由Streptomyces chibanensis中所分離而得的azatyrosine [D,L-β(5-hydroxypyridin-2yl)-alanine]，能夠抑制已活化之人類c-Ha-ras, c- raf, c-ErbB-2致癌基因所引發的轉型NIH3T3細胞(transformed NIH3T3 cells)的生長。然而，azatyrosine的主要缺點是必須在高劑量下方才有活性，推測可能是由於它的胺基酸雙性離子化結構使得通透細胞的能力過低。因此，台大藥學系藥物化學研究者將其結構經過修飾並合成一系列azatyrosine醯胺類衍生物，以增加進入細胞內的能力。本研究試圖探知azatyrosinamides : CHY[D,L-N-propyl 3-(5-Benzyloxypyridin-2-yl)-3-(N-t-butoxycarbonylamido)propionamide]; NPR[D,L-N-cyclohexyl 3-(5-Benzyloxypyridin-2-yl)-3-(N-t-butoxy carbonylamido)propionamide]是否具有毒殺細胞的能力，同時並試圖了解其毒殺細胞的機制為何。我們先選定了NIH3T3 wild type、NIH3T3-ras mutant二種細胞株為研究對象。分別作了azatyrosine, NPR 與CHY對上述兩株細胞之細胞毒性(IC50)測試，軟瓊脂膠生長分析(soft agar growth anylasis )，聚落形成分析(plating efficiency analysis)，azatyrosinamides與tyrosine競爭能力分析，DNA合成抑制分析，蛋白質合成抑制分析，細胞核型態觀察(Hoechst stain)，Merocyanine 540 染色，細胞週期分析(cell cycle anylasis)，及DNA片段分析(DNA fragmentation)等。實驗結果顯示: azatyrosine及NPR與CHY，對NIH3T3 wild type、NIH3T3-ras mutant二種細胞株的細胞毒性(IC50)是有差別的。低濃度的NPR與CHY，除了會抑制NIH3T3-ras mutant細胞在軟瓊脂膠(soft agar)中的生長之外，也可以抑制NIH3T3-wild type細胞形成聚落。 NPR與CHY會抑制DNA與蛋白質的合成，但NPR與CHY不會與tyrosine競爭結合至蛋白質上。根據細胞核型態觀察(Hoechst stain)，Merocyanine 540染色，細胞週期分析(cell cycle anylasis)，及DNA片段分析(DNA fragmentation)等結果顯示NPR與CHY對於NIH3T3 wild type、NIH3T3-ras mutant二種細胞株所產生的細胞毒殺作用，可能是藉由細胞凋亡(apoptosis)而來的。

Azatyrosine [L-β-(5-hydroxypyrid-2-yl)-alanine], an antibiotic agent isolated from Streptomyces chibanesis, has been reported to exhibit growth inhibitions of c-Ha-ras,c-raf,c-erB-2 transformed NIH3T3 cells. However, to exert its cytotoxicity azatryrosine should be used at a fairly high concentration. We therefore explore the possibility of using its amide analogues as anticancer agents. Two analogues, designed HPW021×314 NPR and HPW021×314CHY, were found to posses low IC50 values against both NIH3T3 wild type and ras-transformed cell lines and, therefore, were subjected to a series of characterizations. The results are as the following. (1) The IC50 values of the two analogues on both cell lines are much lower than that of azatyrosine, being 29- fold to 101-fold lowers on the wild-type and 86- to 226- fold lower on the ras- transformed cells, respectively. (2) The analogues at very low concentrations are enough to inhibit the growth of the ras-transformed cells on soft agar and the colony formation of the wild-type cells on culture dish. (3) The analogues can inhibit both the syntheses of DNA and protein and tyrosine at high concentrations can not reverse the cytotoxcicities of the analogues on both cell lines, suggesting that the analogues do not compete with tyrosine for incorporation into protein. Hence the analogues have a different action mechanism from that of azatyrosine. (5) The results of Hoechst staining, Merocyanine 540 staining, cell cycle analysis, and DNA fragmentation indicate that the analogues may owe their cytotoxicities to their induction of apoptosis on both cell lines.

