進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-1907201216534600
論文名稱(中文) 研究Eps8-Ku70交互作用在癌細胞增生中所扮演的角色
論文名稱(英文) Participation of Eps8-Ku70 interaction in cancer cell proliferation
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 100
學期 2
出版年 101
研究生(中文) 許涓藍
研究生(英文) Chuan-Lan Hsu
學號 s26994100
學位類別 碩士
語文別 中文
論文頁數 81頁
口試委員 指導教授-呂增宏
口試委員-賴明德
口試委員-馬明琪
口試委員-張雋曦
中文關鍵字 Eps8  Ku70  EGFR  STAT3  細胞增生  HeLa cell 
英文關鍵字 Eps8  Ku70  EGFR  Stat3  cell proliferation  HeLa cell 
學科別分類
中文摘要 Ku70影響許多細胞生理功能,像是V(D)J recombination、DNA修復、telomere maintenance和細胞凋亡。然而,近年來Ku70的研究大多都是DNA修復方面的研究,至於在細胞生長分裂的角色則不清楚。近年來的研究發現 (1) 在EGFR signaling被抑制的時候,Ku70會和EGFR、DNA-PK產生複合體; (2) Eps8 (EGF receptor pathway substrate No.8)為EGFR的受質,且大量表現Eps8會促進EGF調控細胞增生作用; (3) Eps8在大腸直腸癌細胞和子宮頸癌細胞會有大量表現的情形,若專一抑制Eps8的表達則可以抑制腫瘤的形成。由於在yeast two-hybrid system中, 我們發現Eps8 和Ku70會有交互作用的產生,因此,我們認為Ku70可能參與Eps8引發的癌細胞形成。首先,我們利用siRNA技術 knockdown Ku70,發現knockdown Ku70會抑制細胞生長和colony 形成,且在Ku70 knockdown的細胞送入Eps8會使此現象有回復的情形。另外,在EGF刺激下,我們發現Ku70 knockdown會使EGFR下游的STAT3磷酸化被抑制,且此現象也會被外送的Eps8回復。進一步我們發現Ku70 knockdown所造成的STAT3活化被抑制是因為Eps8和IRSp53的交互作用被抑制所導致。總結以上,我們發現Ku70 knockdown會造成Eps8和IRSp53交互作用被抑制,進而減少STAT3的活化,導致細胞增生被抑制。
英文摘要 The DNA end-joining protein Ku70 is important in the maintenance of genomic DNA integrity and is involved in many cellular processes, including V(D)J recombination, DNA repair, telomere maintenance and cell death. However, the role of Ku70 in cell proliferation is still vague. Given that EGFR interacts with both DNA-PK and Ku70 when EGFR-mediated signaling is inhibited, Ku70 might participate in EGFR-mediated mitogenesis. In a yeast two-hybrid screening, we have identified Ku70 as an Eps8-interacting protein. In addition, we observed that attenuation of Ku70 in HeLa cells inhibited the cell proliferation and colony formation in soft agar, which could be rescued by ectopically expressed Eps8. Meanwhile, attenuation of Ku70 abolished EGF-mediated Stat3 activation. And, ectopic Eps8 could recover this defect. Finally, Ku70 knockdown decreased the IRSp53-Eps8 interaction in EGF-stimulated HeLa cells. Taken together, our data suggested that Ku70 attenuation reduced the interaction between Eps8 and IRSp53 which resulted in the suppression of EGF receptor-mediated Stat3 activation and the following cell proliferation.
論文目次 中文摘要 1
英文摘要 3
縮寫簡表 7
第一章 緒論 9
第二章 實驗材料與方法 18
第一節 實驗材料 19
第二節 實驗方法 22
第三章 實驗結果 41
第四章 討論 51
第五章 圖表 57
參考文獻 76

參考文獻 Araki, R., Fukumura, R., Fujimori, A., Taya, Y., Shiloh, Y., Kurimasa, A., Burma, S., Li, G.C., Chen, D.J., Sato, K., et al. (1999). Enhanced phosphorylation of p53 serine 18 following DNA damage in DNA-dependent protein kinase catalytic subunit-deficient cells. Cancer Res 59, 3543-3546.

Avraham, R., and Yarden, Y. (2011). Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nat Rev Mol Cell Biol 12, 104-117.

Bandyopadhyay, D., Mandal, M., Adam, L., Mendelsohn, J., and Kumar, R. (1998). Physical interaction between epidermal growth factor receptor and DNA-dependent protein kinase in mammalian cells. J Biol Chem 273, 1568-1573.

