摘 要
Her-2/neu 致癌基因屬erbB族群的成員之一，其蛋白質產物為185kDa的穿膜受體(transmembrane receptor) p185erbB2，具有酪氨酸激脢(tyrosine kinase)活性，對於控制細胞生長、分化等扮演重要角色，在特定癌細胞中，如非小球性肺癌(Non-small cell lung cancer)、乳癌即常發現neu的大量表現。臨床及基礎研究中指出此基因之大量表現常導致細胞對化學治療劑的藥物拮抗性。
本實驗室先前以含活化型(Val664→Glu)及原生型neu之質體轉染一株P185 erbB2表現量極低的膀胱癌細胞株TCCSUP，分別獲得兩株具活化態neu轉染細胞NA1、NA2及三株正常態之neu轉染細胞N5、N10、N14。在分析其對化學治療劑methotrexate之毒殺曲線時，發現正常態neu大量表現之轉染細胞具有明顯較高的藥物敏感性，並可能經由細胞凋亡機轉造成細胞死亡。
為探討細胞分子層次上，methotrexate造成HER-2/neu轉染細胞株死亡的可能機制，目前我們的研究著重於分析methotrexate處理下的轉染細胞株其內在HER-2/neu 下游各種訊息傳遞途徑(signal transduction)各主要的變化，我初步選定分析的對象包括： Mitogen-activated protein kinase superfamily中的 ERK MAPK激脢途徑(主要之訊息傳遞角色為細胞之生長及分化)；p38激脢途徑(主要之角色為應付細胞對於外界壓力，藥物反應之相關基因表現)。實驗結果顯示（1）Neu之轉染細胞株的Ras/Raf/ERK MAPK途徑擁有較高基礎活性。（2）ERK MAPK活性在MTX處理時，可能和細胞生長密度（cell density）有關，正常態neu的轉染細胞在低細胞密度時ERK磷酸化不受MTX影響，高細胞密度時則呈少量磷酸化的反應。（3）ERK，p38 MAPK兩激脢途徑在參與MTX所造成neu轉染細胞株的細胞死亡途徑可能僅佔有非決定性影響力（Non-determining factor）。更進一步地，我們將繼續研究apoptosis之引發與其他訊息傳遞途徑之關係。

Abstract
HER-2/Neu, a proto-oncogene encodes a 185-kDa protein, which belongs to type I receptor tyrosine kinase family, and is essential for cell growth, differentiation regulation. Overexpression of Neu is frequently observed in many tumor types such as Non-small-cell-lung-cancer (NSCLC), breast cancer where it correlates with intrinsic chemotherapeutic resistance.
To investigate the role of Neu in bladder cancer, both wild-type and an active version of HER-2/Neu (Val664-to-Glu) transfectant were established in TCCSUP human bladder transitional carcinoma cell line previously, the cell cytotoxic characteristics to anti-cancer drugs of these transfectants were examined. We found that the chemosensitivity to methotrexate (MTX), but not cisplatin or adriamycin observed in TCCSUP-Neu transfectants is higher than both parental and control cell lines. The cell death of TCCSUP-Neu transfectants during MTX treat is prone to undergo apoptosis.
To understand the underlying mechanisms, we examined the major signal transduction pathways downstream HER-2/neu during MTX treatment, our results indicate that：
(1) TCCSUP-Neu transfectants contain high basal ERK MAPK activity. (2) The response of ERK MAPK to MTX may be dependent on cell density. ERK activation is only observed at confluent cell density. (3) The activation of ERK and p38 MAPK is not essential for MTX-induced cell death.

目 錄
一、 授權書
二、 考試合格證明
三、 誌謝感言
四、 目錄 1
五、 摘要
(1) 中文摘要 2
(2) 英文摘要 4
六、 縮寫表 5
七、 圖表錄 7
八、 緒論 9
九、 材料與方法 16
十、 結果 45
十一、 討論 54
十二、 參考文獻 59
十三、 圖 65
十四、 附錄 86
十五、 自述 88

參考文獻
Alimandi M, et al. Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene. 1995 May 4; 10(9): 1813-21.
Allred DC, et al. HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol. 1992 Apr; 10(4): 599-605.
Arlene F. et al. Urinary tract infection and risk of bladder cancer. Am. J Epidemiol 1984, 119:510-15.
Avishay Sella, M.D. Radiation therapy-associated invasive bladder tumors. Urology 1989, 33:185-88.
Bacus, S.S., Chen X, et al. An immunological approach reveals biological differences between the two NDF/heregulin receptors, ErbB-3 and ErbB-4.
