Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23684
標題: 過量表現 gelsolin 對 MDA-MB-231人類乳癌細胞內肌動蛋白絲重組和細胞增生的影響
Effects of gelsolin overexpression on actin filament reorganization and cell proliferation in MDA-MB-231 human breast cancer cells
作者: 邱冠穎
Chiu, Kuan-Ying
關鍵字: breast cacner
乳癌
MDA-MB-231
actin binding proteins gelsolin
cell proliferation
乳癌細胞株MDA-MB-231
肌動蛋白結合蛋白 gelsolin
細胞增生
出版社: 生命科學系所
引用: Asch, H.L., Head, K., Dong, Y., Natoli, F., Winston, J.S., Connolly, J.L. & Asch, B.B. (1996) Widespread loss of gelsolin in breast cancers of humans, mice, and rats. Cancer Res, 56, 4841-4845. Azuma, T., Koths, K., Flanagan, L. & Kwiatkowski, D. (2000) Gelsolin in complex with phosphatidylinositol 4,5-bisphosphate inhibits caspase-3 and -9 to retard apoptotic progression. J Biol Chem, 275, 3761-3766. Baron, A., Migita, T., Tang, D. & Loda, M. (2004) Fatty acid synthase: a metabolic oncogene in prostate cancer? J Cell Biochem, 91, 47-53. Bernofsky, C. (1980) Nicotinic acid adenine dinucleotide phosphate (NAADP+). Methods Enzymol, 66, 105-112. Berridge, M.J. (1993) Inositol trisphosphate and calcium signalling. Nature, 361, 315-325. Berridge, M.J., Lipp, P. & Bootman, M.D. (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol, 1, 11-21. Bharadwaj, S., Hitchcock-DeGregori, S., Thorburn, A. & Prasad, G.L. (2004) N terminus is essential for tropomyosin functions: N-terminal modification disrupts stress fiber organization and abolishes anti-oncogenic effects of tropomyosin-1. J Biol Chem, 279, 14039-14048. Bharadwaj, S., Thanawala, R., Bon, G., Falcioni, R. & Prasad, G.L. (2005) Resensitization of breast cancer cells to anoikis by tropomyosin-1: role of Rho kinase-dependent cytoskeleton and adhesion. Oncogene, 24, 8291-8303. Burtnick, L.D., Koepf, E.K., Grimes, J., Jones, E.Y., Stuart, D.I., McLaughlin, P.J. & Robinson, R.C. (1997) The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation. Cell, 90, 661-670. Carlsson, L., Nystrom, L.E., Sundkvist, I., Markey, F. & Lindberg, U. (1977) Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells. J Mol Biol, 115, 465-483. Chaponnier, C., Yin, H.L. & Stossel, T.P. (1987) Reversibility of gelsolin/actin interaction in macrophages. Evidence of Ca2+-dependent and Ca2+-independent pathways. J Exp Med, 165, 97-106. Choe, H., Burtnick, L.D., Mejillano, M., Yin, H.L., Robinson, R.C. & Choe, S. (2002) The calcium activation of gelsolin: insights from the 3A structure of the G4-G6/actin complex. J Mol Biol, 324, 691-702. Cooley, B.C. & Bergtrom, G. (2001) Multiple combinations of alternatively spliced exons in rat tropomyosin-alpha gene mRNA: evidence for 20 new isoforms in adult tissues and cultured cells. Arch Biochem Biophys, 390, 71-77. Cunningham, C.C., Stossel, T.P. & Kwiatkowski, D.J. (1991) Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science, 251, 1233-1236. Dabrowska, R., Hinssen, H., Galazkiewicz, B. & Nowak, E. (1996) Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg(2+)-ATPase activity. Biochem J, 315 ( Pt 3), 753-759. Dosaka-Akita, H., Hommura, F., Fujita, H., Kinoshita, I., Nishi, M., Morikawa, T., Katoh, H., Kawakami, Y. & Kuzumaki, N. (1998) Frequent loss of gelsolin expression in non-small cell lung cancers of heavy smokers. Cancer Res, 58, 322-327. Eves, R., Webb, B.A., Zhou, S. & Mak, A.S. (2006) Caldesmon is an integral component of podosomes in smooth muscle cells. J Cell Sci, 119, 