Castle, V., Kwok, R., Opipari, A., and Subramanian, C. (2010). Ku70 acetylation in neuroblastoma pathogenesis and therapy. Trans Am Clin Climatol Assoc 121, 183-191; discussion 191.

Chen, Y.J., Shen, M.R., Maa, M.C., and Leu, T.H. (2008). Eps8 decreases chemosensitivity and affects survival of cervical cancer patients. Mol Cancer Ther 7, 1376-1385.

Cohen, H.Y., Lavu, S., Bitterman, K.J., Hekking, B., Imahiyerobo, T.A., Miller, C., Frye, R., Ploegh, H., Kessler, B.M., and Sinclair, D.A. (2004). Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis. Molecular Cell 13, 627-638.

Ding, X., Yang, Z., Zhou, F., Hu, X., Zhou, C., Luo, C., He, Z., Liu, Q., Li, H., Yan, F., et al. (2012). Human Intersectin 2 (ITSN2) binds to Eps8 protein and enhances its degradation. BMB Rep 45, 183-188.

Fazioli, F., Minichiello, L., Matoska, V., Castagnino, P., Miki, T., Wong, W.T., and Di Fiore, P.P. (1993). Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals. EMBO J 12, 3799-3808.

Hacker, S., Karl, S., Mader, I., Cristofanon, S., Schweitzer, T., Krauss, J., Rutkowski, S., Debatin, K.M., and Fulda, S. (2011). Histone deacetylase inhibitors prime medulloblastoma cells for chemotherapy-induced apoptosis by enhancing p53-dependent Bax activation. Oncogene 30, 2275-2281.

Herbst, R.S. (2004). Review of epidermal growth factor receptor biology. Int J Radiat Oncol Biol Phys 59, 21-26.

Husseini, G.A., El-Fayoumi, R.I., O'Neill, K.L., Rapoport, N.Y., and Pitt, W.G. (2000). DNA damage induced by micellar-delivered doxorubicin and ultrasound: comet assay study. Cancer Lett 154, 211-216.

Hynes, N.E., and Lane, H.A. (2005). ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 5, 341-354.

Jin, S., and Weaver, D.T. (1997). Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions. EMBO J 16, 6874-6885.

Koike, M. (2002). Dimerization, translocation and localization of Ku70 and Ku80 proteins. J Radiat Res (Tokyo) 43, 223-236.

Kurimasa, A., Kumano, S., Boubnov, N.V., Story, M.D., Tung, C.S., Peterson, S.R., and Chen, D.J. (1999). Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining. Molecular and Cellular Biology 19, 3877-3884.

Lanzetti, L., Rybin, V., Malabarba, M.G., Christoforidis, S., Scita, G., Zerial, M., and Di Fiore, P.P. (2000). The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5. Nature 408, 374-377.

Leu, T.H., Yeh, H.H., Huang, C.C., Chuang, Y.C., Su, S.L., and Maa, M.C. (2004). Participation of p97Eps8 in Src-mediated transformation. J Biol Chem 279, 9875-9881.

Li, B., Reddy, S., and Comai, L. (2011). Depletion of Ku70/80 reduces the levels of extrachromosomal telomeric circles and inhibits proliferation of ALT cells. Aging (Albany NY) 3, 395-406.
Liu, P.S., Jong, T.H., Maa, M.C., and Leu, T.H. (2010). The interplay between Eps8 and IRSp53 contributes to Src-mediated transformation. Oncogene 29, 3977-3989.

Maa, M.C., Hsieh, C.Y., and Leu, T.H. (2001). Overexpression of p97Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97Eps8-mediated ERK activation. Oncogene 20, 106-112.

Maa, M.C., Lee, J.C., Chen, Y.J., Lee, Y.C., Wang, S.T., Huang, C.C., Chow, N.H., and Leu, T.H. (2007). Eps8 facilitates cellular growth and motility of colon cancer cells by increasing the expression and activity of focal adhesion kinase. J Biol Chem 282, 19399-19409.

Mahaney, B.L., Meek, K., and Lees-Miller, S.P. (2009). Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. Biochem J 417, 639-650.

Mimori, T., Hardin, J.A., and Steitz, J.A. (1986). Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. J Biol Chem 261, 2274-2278.

