon, G., and Lipson, K. E. (2003). Potent and selective inhibitors of the Met [hepatocyte growth factor/scatter factor (HGF/SF) receptor] tyrosine kinase block HGF/SF-induced tumor cell growth and invasion. Mol Cancer Ther 2(11): 1085-92.
Weidner, K. M., Arakaki, N., Hartmann, G., Vandekerckhove, J., Weingart, S., Rieder, H., Fonatsch, C., Tsubouchi, H., Hishida, T., Daikuhara, Y., and Birchmeier, W. (1991). Evidence for the identity of human scatter factor and human hepatocyte growth factor. Proc Natl Acad Sci U S A 88(16): 7001-5.
Weidner, K. M., Behrens, J., Vandekerckhove, J., and Birchmeier, W. (1990). Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J Cell Biol 111(5 Pt 1): 2097-108.
Xiao, G. H., Jeffers, M., Bellacosa, A., Mitsuuchi, Y., Vande Woude, G. F., and Testa, J. R. (2001). Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. Proc Natl Acad Sci U S A 98(1): 247-52.
Zhang, Y. W., and Vande Woude, G. F. (2003). HGF/SF-met signaling in the control of branching morphogenesis and invasion. J Cell Biochem 88(2): 408-17.