由早先的研究得知，Eps8 (EGF receptor pathway substract NO.8) 同時為一些receptor tyrosine kinases與 cytoplasmic tyrosine kinase Src 的受質之一。在許多不同的細胞株中，Eps8都有分子量97-kDa 和68-kDa 兩種isoforms。在我們先前的研究指出p97Eps8的過度表達不但能促使細胞forcus的形成，在動物實驗中也能促進腫瘤的形成。此外在經v-Src轉型的細胞 (IV5) 中，我們也發現p97Eps8 與 p68Eps8不論在蛋白的表現量或其酪胺酸的磷酸化都有大量增加的情形。為了更進一步了解Eps8 在v-Src造成細胞轉型作用中所扮演的角色，我們利用siRNAs 的技術，專一性抑制Eps8的表達，來研究v-Src造成細胞轉型作用是否受到影響。首先，我們挑出表現eps8 siRNA的IV5細胞株 (eps8 siRNA-1與eps8 siRNA-2) 以及表現negative control的細胞株 (Ctrl-1與Ctrl-2)。我們證實eps8 siRNA-1與eps8 siRNA-2細胞內Eps8的表達有受到抑制的現象，而且不論在細胞的生長上或在動物腫瘤的形成上，都會受到明顯的抑制。我們進一步表達出含有p97eps8-specific siRNA的IV5細胞株 (p97eps8 siRNA-25與p97eps8 siRNA-31)，可以發現這些細胞生長速度與動物腫瘤的形成都同樣有降低的結果。我們也證實細胞生長減緩的結果並非由於v-Src kinase 活性下降所致。接著我們以fluorescence activated cell sorting (FACS)分析細胞的cell cycle，從實驗結果發現eps8 siRNA細胞株與p97eps8 siRNA細胞株有G1 phase-retarded及S-phase減少的現象。最後，我們證實了p97eps8 siRNA細胞株生長速度的下降可能是藉由抑制AKT的活化。因此，p97Eps8主要扮演在v-Src-mediated growth acceleration上，而影響細胞的轉型作用。

　　Eps8 (EGF receptor pathway substrate NO.8) is a common substrate of various receptor tyrosine kinases and cytoplasmic tyrosine kinase Src. It exists in two isoforms, p97Eps8 and p68Eps8 in many cell lines. Our previous studies have indicated that C3H10T1/2 fibroblast overexpressing p97Eps8 not only exhibits the ability of focus formation in cell culture but also promotes the tumor formation in mice as compared to the control cells. Since both tyrosyl phosphorylation and protein expression of Eps8 are elevated in v-Src transformed cell IV5, we wonder whether Eps8 may participate in v-Src-mediated transformation. By utilizing small interference RNA (siRNA) technology, we have generated IV5 cell lines with reduced Eps8 (both 97-kDa and 68-kDa proteins) expression. The cell growth (in both culture dish and animal) of these cell lines is significantly slower than the control cells. Furthermore, we have generated p97eps8-specific siRNA cell lines. Similarly, p97eps8 siRNA cells have slower growth rate than control cells. We also have demonstrated that the remarkable effect was not result form down–regulation of v-Src kinase activity. FACS analysis indicitated that these cells exhibit retardation of G1 phase progresssion as compared to the control cells. Finally, We have demonstrated that inhibition of v-Src-mediated transformation in p97eps8 siRNA cells may result form down-regulation of AKT activation. Thus, p97Eps8 may play an important role in v-Src-mediated cell cycle progression and transformation.

Abram CL and Courtneidge SA. (2000). Src family tyrosine kinases and growth factor signaling. Exp. Cell Res., 254, 1-13.

Bernstein E, Causy AA, Hammond SM and Hannon GJ. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409, 363-366.

Biesova Z, Piccoli C and Wong WT. (1997).Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth. Oncogene, 14, 233- 241.

Brown MT and Cooper JA. (1996). Regulation, substrates and functions of src. Biolchim. Biophys. Acta, 1287, 121-149.

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.

Fire EW, Siqun XU, Montgomery MK, Kostas SA, Driver SE and Mello CC. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806-811.

Hammond SM, Boettcher S, Caudy AA, Kobayashi R and Hannon GJ. (2001). Argonaute 2, a link between genetic and biobhemical analyses of RNAi. Science, 293, 1146-1150.

Hanada M, Feng J and Hemmings BA. (2004). Structure, regulation and function of PKB/Akt- a major therapeutic target. Biochim. Biophys. Acta, 1697, 3-16.

Hauck CR, Hunter T and Schlaepfer DD. (2001). The v-Src SH3 domain facilitates a cell adhesion-independent association with focal adhesion kinase. J Biol Chem., 276, 17653-62.

Innocenti M, Frittoli E, Ponzanelli I, Falck JR, Bracmann SM, Di Fiore P P and Scita G. (2003). Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1. J. Cell Biol., 160, 17-23.