J Biol Chem. 1996 Mar 29; 271(13): 7620-9.
Berman E. et al. Amplification of the dihydrofolate reductase gene is a mechanism of acquired resistance to methotrexate in patients with acute lymphoblastic leukemia and is correlated with p53 gene mutations. Blood 1995, 86(2):677-84.
Borg A, et al. HER-2/neu amplification predicts poor survival in node-positive breast cancer. Cancer Res. 1990 Jul 15; 50(14): 4332-7.
Boulikas T. Phosphorylation of transcription factors and control of the cell cycle. Crit Rev Eukaryot Gene Expr. 1995;5(1):1-77. Review.
Brenuinger LM, Paul S, Gaughan K, Miki T, Chan A, Aaronson SA, Kruh GD. Expression of multidrug resistance-associated protein in NIH/3T3 cells confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution. Cancer Res 1995, 55:5342-47.
Chin BY, et al. Transforming growth factor beta1 rescues serum deprivation-induced apoptosis via the mitogen-activated protein kinase (MAPK) pathway in macrophages. J Biol Chem. 1999 Apr 16;274(16):11362-8.
Collins K, et al. The cell cycle and cancer. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2776-8.
David P. Wood, JR.,Carlos Cordon-Cardo. DNA, RNA and immunohistochemical characterization of the HER-2/neu oncogene in transitional cell carcinoma of the bladder. The Journal of Urology 1991, 146:1398-1401.
Dominique Lallemand, et al. Stress-activated protein kinases are negatively regulated by cell density. EMBO J. 1998 Oct 1;17(19):5615-26.
Eijdems EW, de Haas M, Timmerman AJ, Nan der Schans GP, et al. Reduced topoisomerase II activity in multidrug-resistant human non-small cell lung cancer cell line. Br. J Cancer 1995, 71:40-47.
Eliopoulos AG, Kerr DJ, Herod J, Hodgkins L, Reed JC. The control of apoptosis and drug resistance in ovarian cancer: influence of p53 and bcl-2. Oncogene 1995, 11:1217-228.
Evans DL, et al. Differential sensitivity to the induction of apoptosis by cisplatin in proliferating and quiescent immature rat thymocytes is independent of the levels of drug accumulation and DNA adduct formation.
Cancer Res. 1994 Mar 15;54(6):1596-603.
Fan S, EI-Deiry WS, Bae I, Freeman J, Jondle D, Bhatia K, Fornace AJ. p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. Cancer Res 1994, 54:5824-830.
Feldhoff PW, MirskiSE, Cole SP, Sullivan DM. Altered subcellular distribution of topoisomerase II alpha in a drug-resistant human small cell lung cancer cell line. Cancer Res 1994, 54:756-62.
Goker E. Waltham M. et al. Amplification of the dihydrofolate reductase gene is a mechanism of acquired resistance to methotrexate in patients with acute lymphoblastic leukemia and is correlated with p53 gene mutations. Blood 1995, 86(2):677-84.
Gusterson BA, et al. Prognostic importance of c-erbB-2 expression in breast cancer. International (Ludwig) Breast Cancer Study Group. J Clin Oncol. 1992 Jul;10(7):1049-56.
Gupta K, et al. VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling.
Exp Cell Res. 1999 Mar 15;247(2):495-504.
Hochnauser D. Schnieders B. Bertino JR. Effect of cyclin D1 overexpression on drug sensitivity in a human fibrosarcoma cell line. Journal of the National Cancer Institute 1996, 88(18): 1269-75.
Hynes NE, et al. The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta. 1994 Dec 30; 1198(2-3):165-84. Review.
Kelly J.D. Apoptosis and its clinical significance for bladder cancer. BJU international 1999, 83, 1-10
Kern JA, et al. p185neu expression in human lung adenocarcinomas predicts shortened survival. Cancer Res. 1990 Aug 15; 50(16): 5184-7.
Kerr JF, et al. Apoptosis. Its significance in cancer and cancer therapy. Cancer. 1994 Apr 15; 73(8): 2013-26. Review.
Karin M, et al. Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr Biol. 1995 Jul 1; 5(7):747-57. Review.
Koleske AJ Essential roles for the Abl and Arg tyrosine kinases in eurulation.
Neuron. 1998 Dec; 21(6):1259-72.
Kruh GD, Chan A, Myers K, Gaughan K. Expression complementary DNA library transfer establishes mrp as a multidrug resistance gene. Cancer Res 1994, 54:1649-52.