一. 授權書
二. 考試合格證明
三. 誌謝感言
四. 摘要
A. 中文摘要
B. 英文摘要 i
ii
iv
五. 目錄
六. 圖表錄 v
vii
七. 縮寫表 ix
八. 研究背景 1
九. 研究目的 10
十. 研究方法
A. 細胞培養 11
B. 生長速率分析 11
C. 細胞洋菜膠生長分析 12
D. 細胞毒殺試驗 13
E. 聚落形成能力分析 14
F. DNA片段之純化及電泳分析 15
G. 細胞週期之分析 17
H. 細胞核型態之觀察 18
I. 蛋白質電泳及西方點墨 19
J. 3H-thymidine Incorporation 24
K. 35S-methionine Incorporation 25
十一. 結果 27
十二. 討論 32
十三. 參考文獻 35
十四. 圖表 42
十五. 自述 70

1.Adrens, M. J., Mcgercor, N. J. and Wyllie, A.H. Apoptosis is inversely related to necrosis and determines net growth in tumors bearing constitutively expressed myc, ras and HPV oncogenes. Amer J Path 144, 1045-1057 (1994).
2.Adrens, M. J., Mcgercor, N. J., Brown, E. J. and Wyllie, A.H. Susceptibility to apoptosis is differentially regulated by c-myc and Ha-ras oncogenes and is associated with endonuclease availability. Br J Cancer 68, 1127-1133 (1993).
3.Barbacid, M. ras genes. Ann Rev Biochem 56, 779-827 (1987).
4.Beitel, G. J., Clark, S. G. and Horvitz, H. R. Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348, 503-509 (1990).
5.Benito, M., Porras, A., Nebreda, A. R. and Santos. E. Differentiation of 3T3-11 fibroblasts to adopocytes induced by transfection of ras oncogenes. Science 253, 565-567 (1990).
6.Benoit, R. M., Eiseman, J., Jacobs, S. C. and Kyprianou, N. Reversion of human prostate tumorigenic growth by azatyrosine. Urology 46, 370-7 (1995).
7.Buday, L. and Downward, J. Epidermal growth factorregulates p21ras through the formation of a complex of receptor, Grb2 adaptor protein, and nucleotide exchange factor. Cell 73, 611-620 (1993).
8.Buno, B. J., White, S. M., Williamson, P. L. and Schlegel, R.A. Plasma membrane lipid organization and the adherence of differentiating lymphocytes to macrophages. J Cellular Physiology 138, 61-69 (1989).
9.Chung, D. L., Brandt-Rauf, P., Murphy, R. B., Nishimura, S., Yamaizumi, Z, Weinstein, I. B. and Picus, M. R. A peptide from the gap-binding domain of the ras-p21 protein and azatyrosine block ras-induced maturation of xenopus oocytes. Anticancer Res 11, 1373-8 (1991).
10.Chung, D. L., Joran, A., Friedman, F., Robinson, R., Brandt-Rauf, P. W., Murphy, R. B., Ronai, Z., Baskin, L., Dykes, D. C., Murphy, R. B., Nishimura, S., Yamaizumi, Z., Weinstein, I. B. and Picus, M. R. Evidence that oocyte maturation induced by an oncogenic ras-p21 protein and insulin is mediated by overlapping yet distinct mechanisms. Exp Cell Res 203, 329-35 (1992).
11.Cohen, J. J. Apoptosis. Immunology Today 14, 126-130 (1993).
12.Cory, S. Regulation of lymphocyte survival by bcl-2 gene family. Ann Rev Immunol 13, 513-543 (1995).
13.Datta, S. R., Dudex, H., Tao, X., Masters, S. and Greenberg, H. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231-241 (1997).