1691-1702. Fanning, A.S., Wolenski, J.S., Mooseker, M.S. & Izant, J.G. (1994) Differential regulation of skeletal muscle myosin-II and brush border myosin-I enzymology and mechanochemistry by bacterially produced tropomyosin isoforms. Cell Motil Cytoskeleton, 29, 29-45. Fedorov, A.A., Magnus, K.A., Graupe, M.H., Lattman, E.E., Pollard, T.D. & Almo, S.C. (1994) X-ray structures of isoforms of the actin-binding protein profilin that differ in their affinity for phosphatidylinositol phosphates. Proc Natl Acad Sci U S A, 91, 8636-8640. Franzen, B., Linder, S., Uryu, K., Alaiya, A.A., Hirano, T., Kato, H. & Auer, G. (1996) Expression of tropomyosin isoforms in benign and malignant human breast lesions. Br J Cancer, 73, 909-913. Frisch, S.M. & Francis, H. (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol, 124, 619-626. Furukawa, K., Fu, W., Li, Y., Witke, W., Kwiatkowski, D.J. & Mattson, M.P. (1997) The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J Neurosci, 17, 8178-8186. Gabrielson, E.W., Pinn, M.L., Testa, J.R. & Kuhajda, F.P. (2001) Increased fatty acid synthase is a therapeutic target in mesothelioma. Clin Cancer Res, 7, 153-157. Garavito-Aguilar, Z.V., Recio-Pinto, E., Corrales, A.V., Zhang, J., Blanck, T.J. & Xu, F. (2004) Differential thapsigargin-sensitivities and interaction of Ca2+ stores in human SH-SY5Y neuroblastoma cells. Brain Res, 1011, 177-186. Gettemans, J., Van Impe, K., Delanote, V., Hubert, T., Vandekerckhove, J. & De Corte, V. (2005) Nuclear actin-binding proteins as modulators of gene transcription. Traffic, 6, 847-857. Graether, S.P., Heinonen, T.Y., Raharjo, W.H., Jin, J.P. & Mak, A.S. (1997) Tryptophan residues in caldesmon are major determinants for calmodulin binding. Biochemistry, 36, 364-369. Grynkiewicz, G., Poenie, M. & Tsien, R.Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem, 260, 3440-3450. Gunning, P., O''Neill, G. & Hardeman, E. (2008) Tropomyosin-based regulation of the actin cytoskeleton in time and space. Physiol Rev, 88, 1-35. Gunning, P.W., Schevzov, G., Kee, A.J. & Hardeman, E.C. (2005) Tropomyosin isoforms: divining rods for actin cytoskeleton function. Trends Cell Biol, 15, 333-341. Hayashi, K., Fujio, Y., Kato, I. & Sobue, K. (1991) Structural and functional relationships between h- and l-caldesmons. J Biol Chem, 266, 355-361. Heng, Y.W. & Koh, C.G. (2010) Actin cytoskeleton dynamics and the cell division cycle. Int J Biochem Cell Biol, 42, 1622-1633. Holmes, K.C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Atomic model of the actin filament. Nature, 347, 44-49. Honore, B., Madsen, P., Andersen, A.H. & Leffers, H. (1993) Cloning and expression of a novel human profilin variant, profilin II. FEBS Lett, 330, 151-155. Howard, T., Chaponnier, C., Yin, H. & Stossel, T. (1990) Gelsolin-actin interaction and actin polymerization in human neutrophils. J Cell Biol, 110, 1983-1991. Hsu, Y.C. & Liou, Y.M. (2010) The anti-cancer effects of (-)-epigalocathine-3-gallate on the signaling pathways associated with membrane receptors in MCF-7 cells. J Cell Physiol. Hu, E., Chen, Z., Fredrickson, T. & Zhu, Y. (2001) Molecular cloning and characterization of profilin-3: a novel cytoskeleton-associated gene expressed in rat kidney and testes. Exp Nephrol, 9, 265-274. Hughes, J.A., Cooke-Yarborough, C.M., Chadwick, N.C., Schevzov, G., Arbuckle, S.M., Gunning, P. & Weinberger, R.P. (2003) High-molecular-weight