腫瘤的轉移是造成癌症病人治療失敗及死亡的主要原因。RECK是個新發現的腫瘤抑制基因，它是在v-Ki-ras轉型(transformed)的NIH3T3細胞，藉轉殖(transfect)入纖維母細胞cDNA庫的cDNA colony，而篩選出可以使NIH3T3回復成平坦細胞型態的新基因。RECK可以抑制MMP-2跟MMP-9的釋放及活化，在in vitro可以抑制細胞的侵犯能力，此外RECK在動物實驗亦證實可以抑制腫瘤轉移，因此稱RECK是個轉移抑制基因，然而RECK是個普遍被致癌訊息傳導所負向調節的標的物。c-Myc是最常被活化的致癌基因之一，可促進腫瘤新生中的許多過程。在我的實驗裡，轉殖c-Myc的表現載體到NIH3T3細胞，可以抑制RECK表現，而啟動子活性試驗指出c-Myc可抑制RECK的轉錄。利用DNA affinity precipitation assay跟chromatin immunoprecipitation assay，證實c-Myc可能是藉由與RECK近端啟動子上的SP1結合位作用，進而抑制RECK。c-Myc可能藉由抑制這個轉移及血管新生的抑制者RECK，進而增加細胞的侵犯性，在Myc表現的細胞中，重新回復RECK的表現，可以有效地抑制細胞的侵犯性，這也許可以作為抑制Myc所誘導的癌症侵犯的一種治療策略。

The major cause of therapy failure and death of cancer patients is metastasis. RECK is a newly identified gene which was isolated by screening for human fibroblast cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH3T3 cells. It can inhibit the release and activation of MMP-2 and MMP-9, and prevent cell invasion in vitro. In addition, RECK can repress tumor metastasis in experimental animal. Thus, RECK is a metastasis suppressor gene. However, RECK gene is a common target that is negatively regulated by oncogenic signals. Overexpression of c-myc protooncogene is frequently found in several types of human cancer and contributes to multiple steps of tumorigenesis. Ecto-expression of c-Myc in NIH3T3 cells inhibited RECK expression. Promoter activity assay suggested c-Myc repressed RECK at transcriptional level. By using DNA affinity precipitation assay and chromatin immunoprecipitation assay, we found that oncogenic c-Myc bound to the SP1 sites of RECK promoter in vitro and in vivo. It is possible that c-Myc could repress RECK expression via SP1. Our data suggest that c-Myc may inhibit the metastatic/angiogenic suppressor RECK to enhance cell invasiveness and restoration of RECK may be a novel strategy to inhibit c-Myc-mediated invasion.

中文摘要 ──────────────────────── 2
英文摘要 ──────────────────────── 3
縮寫語 ───────────────────────── 4
前言 ────────────────────────── 5
研究方向 ──────────────────────── 10
實驗材料與方法 ───────────────────── 11
實驗結果 ──────────────────────── 33
討論 ────────────────────────── 39
實驗結果之圖表 ───────────────────── 42
參考文獻 ──────────────────────── 56
附圖 ────────────────────────── 60
附表 ────────────────────────── 67

