621. Translational Restriction of Protein Expression of a Modifed Suicide Gene and Cytotoxicity in Human Mammary Tumor Cells Expressing High Levels of the Translation Initiation Factor eIF4E

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Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficent translation to cells that express high levels of the translation initiation factor, eIF4E. Previous study data have shown that most tumor cells express elevated levels of eIF4E. Gene delivery was optimized by incorporation of the modified suicide gene (HSV-UTk) into an adenovirus vector (Ad) that infects a broad range of cells. The goal of this project in the development of Ad-HSV-UTk was to target infection of a broad population of cells and yet limit translation of the suicide gene to those cells expressing elevated levels of eIF4E. The efficacy of this novel approach of targeting suicide gene expression and cytotoxicity by means of translational restriction was tested in vitro with the use of the human mammary tumor cells [MCF-7, MDA-MB435, ZR 75-1, MCF-10A and MCF10A cells that over express eIF4E (MCF10A-4E)], as well as primary cultures of human mammary epithelial cells (HMEC) and human mammary stromal cells (HMSC) that express relatively low levels of eIF4E. Real time rt-PCR was used to quantify transcription levels of HSV-Tk for both Ad-HSV-Tk and Ad-HSV-UTk infected cells, as well as the translation initiation factor eIF4E. Translation of HSV-Tk in both Ad-HSV-Tk and Ad-HSV-UTk infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined by assessing cell viability following treatment with the prodrug GCV in Ad-HSV-Tk and Ad-HSV-UTk infected cells with the use of the reductase activity indicator MTT. These data demonstrated enhanced cytotoxicity for the Ad-HSV-UTk /GCV treatment in high eIF4E expressing cells compared to low eIF4E expressing cells, whereas the Ad-HSV-Tk/GCV treatment showed similar cytotoxicity in both high and low eIF4E expressing cells. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro is effective and may provide a platform for enhanced cancer gene therapy specificity in vivo.