tropomyosins localize to the contractile rings of dividing CNS cells but are absent from malignant pediatric and adult CNS tumors. Glia, 42, 25-35. Ishikawa, R., Yamashiro, S. & Matsumura, F. (1989) Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of Ca2+. Potentiation of the annealing process by caldesmon. J Biol Chem, 264, 16764-16770. Janmey, P.A., Stossel, T.P. & Allen, P.G. (1998) Deconstructing gelsolin: identifying sites that mimic or alter binding to actin and phosphoinositides. Chem Biol, 5, R81-85. Kamada, S., Kusano, H., Fujita, H., Ohtsu, M., Koya, R.C., Kuzumaki, N. & Tsujimoto, Y. (1998) A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci U S A, 95, 8532-8537. Kiselar, J.G., Janmey, P.A., Almo, S.C. & Chance, M.R. (2003) Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting. Proc Natl Acad Sci U S A, 100, 3942-3947. Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T.J., Kirschner, M.W., Koths, K., Kwiatkowski, D.J. & Williams, L.T. (1997) Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science, 278, 294-298. Koya, R.C., Fujita, H., Shimizu, S., Ohtsu, M., Takimoto, M., Tsujimoto, Y. & Kuzumaki, N. (2000) Gelsolin inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c release. J Biol Chem, 275, 15343-15349. Kuhajda, F.P. (2000) Fatty-acid synthase and human cancer: new perspectives on its role in tumor biology. Nutrition, 16, 202-208. Kuhajda, F.P. (2006) Fatty acid synthase and cancer: new application of an old pathway. Cancer Res, 66, 5977-5980. Kuhajda, F.P., Pizer, E.S., Li, J.N., Mani, N.S., Frehywot, G.L. & Townsend, C.A. (2000) Synthesis and antitumor activity of an inhibitor of fatty acid synthase. Proc Natl Acad Sci U S A, 97, 3450-3454. Kuznetsov, G., Brostrom, M.A. & Brostrom, C.O. (1993) Role of endoplasmic reticular calcium in oligosaccharide processing of alpha 1-antitrypsin. J Biol Chem, 268, 2001-2008. Kwiatkowski, D.J. (1999) Functions of gelsolin: motility, signaling, apoptosis, cancer. Curr Opin Cell Biol, 11, 103-108. Kwiatkowski, D.J., Mehl, R. & Yin, H.L. (1988) Genomic organization and biosynthesis of secreted and cytoplasmic forms of gelsolin. J Cell Biol, 106, 375-384. Kwon, H.J., Yoshida, M., Nagaoka, R., Obinata, T., Beppu, T. & Horinouchi, S. (1997) Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression. Oncogene, 15, 2625-2631. Lambrechts, A., Verschelde, J.L., Jonckheere, V., Goethals, M., Vandekerckhove, J. & Ampe, C. (1997) The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences. EMBO J, 16, 484-494. Lange, K. & Brandt, U. (1996) Calcium storage and release properties of F-actin: evidence for the involvement of F-actin in cellular calcium signaling. FEBS Lett, 395, 137-142. Lange, K. & Gartzke, J. (2006) F-actin-based Ca signaling-a critical comparison with the current concept of Ca signaling. J Cell Physiol, 209, 270-287. Lee, J.S., Lee, M.S., Oh, W.K. & Sul, J.Y. (2009) Fatty acid synthase inhibition by amentoflavone induces apoptosis and antiproliferation in human breast cancer cells. Biol Pharm Bull, 32, 1427-1432. Lee, Y.J. & Keng, P.C. (2005) Studying the effects of actin cytoskeletal destabilization on cell cycle by cofilin overexpression. Mol Biotechnol, 31, 1-10. Liao, C.J., Wu, T.I., Huang, Y.H., Chang, T.C., Wang, C.S., Tsai, M.M., Hsu, C.Y., Tsai, M.H., Lai, C.H. & Lin, K.H. (2011) Overexpression of gelsolin in human cervical carcinoma and its