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系統識別號 U0026-0508201321272900
論文名稱(中文) 探討癌細胞中Eps8於Ku70調控的DNA雙股斷裂修復所扮演的角色
論文名稱(英文) The Role of Eps8 in Ku70-mediated DNA Double Strand Break Repair in Cancer Cells
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 101
學期 2
出版年 102
研究生(中文) 蕭潔君
研究生(英文) Chieh-Chun Hsiao
學號 s26004044
學位類別 碩士
語文別 英文
論文頁數 55頁
口試委員 指導教授-呂增宏
口試委員-馬明琪
口試委員-劉校生
口試委員-張雋曦
中文關鍵字 Eps8  Ku70  γ-H2AX  DNA 修復 
英文關鍵字 Eps8  Ku70  HeLa cell  DNA damage repairing 
學科別分類
中文摘要 舉凡鹼基配對缺失或單雙股的斷裂等的DNA受損,皆可能會導致細胞凋亡或是癌症的產生。當癌症發生時,一般會以外科手術或放射性照射合併抗癌藥物來治療。然而,此舉常會使病人產生多重抗藥性。癌細胞會藉由活化non-homologous end-joining (NHEJ)或homologous recombination (HR)來對抗藥物所導致的雙股斷裂。Ku70蛋白主要是透過前者的路徑來修復DNA斷裂。先前實驗室經由yeast two-hybrid system,發現Eps8與Ku70之間是具有交互作用存在。因此,我們進一步想了解,Eps8是否會影響Ku70所調控的DNA修復。首先,給予HeLa細胞抗癌藥物doxorubicin以及cisplatin,使其產生雙股斷裂。接著,利用MTT Assay以及Comet Assay觀察細胞存活率及修復DNA的能力。我們發現分別knockdown Eps8與Ku70皆會使細胞的化學敏感性增加,但當在Ku70低表達的細胞內大量表達Eps8時,則會使細胞具抗藥性。knockdown Eps8與Ku70的細胞內,其DNA受損情形,皆較控制組為嚴重。然而,在Ku70減低表達細胞內表達外送的Eps8,反而會抑制細胞修復DNA的能力。這些結果皆顯示,Ku70與Eps8可能經由不同訊息傳遞路徑來修復DNA。此外,在knockdown Eps8的細胞中,γ-H2AX的表現量相較於控制組,在短時間內有急遽的改變。綜合上述,Eps8與Ku70可能循不同的訊息傳遞路徑,調控DNA的修復。
英文摘要 DNA damage, such as base damage and DNA strand breaks, might result in cell apoptosis or cancer formation. Double strand breaks (DSBs) are the primary cytotoxic lesion induced by ionizing radiation (IR) and DNA-targeting anti-cancer drugs. The conventional chemotherapeutic drugs are used together with surgery or radiation therapy to treat metastatic cancers, but usually cause multidrug resistance (MDR). DNA repair ability might be one of the causes of anti-cancer drug resistance. Cancer cells may activate non-homologous end-joining (NHEJ) or homologous recombination (HR) to repair DSBs against DNA-damaging agents. The Ku heterodimer (Ku70/Ku80) is one of the main component of the NHEJ pathway that repairs DNA double-strand breaks (DSBs). We are interested in studying Ku70-interacting proteins, such as Eps8, in DSBs repair. First, we generate HeLa cell expressing Eps8-siRNA or Ku70-siRNA with/without ectopic Eps8. Next, we investigate the participation of Eps8 in the repairing pathway of DNA-damaging agents (doxorubicin and cisplatin) induced DSBs. Single cell electrophoresis (comet assay) is utilized to estimate the degree of DNA damage or DNA repairing ability. We found that doxorubicin- and cisplatin-induced cytotoxicity in Ku70 knockdown cells was decreased by ectopic Eps8. Next, attenuation of Eps8 inhibits DSBs-inudced phosphorylation of H2AX. Intestingly, silencing either Eps8 or Ku70 impaired DSBs repairing. However, ectopically expressed Eps8 further worsen DNA damaging agents-induced DNA damage in Ku70 knockdown cells. This phenomenon can’t be found in cells expressing dominant negative Eps8. In conclusion, Eps8-mediated DSBs repairing might be via a different pathway from Ku70.
論文目次 Abstract 2
中文摘要(Abstract in Chinese) 3
Acknowledgement 4
Table of Contents 5
List of Figures 7
Abbreviations 8
Introduction 9
I: Ku70 10
II: Eps8 act as an oncoprotein 11
III: γ-H2AX: a marker of DSB 12
IV:Tumor suppresor gene P53 13
V:The mode of cytotoxic action and DNA damaging agent 14
Materials and Methods 17
I. Materials 17
A. Cell lines 17
B. Plasmid 17
C. Drugs 17
II. Methods 20
1. Cell culture 20
2. Cell lines generation 20
3. Immunoblotting 21
4. primary antibody and reagents 23
5. MTT assay 23
6. Comet assay 23
7. Statistic analysis 24
Results 25
I. Eps8 interacts with Ku70 in cancer cells 25
II. Attenuation of Eps8 decrease DNA repairing ability in doxorubicin-treated HeLa cells 25
III. Attenuation of Eps8 increases the chemosensitivities of anti-cancer drugs in HeLa cells 26
IV. Participation of Eps8 and Ku70 in DSBs-repair may via different pathways in anti-cancer drug-treated HeLa cells 27
V. Participation of Eps8 and Ku70 in DSBs-repair may alter the phosphorylation of H2AX in anti-cancer drug-treated HeLa cells 28
VI. Attenuation of Eps8 decreases DSB-induced Chk1 phosphorylation in HeLa cells 28
VII. Attenuation of Eps8 increases DSB-induced P53 phosphorylation in HeLa cells 29
Discussion 31
References 34
Figures 39



參考文獻 Andrea Kinner., Wenqi Wu., Christian Staudt and George Iliakis. (2008). r-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Research 17, 5678–5694.

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.

Cheng Q., Barboule N., Frit P., Gomez D., Bombarde O., Couderc B., Ren GS., Salles B., Calsou P.(2011). Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks. Nucleic Acids Research 22, 9605-9619

Chuan-Lan Hsu. (2012) Participation of Eps8-Ku70 interaction in cancer cell proliferation. Master thesis.

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.


Dana Branzei ., Marco Foiani. (2008). Regulation of DNA repair throughout the cell cycle. Molecular cell biology 9, 297-308.

Delmastro, D. A., Li, J., Vaisman, A., Solle, M., and Chaney, S. G. (1997). DNA damage inducible-gene expression following platinum treatment in human ovarian carcinoma cell lines. Cancer chemotherapy and pharmacology 39, 245-253.

Demarcq, C., Bunch, R. T., Creswell, D., and Eastman, A. (1994). The role of cell cycle progression in cisplatin-induced apoptosis in Chinese hamster ovary cells. Cell Growth Differ 5, 983-993.

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.

Hawkins D.S., Demers GW., Galloway DA. (1996).Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents. Cancer Res 56, 892-898.

Hollstein,M., Sidransky, D., Vogelstein, B., and Harris, C. C. (1991). p53 mutations in human cancers. Science , NY 253, 49-53.

Jamieson, E. R., and Lippard, S. J. (1999). Structure, Recognition, and Processing of Cisplatin-DNA Adducts. Chemical reviews 99, 2467-2498.


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.

Ling F.H., Lu V., Svec F., Fréchet J.M. (2002). Effect of multivalency on the performance of enantioselective separation media for chiral HPLC prepared by linking multiple selectors to a porous polymer support via aliphatic dendrons. J Org Chem 67, 1993-2002.

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.

Michael B. Kastan. (2008). DNA Damage Responses: Mechanisms and Roles in Human Disease: 2007 G.H.A. Clowes Memorial Award Lecture. Mol Cancer Research 6, 517-524.

Micheau, O., Solary, E., Hammann, A., Martin, F., and Dimanche-Boitrel, M. T. (1997). Sensitization of cancer cells treated with cytotoxic drugs to fas-mediated cytotoxicity. Journal of the National Cancer Institute 89, 783-789.

Monika Podhorecka., Andrzej Skladanowski., Przemyslaw Bozko. (2010). H2AX Phosphorylation: Its Role in DNA Damage Response and Cancer Therapy. Journal of nucleic acids 6, 673-675

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.

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.

Tang, D., Wu, D., Hirao, A., Lahti, J. M., Liu, L., Mazza, B., Kidd, V. J., Mak, T. W., and Ingram, A. J. (2002). ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53. The Journal of biological chemistry 277, 12710-12717.

Vogelstein, B., Lane, D., and Levine, A. J. (2000). Surfing the p53 network. Nature 408, 307-310.

Vousden, K. H. (2005). Apoptosis p53 and PUMA: a deadly duo. Science New York, NY 309, 1685-1686.

Wang, D., and Lippard, S. J. (2005). Cellular processing of platinum anticancer drugs. Nature reviews 4, 307-320.

Wang H, Perrault AR, Takeda Y, Qin W, Wang H, Iliakis G. (2003). Biochemical evidence for Ku-independent backup pathways of NHEJ. Nucleic Acids Research 31, 5377-5388.

Yap LF, Jenei V, Robinson CM, Moutasim K, Benn TM, Threadgold SP, Lopes V, Wei W, Thomas GJ, Paterson IC. (2009). Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation. Oncogene 27, 2524-34.
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