14.Daum, G., Eisenmann-Tappe, I., Fries, H. W., Troppmair, J. and Rapp, U. R. The ins and outs of Raf kinase. TIBS 19, 474-480 (1994).
15.Delong, L. and Lu-Hai, W. Oncogenes, protein tyrosine kinase, and signal transduction. J Biomed Sci 1, 65-82 (1994).
16Dent, P., Wayne, H., Timothy, A. J. H., Leigh, A.V., Yhmas, M. R. and Thomas, W. S. Activation of mitogen-activated protein kinase by r-raf in NIH3T3 cells and in vitro. Science 257, 1404-1407 (1992).
17.Downard, J. Regulatory mechanism for ras protein. Bioessays 14, 177-184 (1992).
18.Egan, S. E., Gidding, B. W., Brook, M. W., Budy, L., Sizedand, A. W. and Weinberg, R. A. Association of Sos ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature 363, 45-51 (1993).
19.Farinelli, S. E. and Green, L. A. Cell cycle blockers mimosine, ciclopirox and deferoxamine prevent the death of PC12 cells and postmitotic sympathetic neurons after removal of trophic support. J Neuroscience 16, 1150-1162 (1996).
20.Feig, L. A. and Cooper, G.M. Inhibition of NIH3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 8, 3235-3243 (1988).
21.Feramisco, J. R., Gross, M., Kamata, T., Rosenberg, M. and Sweet, R. W. Microinjection of oncogene from of the human Ha-ras (t24) protein results in rapid proliferation of quiescent cells. Cell 38, 109-117 (1984).
22.Fujita-Yoshigaki, J., Yokoyama, S., Shindo-Okada, N. and Nishimura, S. Azatyrosine inhibits neurite outgrowth of PC12 cells induced by oncogenic ras. Oncogene 7, 2019-24 (1992).
23.Hiles, I. D., Otsu, M., Volinia, S., Fry, M. J., Gout, I., Dhand, R., Panayotou, G., Ruiz-Larrea, F., Thompson, A., Totty, N. F., Hsuan, J. J., Courtneidge, S. A., Parker, P. J. and Waterfield, M. D. Phosphatidylinsitol 3-kinase; Structure and expression of the 110 kd catalytic subunit. Cell 70, 419-429 (1992).
24.Hirakawa, T. and Ruley, H. E. Reduced of cells from ras oncogene induced growth arrest by second, complementing, oncogene. Proc Natl Acad Sci (USA) 85, 1519-1523 (1988).
25.Inouye, S., Shomura, T., Tsuroka, T., Ogawa, Y., Watanabe, H., Yoshida, J. and Niida, T. L-beta-(5-Hydroxy-2-pyridyl)-alanine and L-beta-3-(3-Hydroxyureido)-alanine from Streptomyces. Chem Pharm Bull 23, 2669 (1975).
26.Izawa, M., Takayama, S., Shindo-Okada, N., Doi, S., Kimura, M., Katsuki, M. and Nishimura, S. Inhibition of chemical carcinogenesis in vivo by azatyrosine. Cancer Res 52, 1628-30 (1992).
27.Jacobson, M. D., Weil, M. and Raff, M. C. Programmed cell death in animal development. Cell 88, 347-354 (1997).
28.Kauffmann-Zeh, A., Rodriguez-Viciana, P., Ulruch, E., Gilbert, C., Coffer, P., Downward, J. and Evan, G. Suppression of c-Myc-induced apoptosis by ras signalling through PI(3)K and PKB. Nature 385, 544-548 (1997).
29.Kennedy, S.G., Wagner, A. J., Conzen, S. D., Jordan, J., Bellacosa, A., Tsichlis, P. N. and Hay, N. The PI-3 kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev 11, 701-713 (1997).
30.King, K. L. and Cidlowski, J. A. Cell cycle and apoptosis: common pathways to life and death. J Cell Biochem 58 (2), 175-180 (1995).