Takahashi C, Sheng Z, Horan TP, Kitayama H, Maki M, Hitomi K, Kitaura Y, Takai S, Sasahara RM, Horimoto A, Ikawa Y, Ratzkin BJ, Arakawa T, Noda M. Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane anchored glycoprotein RECK. Proc Natl Acad Sci USA. 1998 Oct 27;95(22):13221-6.
Liu LT, Peng JP, Chang HC, Hung WC. RECK is a target of Epstein-Barr virus latent membrane protein 1. Oncogene. 2003 Nov 13;22(51):8263-70.
Chang HC, Cho CY, Hung WC. Silencing of the metastasis suppressor RECK by RAS oncogene is mediated by DNA methyltransferase 3b-induced promoter methylation. Cancer Res. 2006 Sep 1;66(17): 8413-20.
Manes S, Mira E, Barbacid MM, Cipres A, Fernandez-Resa P, Buesa JM, Merida I, Aracil M, Marquez G, Martinez-A C. Identification of insulin-like growth factor-binding protein-1 as a potential physiological substrate for human stromelysin-3. J Biol Chem. 1997 Oct 10;272 (41):25706-12.
Oh J, Takahashi R, Kondo S, Mizoguchi A, Adachi E, Sasahara RM, Nishimura S, Imamura Y, Kitayama H, Alexander DB, Ide C, Horan TP, Arakawa T, Yoshida H, Nishikawa S, Itoh Y, Seiki M, Itohara S, Takahashi C, Noda M. The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell. 2001 Dec 14;107(6):789-800.
Vennstrom B, Sheiness D, Zabielski J, Bishop JM. Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29. J Virol. 1982 42, 773-779.
Nesbit CE, Tersak JM, Prochownik EV. MYC oncogenes and human neoplastic disease. Oncogene. 1999 May 13;18(19):3004-16.
Schlagbauer-Wadl H, Griffioen M, van Elsas A, Schrier PI, Pustelnik T, Eichler HG, Wolff K, Pehamberger H, Jansen B. Influence of increased c-Myc expression on the growth characteristics of human melanoma. J Invest Dermatol. 1999 Mar;112(3):332-6.
Nesbit CE, Tersak JM, Prochownik EV. MYC oncogenes and human neoplastic disease. Oncogene. 1999 May 13;18(19):3004-16. Review.
Claassen GF, Hann SR. Myc-mediated transformation: the repression connection. Oncogene. 1999 May 13;18(19):2925-33. Review.
Adhikary S, Eilers M. Transcriptional regulation and transformation by Myc proteins. Nat Rev Mol Cell Biol. 2005 Aug;6(8):635-45. Review.
Staller P, Peukert K, Kiermaier A, Seoane J, Lukas J, Karsunky H, Moroy T, Bartek J, Massague J, Hanel F, Eilers M. Repression of p15INK4b expression by Myc through association with Miz-1. Nat Cell Biol. 2001 Apr;3(4):392-9.
Vaque JP, Navascues J, Shiio Y, Laiho M, Ajenjo N, Mauleon I, Matallanas D, Crespo P, Leon J. Myc antagonizes Ras-mediated growth arrest in leukemia cells through the inhibition of the Ras-ERK-p21Cip1 pathway. J Biol Chem. 2005 Jan 14;280(2):1112-22.
Izumi H, Molander C, Penn LZ, Ishisaki A, Kohno K, Funa K. Mechanism for the transcriptional repression by c-Myc on PDGF beta-receptor. J Cell Sci. 2001 Apr;114 (Pt 8):1533-44.
He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science. 1998 Sep 4;281(5382):1509-12.
Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A, Verastegui E, Zlotnik A. Involvement of chemokine receptors in breast cancer metastasis. Nature. 2001 Mar 1;410(6824):50-6.
Chiang YC, Teng SC, Su YN, Hsieh FJ, Wu KJ. c-Myc directly regulates transcription of the NBS1 gene involved in DNA double-strand break repair. J Biol Chem. 2003 May 23;278(21):19286-91.
Mauleon I, Lombard MN, Munoz-Alonso MJ, Canelles M, Leon J, Kinetics of myc-max-mad gene expression during hepatocyte proliferation in vivo: differential regulation of mad family and stress-mediated induction of c-myc. Mol Carcinog. 2004 Feb;39(2):85-90.
Gregory MA, Qi Y, Hann SR. Phosphorylation by glycogen synthase kinase-3 controls c-myc proteolysis and subnuclear localization. J Biol Chem. 2003 Dec 19;278(51):51606-12.
Lee SG, Su ZZ, Emdad L, Sarkar D, Fisher PB. Astrocyte elevated gene-1 (AEG-1) is a target gene of oncogenic Ha-ras requiring phosphatidylinositol 3-kinase and c-Myc. Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17390-5.
Pelengaris S, Khan M, Evan GI. Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell. 2002 May 3;109(3):321-34.
Vaque JP, Navascues J, Shiio Y, Laiho M, Ajenjo N, Mauleon I, Matallanas D, Crespo P, Leon J. Myc antagonizes Ras-mediated growth arrest in leukemia cells through the inhibition of the Ras-ERK-p21Cip1 pathway. J Biol Chem. 2005 Jan 14;280(2):1112-22.
Gartel AL, Ye X, Goufman E, Shianov P, Hay N, Najmabadi F, Tyner AL. Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3. Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4510-5.
Gartel AL, Shchors K. Mechanisms of c-myc-mediated transcriptional repression of growth arrest genes. Exp Cell Res. 2003 Feb 1;283(1):17-21. Review.
Staller P, Peukert K, Kiermaier A, Seoane J, Lukas J, Karsunky H, Moroy T, Bartek J, Massague J, Hanel F, Eilers M. Repression of p15INK4b expression by Myc through association with Miz-1. Nat Cell Biol. 2001 Apr;3(4):392-9.
Brenner C, Deplus R, Didelot C, Loriot A, Vire E, De Smet C, Gutierrez A, Danovi D, Bernard D, Boon T, Pelicci PG, Amati B, Kouzarides T, de Launoit Y, Di Croce L, Fuks F. Myc represses transcription through recruitment of DNA methyltransferase corepressor. EMBO J. 2005 Jan 26;24(2):336-46.
Treszl A, Adany R, Rakosy Z, Kardos L, Begany A, Gilde K, Balazs M. Extra copies of c-myc are more pronounced in nodular melanomas than in superficial spreading melanomas as revealed by fluorescence in situ hybridisation. Cytometry B Clin Cytom. 2004 Jul;60(1):37-46