clinicopathological significance. Gynecol Oncol, 120, 135-144. Lim, D., Lange, K. & Santella, L. (2002) Activation of oocytes by latrunculin A. FASEB J, 16, 1050-1056. Lin, J.J., Warren, K.S., Wamboldt, D.D., Wang, T. & Lin, J.L. (1997) Tropomyosin isoforms in nonmuscle cells. Int Rev Cytol, 170, 1-38. Little, J.L., Wheeler, F.B., Fels, D.R., Koumenis, C. & Kridel, S.J. (2007) Inhibition of fatty acid synthase induces endoplasmic reticulum stress in tumor cells. Cancer Res, 67, 1262-1269. Liu, M.C., Marshall, J.L. & Pestell, R.G. (2004) Novel strategies in cancer therapeutics: targeting enzymes involved in cell cycle regulation and cellular proliferation. Curr Cancer Drug Targets, 4, 403-424. Lodish, H.F., Kong, N. & Wikstrom, L. (1992) Calcium is required for folding of newly made subunits of the asialoglycoprotein receptor within the endoplasmic reticulum. J Biol Chem, 267, 12753-12760. Mahadev, K., Raval, G., Bharadwaj, S., Willingham, M.C., Lange, E.M., Vonderhaar, B., Salomon, D. & Prasad, G.L. (2002) Suppression of the transformed phenotype of breast cancer by tropomyosin-1. Exp Cell Res, 279, 40-51. Metzler, W.J., Bell, A.J., Ernst, E., Lavoie, T.B. & Mueller, L. (1994) Identification of the poly-L-proline-binding site on human profilin. J Biol Chem, 269, 4620-4625. Michalak, M., Robert Parker, J.M. & Opas, M. (2002) Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum. Cell Calcium, 32, 269-278. Morgan, K.G. & Gangopadhyay, S.S. (2001) Invited review: cross-bridge regulation by thin filament-associated proteins. J Appl Physiol, 91, 953-962. Nag, S., Ma, Q., Wang, H., Chumnarnsilpa, S., Lee, W.L., Larsson, M., Kannan, B., Hernandez-Valladares, M., Burtnick, L.D. & Robinson, R.C. (2009) Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin. Proc Natl Acad Sci U S A, 106, 13713-13718. Nishimura, K., Ting, H.J., Harada, Y., Tokizane, T., Nonomura, N., Kang, H.Y., Chang, H.C., Yeh, S., Miyamoto, H., Shin, M., Aozasa, K., Okuyama, A. & Chang, C. (2003) Modulation of androgen receptor transactivation by gelsolin: a newly identified androgen receptor coregulator. Cancer Res, 63, 4888-4894. Nusco, G.A., Chun, J.T., Ercolano, E., Lim, D., Gragnaniello, G., Kyozuka, K. & Santella, L. (2006) Modulation of calcium signalling by the actin-binding protein cofilin. Biochem Biophys Res Commun, 348, 109-114. Obermann, H., Raabe, I., Balvers, M., Brunswig, B., Schulze, W. & Kirchhoff, C. (2005) Novel testis-expressed profilin IV associated with acrosome biogenesis and spermatid elongation. Mol Hum Reprod, 11, 53-64. Pawlak, G. & Helfman, D.M. (2001) Cytoskeletal changes in cell transformation and tumorigenesis. Curr Opin Genet Dev, 11, 41-47. Philchenkov, A.A. (2003) Caspases as regulators of apoptosis and other cell functions. Biochemistry (Mosc), 68, 365-376. Phillips, G.N., Jr., Lattman, E.E., Cummins, P., Lee, K.Y. & Cohen, C. (1979) Crystal structure and molecular interactions of tropomyosin. Nature, 278, 413-417. Pittenger, M.F., Kazzaz, J.A. & Helfman, D.M. (1994) Functional properties of non-muscle tropomyosin isoforms. Curr Opin Cell Biol, 6, 96-104. Pizer, E.S., Pflug, B.R., Bova, G.S., Han, W.F., Udan, M.S. & Nelson, J.B. (2001) Increased fatty acid synthase as a therapeutic target in androgen-independent prostate cancer progression. Prostate, 47, 102-110. Pizer, E.S., Thupari, J., Han, W.F., Pinn, M.L., Chrest, F.J., Frehywot, G.L., Townsend, C.A. & Kuhajda, F.P. (2000) Malonyl-coenzyme-A is a potential mediator of cytotoxicity induced by fatty-acid synthase inhibition in human breast cancer cells and xenografts. Cancer Res, 60, 213-218. Pollard, T.D. & Borisy, G.G. (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell, 112, 453-465. Prasad, G.L., Fuldner, R.A. & Cooper, H.L. (1993) Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene. Proc Natl Acad Sci U S A, 90, 7039-7043. Prasad, S.C., Thraves, P.J., Dritschilo, A. & Kuettel, M.R. (1997) Protein expression changes associated with radiation-induced neoplastic progression of human prostate epithelial cells. Electrophoresis, 18, 629-637. Rao, J., Seligson, D., Visapaa, H., Horvath, S., Eeva, M., Michel, K., Pantuck, A., Belldegrun, A. & Palotie, A. (2002) Tissue microarray analysis of cytoskeletal actin-associated biomarkers gelsolin and E-cadherin in urothelial carcinoma. Cancer, 95, 1247-1257. Reshetnikova, G., Barkan, R., Popov, B., Nikolsky, N. & Chang, L.S. (2000) Disruption of the actin cytoskeleton leads to inhibition of mitogen-induced cyclin E expression, Cdk2 phosphorylation, and nuclear accumulation of the retinoblastoma protein-related p107 protein. Exp Cell Res, 259, 35-53. Shieh, D.B., Chen, I.W., Wei, T.Y., Shao, C.Y., Chang, H.J., Chung, C.H., Wong, T.Y. & Jin, Y.T. (2006) Tissue expression of gelsolin in oral carcinogenesis progression and its clinicopathological implications. Oral Oncol, 42, 599-606. Shieh, D.B., Li, R.Y., Liao, J.M., Chen, G.D. & Liou, Y.M. (2010) Effects of genistein on beta-catenin signaling and subcellular distribution of actin-binding proteins in human umbilical CD105-positive stromal cells. J Cell Physiol, 223, 423-434. Silacci, P., Mazzolai, L., Gauci, C., Stergiopulos, N., Yin, H.L. & Hayoz, D. (2004) Gelsolin superfamily proteins: key regulators of cellular functions. Cell Mol Life Sci, 61, 2614-2623. Sobue, K., Muramoto, Y., Fujita, M. & Kakiuchi, S. (1981) Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci U S A, 78, 5652-5655. Stumvoll, M. & Haring, H. (2002) The peroxisome proliferator-activated receptor-gamma2 Pro12Ala polymorphism. Diabetes, 51, 2341-2347. Takahashi, A., Camacho, P., Lechleiter, J.D. & Herman, B. (1999) Measurement of intracellular calcium. Physiol Rev, 79, 1089-1125. Tanaka, H., Shirkoohi, R., Nakagawa, K., Qiao, H., Fujita, H., Okada, F., Hamada, J., Kuzumaki, S., Takimoto, M. & Kuzumaki, N. (2006) siRNA gelsolin knockdown induces epithelial-mesenchymal transition with a cadherin switch in human mammary epithelial cells. Int J Cancer, 118, 1680-1691. Tanaka, M., Mullauer, L., Ogiso, Y., Fujita, H., Moriya, S., Furuuchi, K., Harabayashi, T., Shinohara, N., Koyanagi, T. & Kuzumaki, N. (1995) Gelsolin: a candidate for suppressor of human bladder cancer. Cancer Res, 55, 3228-3232. Thompson, C.C., Ashcroft, F.J., Patel, S., Saraga, G., Vimalachandran, D., Prime, W., Campbell, F., Dodson, A., Jenkins, R.E., Lemoine, N.R., Crnogorac-Jurcevic, T., Yin, H.L. & Costello, E. (2007) Pancreatic cancer cells overexpress gelsolin family-capping proteins, which contribute to their cell motility. Gut, 56, 95-106. Thor, A.D., Edgerton, S.M., Liu, S., Moore, D.H., 2nd & Kwiatkowski, D.J. (2001) Gelsolin as a negative prognostic factor and effector of motility in erbB-2-positive epidermal growth factor receptor-positive breast cancers. Clin Cancer Res, 7, 