31.Khosravi-Far, R. and Der, C. J. The ras signal transduction pathway., Cancer Metast Rev 13, 67-89 (1994).
32.Koch, C. A., Anderson, D., Mpran, M. F., Ellis, H. and Pawson, T. SH2 and SH3 domains: elements that control interact of cytoplasmic signaling protein. Science 252, 668-678 (1991).
33.Korsmeyer, S. J. Bcl-2: an antidote to programmed cell death. Cancer Surv 15, 105-118 (1992).
34.Kulik, G., Klippel, A. and Weber, M. J. Antiapoptotic signaling by the insulin-like growth factor 1 receptor, Phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 17, 1595-1606 (1997).
35.Kyriakis, J. M., App, H., Zhang, X.. F., Banerjee, P., Brantigan, D. L., Rapp, U. R. and Arruch, J. Raf-1 activates MAP kinase-kinase. Nature 358, 417-421 (1992).
36.Kyprinou, N. and Taylor-Papadimitriou, J. Isolation of azatyrosine induced revertants from ras-transformed human mammary Epithelial Cells. Oncogene 7, 2019-24 (1992).
37.Li, N., Batzer, A., Daly, R., Yajnik, V., Skolnik, E., Chardin, P., Bar-Sagi, D., Margolis, B. and Schlessinger, J. Guanin nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to ras signalling. Nature 363, 85-88 (1993).
38.Lowy, D.R., Function and regulation of ras. Ann Rev Biochem 62, 851-891 (1993).
39.Marshall, C.J. How does p21 ras transform cells. Trends Genet 7, 91-95 (1991).
40.Martin, C. R. Social controls on cell survival and cell death. Nature 356, 397-341 (1992).
41.McEvoy, L., Williamson, P. and Schlegel, R. A. Membrane phospho-lipid asymmetry as a determinant of erythrocyte recognition by macrophage. Proc Natl Acad Sci (USA) 83, 3311-3315 (1986).
42.McEvoy, L., Schlegel, R. A., Williamson, P. and Buono, B. J. Merocyanine 540 as a flow cytometric probe of membrane lipid organization in leukocytes. J Leukocyte Biology 44, 337-344 (1988).
43.Meikrantz, w., Gisselbrcht, S., tam, S. W. and Schlegel, R. Activation of cyclin A-dependent protein kinases during apoptosis. Proc Natl Acad Sci (USA) 91, 3754-3758 (1994).
44.Minn, A. J., Rudin, C. M., Boise, L. H. and Thompson, C. B. Expression of Bcl-xl can confer a multidrug resistence phenotype. Blood 86, 1903-1910 (1995).
45.Monden, Y., Nakamura, T., Kojiri, K., Shindo-Okada, N. and Nishimura, S. Azatyrosine inhibits the activation of c-raf-1,c-jun and AP1 but not the activation of ras during signal tranduction triggered by oncogenic c-erbB-2. Oncology Reports 3, 33-40 (1996).
46.Monden, Y., Nakamura, T., Kojiri, K., Shindo-Okada, N. and Nishimura, S. Stimulation of the expression of rhoB contributes to the ability of azatyrosine to convert c-erbB-2-transformed cells to cells with a normal phenotype. Oncology Reports 3, 49-55 (1996).
47.Monden, Y., Nakamura, T., Kojiri, K., Shindo-Okada, N. and Nishimura, S. Azatyrosine is incorporated into proteins instead of tyrosine residues, with the resultant conversion of transformed cells to cells with a normal phenotype. Oncology Reports 3, 625-629 (1996).
48.Mower, D. A., Peckham, D. W., Illera, V. A., Fishbaugh, J. K., Stunz, L. L. and Ashman, R. F. Decreased membrane phopholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis. J Immunol 152, 4832-4842 (1994).
49.Nimual, A. S., Yatsula, B. A. and Bar-Sagi, D. Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos. Science 279, 560-563 (1998).