Li W. Fan J. Bertino JR. Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell line. Proc Natl Acad Sci U S A. 1995, 92(22): 10436-440.
Lisha Zhang and Mien-Chie Hung. Sensitization of HER-2/neu overexpressing non-small cell lung cancer cell to chemotherapeutic drugs by tyrosine kinase inhibitor emodin. Oncogene 1996, 12:571-76.
Lowe SW, Ruley HE, Jacks T, Housman DE. P53-dependent apoptosis modulates the cytotoxicity of anticancer agent. Cell 1993, 74:957-67.
Ludes-Meyers JH, et al. Transcriptional activation of the human epidermal growth factor receptor promoter by human p53. Mol Cell Biol. 1996 Nov; 16(11): 6009-19.
Mc Donald III ER, Wu GS, Waldman T. Repair defects in p21WAF/CIP1-/- human cancer cells. Cancer Res 1996, 56:2250-255.
Megan J Robinson, et al. Mitogen-activated protein kinase pathways.
Curr Opin Cell Biol. 1997 Apr; 9(2):180-6. Review.
Moch H, Sauter G, Moore D, Waldman F. p53 and erbB-2 protein overexpression are associated with early invasion and metastasis in bladder cancer. Virchows Archiv-A, Pathological Anatomy & Histopathology 1993, 423(5): 329-34.
Nakahara S, et al. Induction of apoptosis signal regulating kinase 1 (ASK1) after spinal cord injury in rats: possible involvement of ASK1-JNK and -p38 pathways in neuronal apoptosis. J Neuropathol Exp Neurol. 1999 May; 58(5): 442-50.
Niimi S, Nagakawa K, Yokota J, Nishio K, Terada M. Resistance to drugs in NIH3T3 cell transfected with c-myc and c-Ha-ras genes. Br. J Cancer 1991, 63:237-41.
Peles E, et al. Regulated coupling of the Neu receptor to phosphatidylinositol 3'-kinase and its release by oncogenic activation. J Biol Chem. 1992 Jun 15; 267(17): 12266-74.
Pietras RJ, Fendly BM, Chazin VR, Pegram MD. Antibody to HER-2/neu receptor blocks DNA repair after cisplatin in human breast and ovarian cancer cells. Oncogene 1994, 9:1829-38.
Ram TG, et al. Phosphatidylinositol 3-kinase recruitment by p185erbB-2 and erbB-3 is potently induced by neu differentiation factor/heregulin during mitogenesis and is constitutively elevated in growth factor-independent breast carcinoma cells with c-erbB-2 gene amplification. Cell Growth Differ. 1996 May; 7(5): 551-61.
Roche S, et al. The phosphatidylinositol 3-kinase alpha is required for DNA synthesis induced by some, but not all, growth factors. Proc Natl Acad Sci U S A. 1994 Sep 13; 91(19): 9185-9.
Roulston A, et al. Early activation of c-Jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor alpha.
J Biol Chem. 1998 Apr 24; 273(17): 10232-9.
Samuel R Denmeade. Programmed cell death (apoptosis) and cancer chemotherapy. Cancer control journal 1998, electronics
Slamon DJ. The effect of HER-2/neu overexpressin on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells. Oncogene 1997, 15(5): 537-47.
Steffens J. and Nagel R. Tumors of the Renal Pelvis and Uterus. Br. J Urol. 1988, 61:277-283.
Tanner B, et al. Prognostic significance of c-erB-2 mRNA in ovarian carcinoma. Gynecol Oncol. 1996 Aug; 62(2): 268-77.
Thompson K, et al. Assay for expression of methotrexate-resistant dihydrofolate reductase activity in the presence of drug-sensitive enzyme.
J Pharmacol Toxicol Methods. 1992 Nov; 28(3): 167-73.
Tricker A.R. et al. urinary excretion of nitrate, nitrite and N-nitroso compounds in schistosomiasis and bilharzia bladder cancer patients. Carcinogenesis vol. 1989, 10(3): 547-52.
Tsai C-M, Chang K-T, Wu LH, Chen JY, et al. Correlation between intrinsic chemoresistance and HER-2/neu gene expression, p53 gene mutation, and cell proliferation characteristics in non-small cell lung cancer cell lines. Cancer Res 1996, 56:206-09.
Wang B, et al. subcellular localization of the Arg protein tyrosine kinase.
Oncogene. 1996 Jul 4; 13(1): 193-7.
Weidner U, et al. Inverse relationship of epidermal growth factor receptor and HER2/neu gene expression in human renal cell carcinoma. Cancer Res. 1990 Aug 1; 50(15): 4504-9.