2415-2424. Uchida, K., Masumori, N., Takahashi, A., Itoh, N., Kato, K., Matusik, R.J. & Tsukamoto, T. (2006) Murine androgen-independent neuroendocrine carcinoma promotes metastasis of human prostate cancer cell line LNCaP. Prostate, 66, 536-545. Van den Abbeele, A., De Corte, V., Van Impe, K., Bruyneel, E., Boucherie, C., Bracke, M., Vandekerckhove, J. & Gettemans, J. (2007) Downregulation of gelsolin family proteins counteracts cancer cell invasion in vitro. Cancer Lett, 255, 57-70. Wakil, S.J. (1989) Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry, 28, 4523-4530. Wang, C.L. (2001) Caldesmon and smooth-muscle regulation. Cell Biochem Biophys, 35, 275-288. Wang, F.L., Wang, Y., Wong, W.K., Liu, Y., Addivinola, F.J., Liang, P., Chen, L.B., Kantoff, P.W. & Pardee, A.B. (1996) Two differentially expressed genes in normal human prostate tissue and in carcinoma. Cancer Res, 56, 3634-3637. Wang, H.Q., Altomare, D.A., Skele, K.L., Poulikakos, P.I., Kuhajda, F.P., Di Cristofano, A. & Testa, J.R. (2005) Positive feedback regulation between AKT activation and fatty acid synthase expression in ovarian carcinoma cells. Oncogene, 24, 3574-3582. Wang, Y., Mattson, M.P. & Furukawa, K. (2002) Endoplasmic reticulum calcium release is modulated by actin polymerization. J Neurochem, 82, 945-952. Witke, W. (2004) The role of profilin complexes in cell motility and other cellular processes. Trends Cell Biol, 14, 461-469. Witke, W., Sharpe, A.H., Hartwig, J.H., Azuma, T., Stossel, T.P. & Kwiatkowski, D.J. (1995) Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell, 81, 41-51. Wyllie, A.H. (1987) Apoptosis: cell death in tissue regulation. J Pathol, 153, 313-316. Yang, D.M. & Kao, L.S. (2001) Relative contribution of the Na(+)/Ca(2+) exchanger, mitochondria and endoplasmic reticulum in the regulation of cytosolic Ca(2+) and catecholamine secretion of bovine adrenal chromaffin cells. J Neurochem, 76, 210-216. Yin, H.L., Kwiatkowski, D.J., Mole, J.E. & Cole, F.S. (1984) Structure and biosynthesis of cytoplasmic and secreted variants of gelsolin. J Biol Chem, 259, 5271-5276. Yin, H.L. & Stossel, T.P. (1979) Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature, 281, 583-586. Zemel, M.B. (2002) Regulation of adiposity and obesity risk by dietary calcium: mechanisms and implications. J Am Coll Nutr, 21, 146S-151S. Zemel, M.B., Kim, J.H., Woychik, R.P., Michaud, E.J., Kadwell, S.H., Patel, I.R. & Wilkison, W.O. (1995) Agouti regulation of intracellular calcium: role in the insulin resistance of viable yellow mice. Proc Natl Acad Sci U S A, 92, 4733-4737.
摘要: Gelsolin (GSN)是肌動蛋白結合蛋白(ABP)的成員之一,主要藉由切割和鍵結來調控肌動蛋白絲動態的變化。近年來許多研究都在探討肌動蛋白絲動態的變化與肌動蛋白結合蛋白在惡性腫瘤中所扮演的角色。研究指出:GSN在腫瘤細胞中為促進因子或是抑制因子,可能與腫瘤的階段與轉移的能力有關;然而GSN對於腫瘤細胞增生的影響仍然不是很明確。因此本論文利用過量表現GSN的人類乳癌細胞株MDA-MB-231做為模式,探討GSN在乳癌細胞中對肌動蛋白絲重組以及細胞生長的影響。首先,將帶有GSN全長cDNA 的質體DNA -pcDNA6-GSN轉染進入MDA-MB-231中,利用西方轉漬法和即時定量聚合酶連鎖反應檢測細胞內GSN的表現量;並挑選出穩定過量表現GSN的細胞株。觀察GSN過量表現細胞株的細胞型態,再利用細胞計數的方式計算細胞的細胞倍增生長時間 (PDT),以及螢光顯微鏡術觀察細胞內肌動蛋白絲的分子結構;發現:GSN過量表現會使細胞型態變的較寬較長,生長不容易聚集,並且會減緩細胞生長的速度,還會使胞內的肌動蛋白絲進行去聚合反應。進一步探討GSN過表現抑制細胞生長的原因;實驗中使用螢光光譜儀檢測fura-2並計算胞內鈣離子濃度、使用q-PCR檢測脂質相關基因:脂肪酸生成酶 (FAS) 和過氧化體增生因子 (PPAR-γ 2) 的表現量、使用原子力顯微鏡(AFM)偵測細胞表面的黏著力,最後使用流式細胞儀檢測細胞週期。實驗結果發現:GSN過量表現細胞株,胞內鈣離子濃度提高了56%,FAS的表現量增加,但是降低了PPAR-γ2的表現量,細胞表面的黏附力提升了24%,並且增加細胞處於G1時期的比例,這些現象可能都與肌動蛋白絲分子結構發生改變有關。另外,GSN過量表現細胞株會增加Tm1的表現;利用siRNA 靜默GSN 過量表現細胞株中Tm1,並且使用trypsin蛋白酶 處理檢測細胞貼附力的實驗中,發現降低Tm1 的表現會增加細胞對胞外的貼附力,並且可能與GSN有加成的作用。綜合以上結果:GSN過量表現影響細胞生長可能是經由 (1) 肌動蛋白絲進行去聚合反應直接或間接的使胞內鈣離子濃度提高,影響細胞內鈣離子訊息。(2)改變脂質相關基因表現,影響脂質代謝,部分干擾細胞生長。(3) 肌動蛋白絲t進行去聚合反應,會經由β-catenin/cyclin D1 的訊息路徑,使細胞停滯於G1 階段,來影響細胞週期。另外,(4) GSN過量表現會增加 Tm1 的表現量;藉由增加兩者基因的表現量,共同影響細胞對胞外環境的貼附力,進而抑制細胞生長。
Gelsolin (GSN) is one of actin binding proteins (ABPs) that regulate dynamic actin filament organization by severing and capping. In recent years, many studies have devoted to establish the role of dynamic actin filament formation by ABPs in controlling the proliferation of malignant tumors. These studies showed that GSN might act as a tumor activator or as a tumor suppressor, and that is related to tumor stage and its ability of metastasis. Apparently, the effect of GSN on tumor cell proliferation is still not clear yet. In this study, GSN-overexpressed MDA-MB-231 human breast cancer cell line was utilized as a model to examine the effect of GSN overexpression on actin filament reorganization and cell proliferation in breast cancer cells. Firstly, a pcDNA6-GSN that contains full-length GSN cDNA was transfected into MDA-MB-231, and then the stable GSN overexpression cell lines of MDA-MB-231 confirmed by real time q-PCR and western bolt were cloned subsequently. Trypan blue exclusion assay was used to estimate the population doubling time (PDT) for cell proliferation of GSN-overexpressed MDA-MB-231, and then the cell morphology and intracellular actin filament formation were observed by the phase microscopy and the epi-fluorescent microscopy, respectively. The results showed, GSN overexpression would inhibit cell proliferation and make cell morphology wider as well as longer, and reduce cell aggregation, and promote actin filament depolymerization. A futher study was performed to investigate how GSN-overexpression could inhibit cell proliferation. The methods and results were as follows: (1) Measuring fura-2 F340/F380 fluorescence ratio in the cells indicated 1.56 folds of increases in Ca2+ levels for GSN-overexpressed cells. (2) Detecting lipid-related genes by q-PCR showed GSN-overexpression facilitates the fatty acid synthase (FAS) but attenuates the peroxisome proliferator-activated receptor-γ2 (PPAR-γ2). (3) Detecting by atomic force microscopy (AFM) indicated GSN-overexpression increased cell surface adhesion force. (4) Detecting cell cycle by cytometry showed GSN-overexpression cell increased percentage of the cell population at G1 phase. In addition, GSN-overexpressed cells will cause Tropomyosin-1 (Tm1) gene up-regulation. Using siRNA to silence Tm1 and detecting cell adhesion force showed that silenced Tm1 gene increased the cell adhesion to the extracellular matrix. Taken together, results obtained in this study suggested that GSN overexpression might affect cell proliferation through (1) actin filament depolymerization, directly or indirectly increased intracellular calcium concentration, and then affecting intracellular calcium signal; (2) changing the lipid-related gene expression to affect lipid metabolism; (3) modulating actin filament polymerization/ depolymerization to arrest cells in G1 phase by inhibiting the β-catenin/cyclin D1 signal pathway; (4) upregulating Tm1 gene that might affect cell adhesion to the extracellular force, inhibited cell growth.
URI: http://hdl.handle.net/11455/23684
其他識別: U0005-1108201114485800
Appears in Collections:生命科學系所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.