Mitra, S., Lee, J.S., Cantrell, M., and Van den Berg, C.L. (2011). c-Jun N-terminal kinase 2 (JNK2) enhances cell migration through epidermal growth factor substrate 8 (EPS8). J Biol Chem 286, 15287-15297.

Mongiovi, A.M., Romano, P.R., Panni, S., Mendoza, M., Wong, W.T., Musacchio, A., Cesareni, G., and Di Fiore, P.P. (1999). A novel peptide-SH3 interaction. EMBO J 18, 5300-5309.

Nyati, M.K., Morgan, M.A., Feng, F.Y., and Lawrence, T.S. (2006). Integration of EGFR inhibitors with radiochemotherapy. Nat Rev Cancer 6, 876-885.

Octavia, Y., Tocchetti, C.G., Gabrielson, K.L., Janssens, S., Crijns, H.J., and Moens, A.L. (2012). Doxorubicin-induced cardiomyopathy: From molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol 52, 1213-1225.

Okamura-Oho, Y., Miyashita, T., and Yamada, M. (2001). Distinctive tissue distribution and phosphorylation of IRSp53 isoforms. Biochem Biophys Res Commun 289, 957-960.

Rampakakis, E., Di Paola, D., and Zannis-Hadjopoulos, M. (2008). Ku is involved in cell growth, DNA replication and G1-S transition. J Cell Sci 121, 590-600.

Salomon, D.S., Brandt, R., Ciardiello, F., and Normanno, N. (1995). Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 19, 183-232.

Salti, G.I., Das Gupta, T.K., and Constantinou, A.I. (2000). A novel use for the comet assay: detection of topoisomerase II inhibitors. Anticancer Res 20, 3189-3193.

Santos, S.D., Verveer, P.J., and Bastiaens, P.I. (2007). Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate. Nat Cell Biol 9, 324-330.

Santra, M.K., Wajapeyee, N., and Green, M.R. (2009). F-box protein FBXO31 mediates cyclin D1 degradation to induce G1 arrest after DNA damage. Nature 459, 722-725.

Schmidt-Glenewinkel, H., Reinz, E., Eils, R., and Brady, N.R. (2009). Systems biological analysis of epidermal growth factor receptor internalization dynamics for altered receptor levels. J Biol Chem 284, 17243-17252.

Schulze, W.X., Deng, L., and Mann, M. (2005). Phosphotyrosine interactome of the ErbB-receptor kinase family. Mol Syst Biol 1, 2005 0008.

Scita, G., Nordstrom, J., Carbone, R., Tenca, P., Giardina, G., Gutkind, S., Bjarnegard, M., Betsholtz, C., and Di Fiore, P.P. (1999). EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401, 290-293.

Shaheen, F.S., Znojek, P., Fisher, A., Webster, M., Plummer, R., Gaughan, L., Smith, G.C., Leung, H.Y., Curtin, N.J., and Robson, C.N. (2011). Targeting the DNA double strand break repair machinery in prostate cancer. PLoS One 6, e20311.

Sorkin, A., and Goh, L.K. (2008). Endocytosis and intracellular trafficking of ErbBs. Exp Cell Res 314, 3093-3106.

Subramanian, C., Opipari, A.W., Jr., Bian, X., Castle, V.P., and Kwok, R.P. (2005). Ku70 acetylation mediates neuroblastoma cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci U S A 102, 4842-4847.

Suetsugu, S., Murayama, K., Sakamoto, A., Hanawa-Suetsugu, K., Seto, A., Oikawa, T., Mishima, C., Shirouzu, M., Takenawa, T., and Yokoyama, S. (2006). The RAC binding domain/IRSp53-MIM homology domain of IRSp53 induces RAC-dependent membrane deformation. J Biol Chem 281, 35347-35358.

Tuteja, R., and Tuteja, N. (2000). Ku autoantigen: A multifunctional DNA-binding protein. Critical Reviews in Biochemistry and Molecular Biology 35, 1-33.

Wang, Y.N., Yamaguchi, H., Hsu, J.M., and Hung, M.C. (2010). Nuclear trafficking of the epidermal growth factor receptor family membrane proteins. Oncogene 29, 3997-4006.

Xi, L., Zhu, S.G., Das, A., Chen, Q., Durrant, D., Hobbs, D.C., Lesnefsky, E.J., and Kukreja, R.C. (2012). Dietary inorganic nitrate alleviates doxorubicin cardiotoxicity: Mechanisms and implications. Nitric Oxide 26, 274-284.


論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2022-12-31起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw