DNA受到傷害會造成鹼基的破壞及DNA strand的斷裂，進而產生癌症，oxygen radicals對於人類癌症(例如大腸癌、乳癌及前列腺癌)的形成具有重要的影響。此外，當p97Eps8高度表達在動物實驗中會促進腫瘤的形成，若專一性抑制Eps8的表達則發現可以抑制腫瘤的形成。DNA damage及Eps8對於癌症的形成都有關係，但目前尚未有文獻證實Eps8與DNA傷害及修補之間的直接關係，所以我們想進一步瞭解H2O2所引發的DNA damage是否會與Eps8的表達有關係。本研究利用單細胞電泳法(single cell electrophoresis; comet assay)來研究DNA damage的情形，當細胞受到的DNA傷害的程度增加，comet score也會隨之增加；反之亦然。首先，我們將Eps8的表現量不同的數種大腸癌細胞(SW480, SW620, HCT116, HT29, LS174T, WiDr)處理不同劑量的H2O2以造成DNA的傷害，探討細胞株當中Eps8的表現量的差別是否會影響DNA damage回復的能力，結果我們發現在Eps8表現量較高的SW620、LS174T、WiDr中，其comet score隨時間下降的情形比Eps8表現量較低的SW480、HCT116、HT29來的明顯，由此可知Eps8的表現量與DNA受到傷害後修補的能力具有相關性。接下來，我們想暸解抑制Eps8表現量，是否會影響DNA受到傷害後修補的情形，所以我們利用Eps8 knockdown的細胞株，處理不同劑量的H2O2造成DNA的傷害，觀察不同時間後comet score是否會有差異，由實驗結果顯示抑制Eps8 的表現量，DNA受到傷害之後其修補的能力也會受到抑制。Eps8可能是透過與其他蛋白質之間的作用，來影響DNA傷害的修補作用。

DNA damage (eg. oxidative damage), such as base damage and DNA strand breaks, results in cancer formation. Oxygen radicals are a necessary component of a variety of human cancers including colon, breast and prostatic cancer. In addition, overexpression of p97Eps8 promote the tumor formation in mice as compared to control. Down-regulation of Eps8 suppress v-Src-mediated tumor formation. DNA damage as well as Eps8 are related to cancer formation. But the relationship between Eps8 and DNA damage is not clear. Therefore, we investigate the participation of Eps8 in the repairing pathway of H2O2-induced DNA damage. Single cell electrophoresis (comet assay) is utilized to estimate DNA damage and its repair. Thus, comet score reflects the degree of DNA damage. First, colon cancer cell lines (SW480, SW620, HCT116, HT29, LS174T, WiDr) expressing Eps8 at different levels are used. We treat these cells with various doses of H2O2 to cause their DNA damage. Then, we analyzed the correlation between Eps8 expression and DNA damage repairing ability. We observed that the comet score of the high Eps8 expessing cells (SW620, LS174T and WiDr) is lower than the low Eps8 expessing cells (SW480, HCT116 and HT29) at 24 hr after H2O2 treatment. Therefore, expression of Eps8 is related to DNA damage repairing ability. Then, we investigate whether inhibition of Eps8 would affect cellular ability of DNA repair. We treat Eps8-knockdown cells with various doses of H2O2. The result indicated that inhibition of Eps8 mitigate DNA damage repairing ability. Eps8 may affect DNA damage repairing ability through interacting with cellular repairing machinery.

Abdel-Rahman WM, Mecklin JP, Peltomaki P. (2006) The genetics of HNPCC: Application to diagnosis and screening. Crit Rev Oncol Hematol. 58: 208-20.

Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. (2002) Molecular Biology of the Cell (Fourth Edition). Garland Science Publishing.

Aquilina G, Bignami M. (2001) Mismatch repair in correction of replication errors and processing of DNA damage. J Cell Physiol. 187: 145-54. Review.

Baker SM, Bronner CE, Zhang L, Plug AW, Robatzek M, Warren G, Elliott EA, Yu J, Ashley T, Arnheim N, Flavell RA, Liskay RM. (1995) Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis. Cell 82: 309-19.

Ban C, Yang W. (1998) Crystal structure and ATPase activity of MutL: implications for DNA repair and mutagenesis. Cell. 95: 541-52.

Ban C, Junop M, Yang W. (1999) Transformation of MutL by ATP binding and hydrolysis: a switch in DNA mismatch repair. Cell. 97: 85-97.

Biesova Z, Piccoli C, Wong WT. (1997) Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth. Oncogene. 14: 233-41.

Buermeyer AB, Deschenes SM, Baker SM, Liskay RM. (1999) Mammalian DNA mismatch repair. Annu Rev Genet. 33: 533-64. Review.

Calderwood DA, Fujioka Y, de Pereda JM, Garcia-Alvarez B, Nakamoto T, Margolis B, McGlade CJ, Liddington RC, Ginsberg MH. (2003) Integrin beta cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling. Proc Natl Acad Sci U S A. 100: 2272-7.

Cannavo E, Marra G, Sabates-Bellver J, Menigatti M, Lipkin SM, Fischer F, Cejka P, Jiricny J. (2005) Expression of the MutL homologue hMLH3 in human cells and its role in DNA mismatch repair. Cancer Res. 65: 10759-66.

Castagnino P, Biesova Z, Wong WT, Fazioli F, Gill GN, Di Fiore PP. (1995) Direct binding of eps8 to the juxtamembrane domain of EGFR is phosphotyrosine- and SH2-independent. Oncogene 10: 723-9.

Chen PC, Dudley S, Hagen W, Dizon D, Paxton L, Reichow D, Yoon SR, Yang K, Arnheim N, Liskay RM, Lipkin SM. (2005) Contributions by MutL homologues Mlh3 and Pms2 to DNA mismatch repair and tumor suppression in the mouse. Cancer Res. 65: 8662-70.

Di Fiore PP, Scita G. (2002) Eps8 in the midst of GTPases. Int J Biochem Cell Biol. 34: 1178-83. Review.

Dutta R, Inouye M. (2000) GHKL, an emergent ATPase/kinase superfamily.
Trends Biochem Sci. 25: 24-8. Review.

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

Ferguson KM, Lemmon MA, Schlessinger J, Sigler PB. (1995) Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell 83: 1037-46.

Flores-Rozas H, Kolodner RD. (1998) The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations. Proc Natl Acad Sci U S A. 95: 12404-9.

Friedberg EC, Walker GC and Siede W. (1995) DNA Repair and Mutagenesis. ASM Press, Washington, DC.

Guarne A, Ramon-Maiques S, Wolff EM, Ghirlando R, Hu X, Miller JH, Yang W. (2004) Structure of the MutL C-terminal domain: a model of intact MutL and its roles in mismatch repair. EMBO J. 23: 4134-45.

Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JP, Markowitz S, Willson JK, Hamilton SR, Kinzler KW, Kane MF, Kolodner RD, Vogelstein B, Kunkel TA, Baylin SB. (1998) Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A. 95: 6870-5.

Imlay JA, Linn S. (1988) DNA Damage and Oxygen Radical Toxicity. Science 240: 1302-1309.

Imlay JA. (2003) Pathways of oxidative damage. Annu Rev Microbiol. 57:395-418.

Jiricny J. (2000) Mediating mismatch repair. Nat Genet. 24: 6-8.

Jiricny J, Nystrom-Lahti M. (2000) Mismatch repair defects in cancer. Curr Opin Genet Dev. 10: 157-61. Review.

Jiricny J, Marra G. (2003) DNA repair defects in colon cancer. Curr Opin Genet Dev. 13: 61-9. Review.

Junop MS, Yang W, Funchain P, Clendenin W, Miller JH. (2003) In vitro and in vivo studies of MutS, MutL and MutH mutants: correlation of mismatch repair and DNA recombination. DNA Repair (Amst). 2: 387-405.

Karlsson T, Songyang Z, Landgren E, Lavergne C, Di Fiore PP, Anafi M, Pawson T, Cantley LC, Claesson-Welsh L, Welsh M. (1995) Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins.
Oncogene 10: 1475-83.

Kishan KV, Scita G, Wong WT, Di Fiore PP, Newcomer ME. (1997) The SH3 domain of Eps8 exists as a novel intertwined dimer. Nat Struct Biol. 4: 739-43.

Kolodner RD, Marsischky GT. (1999) Eukaryotic DNA mismatch repair.
Curr Opin Genet Dev. 9: 89-96. Review.

Lengauer C, Kinzler KW, Vogelstein B. (1998) Genetic instabilities in human cancers. Nature 396: 643-9. Review.

Leu TH, Yeh HH, Huang CC, Chuang YC, Su SL, Maa MC. (2004) Participation of p97Eps8 in Src-mediated transformation. J Biol Chem. 279: 9875-81.

Lipkin SM, Wang V, Jacoby R, Banerjee-Basu S, Baxevanis AD, Lynch HT, Elliott RM, Collins FS. (2000) MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability. Nat Genet. 24: 27-35.

Maa MC, Lai JR, Lin RW, Leu TH. (1999) Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src. Biochim Biophys Acta. 1450: 341-51.

Maa MC, Hsieh CY, Leu TH. (2001) Overexpression of p97Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97Eps8-mediated ERK activation. Oncogene. 20: 106-12.

Marra G, Iaccarino I, Lettieri T, Roscilli G, Delmastro P, Jiricny J. (1998) Mismatch repair deficiency associated with overexpression of the MSH3 gene.
Proc Natl Acad Sci U S A. 95: 8568-73.

Matoskova B, Wong WT, Salcini AE, Pelicci PG, Di Fiore PP. (1995) Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation. Mol Cell Biol. 15: 3805-12.

Matoskova B, Wong WT, Nomura N, Robbins KC, Di Fiore PP. (1996) RN-tre specifically binds to the SH3 domain of eps8 with high affinity and confers growth advantage to NIH3T3 upon carboxy-terminal truncation.
Oncogene 12: 2679-88.

Mitra S, Boldogh I, Izumi T, Hazra TK. (2001) Complexities of the DNA base excision repair pathway for repair of oxidative DNA damage. Environ Mol Mutagen. 38: 180-90. Review.

Mongiovi AM, Romano PR, Panni S, Mendoza M, Wong WT, Musacchio A, Cesareni G, Di Fiore PP. (1999) A novel peptide-SH3 interaction. EMBO J. 18: 5300-9.

Peltomaki P. (2001) DNA mismatch repair and cancer. Mutat Res. 488: 77-85. Review.

Pitcher JA, Touhara K, Payne ES, Lefkowitz RJ. (1995) Pleckstrin homology domain-mediated membrane association and activation of the beta-adrenergic receptor kinase requires coordinate interaction with G beta gamma subunits and lipid. J Biol Chem. 270: 11707-10.

Santucci-Darmanin S, Neyton S, Lespinasse F, Saunieres A, Gaudray P, Paquis-Flucklinger V. (2002) The DNA mismatch-repair MLH3 protein interacts with MSH4 in meiotic cells, supporting a role for this MutL homolog in mammalian meiotic recombination. Hum Mol Genet. 11: 1697-706.

Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M, Di Fiore PP. (2001) An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine. J Cell Biol. 154: 1031-44.

Slupphaug G, Kavli B, Krokan HE. (2003) The interacting pathways for prevention and repair of oxidative DNA damage. Mutat Res. 531: 231-51. Review.

Thomas DC, Roberts JD, Kunkel TA. (1991) Heteroduplex repair in extracts of human HeLa cells. J Biol Chem. 266: 3744-51.

Troll W. (1991) Prevention of cancer by agents that suppress oxygen radical formation. Free Radic Res Commun. 12-13 Pt 2: 751-7.

Wang TF, Kleckner N, Hunter N. (1999) Functional specificity of MutL homologs in yeast: evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction. Proc Natl Acad Sci U S A. 96: 13914-9.

Wong WT, Carlomagno F, Druck T, Barletta C, Croce CM, Huebner K, Kraus MH, Di Fiore PP. (1994) Evolutionary conservation of the EPS8 gene and its mapping to human chromosome 12q23-q24. Oncogene 9: 3057-61.

Wu X, Platt JL, Cascalho M. (2003) Dimerization of MLH1 and PMS2 limits nuclear localization of MutL. Mol Cell Biol. 23: 3320-8.