Eps8(EGF receptor pathway substrate NO.8)是EGFR與Src的受質，具有97KDa及68KDa兩種isoforms。先前我們實驗室發現在人類的大腸癌細胞株(SW620)中，p97Eps8有大量表達的情形，並且p97Eps8會促進大腸癌細胞SW620的細胞生長。於是我們想要瞭解Eps8之結合蛋白質IRSp53是否也參與在Eps8所調控之大腸癌細胞的細胞增生過程中，首先我們觀察在人類的大腸癌細胞株中IRSp53的表現情形，發現在一些大腸癌細胞株中(包括SW620)有不同的IRSp53表達量，接著我們利用Co-IP實驗方法進一步確認了在SW620和WiDr細胞中，IRSp53的確會與Eps8 結合。此外，利用外送IRSp53，IRSp58以及IRSp53-△363 (缺乏C端和Eps8結合區域之序列)到NIH3T3細胞中，發現IRSp53和IRSp58會促進細胞在soft agar上之 colonies的形成而IRSp53-△363則不會，而我們同時送入Eps8和IRSp53則發現Eps8可以加成此作用。結果顯示IRSp53和Eps8的交互作用對於細胞轉型能力是重要的。接著我們進一步將表達IRSp53 siRNA的DNA plasmid 送入SW620大腸癌細胞中去建立IRSp53 siRNA的SW620細胞株，觀察細胞在IRSp53 down-regulation 之下，其對於細胞的生長及致癌能力的作用有何影響。我們發現當SW620細胞中的IRSp53表現受抑制時， Akt Pi-Ser473，cyclin D1和cyclin E也有顯著的下降，並且也發現表現IRSp53 siRNA之SW620細胞的生長不論在culture dish，或在soft agar上都會受到明顯抑制。因此，由以上結果我們認為IRSp53 可能參與在調控人類大腸癌細胞SW620之細胞生長的作用中。

Eps8 (EGF receptor pathway substrate NO.8) is a substrate of both EGFR and Src. It exists in two isoforms p97EPS8 and p68Eps8 in many cell lines. Our previous studies indicated that only the 97-kDa isoform was expressed and promoted cell proliferation of SW620 cells. To understand whether Eps8-interacting protein, IRSp53 is involved in Eps8-mediated cell proliferation of colon cancer cells, first, we observed that expression of IRSp53 is varied in several colon cancer cell lines including SW620. Then we confirmed the interaction between Eps8 and IRSp53 in SW620 and WiDr cells by co-immunoprecipitation. In addition, transient overexpression of myc-tagged IRSp53 and IRSp58 but not IRSp53-△363 (Eps8 binding region deletion) in NIH3T3 cells increased colony formation in soft agar. We also observed that co-transfection of Eps8 and IRSp53 into NIH3T3 cells could synergistically enhanced the number of colony formed in soft agar. These data indicated that the association between IRSp53 and Eps8 could be important for cell transforming ability. Moreover, we utilized small interference RNA (siRNA) technology to generate SW620 cell lines stably expressing IRSp53 siRNA or nonspecific control siRNA and observed that the expression of IRSp53, Akt Pi-Ser473, cyclin D1 and cyclin E is reduced in these cells. Furthermore, the growth rate and the ability of anchorage independent growth were reduced in IRSp53 siRNA expressing cells. Therefore, our results indicated that IRSp53 may participate in cell proliferation of human colon cancer cell SW620.

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