Irby RB, Mao W, Coppola D, Kang J, Loubeau JM, Trudeau W, Kari R, Fujitj DJ, Jove R and Yeatman TJ. (1999). Activating SRC mutation in a subset of advanced human colon cancers. Nat. Genetics, 21, 187-190.

Irby RB and Yeatman TJ. (2001). Role of Src expression and activation in human cancer. Oncogene, 19, 5636-5642.

Jiang Z and Zacksenhaus E. (2002). Activation of retinoblastoma protein in mammary gland leads to ductal growth suppression, precocious differentiation, and adenocarcinoma. J. Cell Biol., 156, 185-198.

Johnson D, Frame MC and Wyke JA. (1998). Expression of he v-Src oncoprotein in fibroblasts disrupts normal regulation of the CDK inhibitor p27 and inhibits quiescence. Oncogene, 16, 2017-2028.

Karlsson T, Songyang Z, Landgren E, Lavergne C, Di Fiore PP, Anafi M, Pawson T, Cantley LC, Claesson-Welsh L and 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, 1475-1483.

Kypta RM, Goldberg Y, Ulug ET and Courtneidge SA. (1990). Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell, 10, 481-492.

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.

Lutz MP, Esser IB, Flossmann-Kast BB, Vogelmann R, Luhrs H, Friess H, Buchler MW and Adler G. (1998). Biochem. Biophys. Res. Commun., 243, 503-508.

Maa M-C, Leu T-H, Maccarley DJ, Schatzman RC and Parsons SJ. (1995). Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src: implications for the etiology of multiple human cancers. Proc. Natl. Acad. Sci. USA, 92, 6981-6985.

Maa M-C, Lai J-R, Lin R-W and Leu T-H. (1999). Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src. Biochim. Biophys. Acta, 1450, 341-351.

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, 19, 106-112.

Martin GS. (2001). The hunting of the Src. Nature Mol. Cell Biol., 2, 467-475.

Matoskova B, Wong WT, Salcini AE, Pelicci PG and Di Fiore PP. (1995). Constitutive phosphorylation of eps8 in tumor cell lines：relevance to malignant transformation. Mol Cell Biol., 15, 3805-3812.

Matoskova B, Wong WT, Seki N, Nagase T, Nomura N, Robbins KC and Di Fiore PP. (1996). RN-tre identifies a family of tre-related proteins displaying a novel potential protein binding domain. Oncogene, 12, 2563-2571.

McManus MT and Sharp PA., (2002). Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet., 3, 737-747.

Nicholson KM and Anderson NG. (2002). The protein kinase B/Akt signaling pathway in human malignancy. Cell Signal., 14, 381-395.

Ohtsubo M, Theodoras AM, Schumacher J, Roberts JM and Pahano M. (1995). Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol. Cell Biol., 15, 2612.

Piwnica WH, Saunders KB, Roberts TM, Smith AE and Cheng SH. (1987). Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell, 49, 75-82.
Riley D, Carragher NO, Frame MC and Wyke JA. (2001). The mechanism of cell cycle regulation by v-Src. Oncogene, 20, 5491-5950.

Scita G, Nordstrom J, Carbone R, Tenca P, Giardina G, Gutkind S, Bjarnegard M, Betsholtz C and Di Fiore PP. (1999). Eps8 and E3B1 transduce signals from Ras to Rac. Nature, 401, 290-293.

Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M and 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, 1031-1044.

Shimakage M, Kawahara K, Kikkawa N, Sasagawa T, Yutsudo M and Inoue H. (2000). Down-regulation of drs mRNA in human colon adenocarcinomas. Int. J. Cancer, 87, 5-11.

Smith MR, DeGudicibus SJ and Stacey DW. (1986) Requirement for c-src proteins during viral oncogene transformation. Nature, 320, 540-543.

Sugimoto Y, Whitman M, Cantley LC and Erikson L. (1984). Evidence that Rous sarcoma virus transforming gene product phosphorylates phosphatidyinositol and diacylglycerol. Proc. Natl. Acad. Sci. USA, 81, 2117-2121.

Vara JA, Casado E, Castro JD, Cejas P, Belda C and Baron M. (2004). PI3K/Akt signaling pathway and cancer. Cancer Treat Rev., 30, 193-204.
Vermeulen K, Van Bockstaele DR and Berneman ZN. (2003). The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif., 36, 131-149.

Wong WT, Carlomagno F, Druck T, Barletta C, Croce CM, Huebner K, Kraus MH and Di Fiore PP. (1994). Evolutionary conservation of the eps8 gene and its mapping to human chromosome 12q23-q24. Oncogene, 9, 3057-3061.

Yu CL, Meyer DJ, Campbell GS, Larner AC, Carter-Su C, Schwartz J and Jove R. (1995). Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science, 269, 81-83.

Zamore PD, Tuschl T, Sharp PA and Bartel DP. (2000). RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 101, 25-33.