50.Nomura, T., Ryoyama, K., Okada, G., Matano, S., Nakamura, S.and Kameyama, T. Non-transformed, but not ras/myc-transformed, serum-free mouse embryo cells recover from growth suppression by azatyrosine. Jap J Cancer Res 83, 851-8 (1992).
51.Parrizas, M., Saltiel, A. R. and LeRoith, D. Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. J Biol Chem 272, 154-161 (1997).
52.Peso, L. D., Gonzales-Garcia, M., Page, C., Herrera, R. and Nunez, G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278, 687-689 (1997).
53.Raff, M. C. Size control:The regulation of cell numbers in animal development. Cell 86, 173-175 (1996).
54.Reddy, S. A., Huang, J. H. and Liao, W. S. Phosphatidylinositol 3-kinase in interleukin 1 signaling. Physical interaction with the interleukin 1 receptor and requirement in NFκB and AP-1 activation. J Biol Chem 272, 29167-29173 (1997).
55.Ridley, A. J., Patersan, H. F., Nobel, M. and Land, H. Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation. EMBO 7, 1635-1645 (1988).
56.Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaessbroeck, B., Gout, I., Fry, M. J., Waterfield, M. D. and Downward, J. Phosphatidyl-inositol-3-OH kinase as a direct target of Ras. Nature 370, 527-532 (1994).
57.Ruch, R. J., Madhukar, B. V., Trosko, J. E. and Klauning, J. E. Reversal of ras-induced inhibition of gap-junctional intercellular communication, transformation, and tumorigenesis by lovastatin. Mol Carcino genesis 267, 24149-24152 (1992).
58.Rubin, L. L., Philpott, K. L. and Brooks, S. F. The cell cycle and cell death. Curr Biol 3, 391-394 (1993).
59.Sakai, N., Ogiso, Y., Fujita, H., Watari, H., Koike, T. and Kuzumaki, N. Induction of apoptosis by a dominant negative Ha-ras mutant (116Y) in K562 cells. Exp Cell Res 215, 131-136 (1994).
60.Satoh, M., Nakafuka, Y. and Kazio, Y. Function of Ras as molecular switch in signal transduction. J Biol Chem 267, 24149-24152.
61.Schegel, R. and Meikratz, W. Apoptosis and the cell cycle. J Cell Biochem 58 (2), 160-174 (1995).
62.Shindo-Okada, N., Makabe, O. and Nishimura, S. Permanent Conversion of mouse and human cells transformed by activated ras or ref genes to apparently normal cells by treatment with the antibiotic azatyrosine. Mol Carcinogenesis 2, 59-67 (1989).
63.Stacey, D. W. and Kung, H. F. Transforming of NIH3T3 cells by microinjection of Ha-ras p21 protein. Nature 310, 508-511 (1984).
64.Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Sciense 267, 1456-1461 (1995).
65.Vaux, D. L., Aguila, H. L. and Weissman, I. L. Bcl-2 pervents death of factor deprived cells but fails to prevent apoptosis in targets of cell mediated killing. Int Immunol 4 (7), 821-824 (1992).
66.Vojiek, A.B.; Hollenberg, S.M. and Cooper, J. A. Mammalian ras interacts directly with the serine/threonine kinase Raf. Cell 74, 205-214 (1993).
67.Wang, H. G., Rapp, U. R. and Reed, J. C. Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell 87, 629-638 (1996).
68.Yu, J., Zhang, Y., Mcilroy, J., Rordorf-Nikolic, T., Orr, G. A. and Backer, J. M. Regulation of the p85/p110 phosphatidylinsitol 3-kinase:stabilization and inhibition of the p110 catalytic subunit by the p85 regulatory subunit. Mol Cell Biol 18, 1379-1387 (1998).
69.李金鳳 PI(3)kinase 及Bcl-2對Ha-ras致癌基因誘導細胞記劃性死亡之影響。 成功大學碩士論文 (1998).