680. TransfeX-Mediated HSV-tk/Ganciclovir Suicide Gene Therapy in HeLa Cervical Carcinoma and HSC-3, FaDu, and H357 Oral Cancer Cells

Abstract

INTRODUCTION: Herpes Simplex Virus thymidine kinase/ganciclovir (HSV-tk/GCV)-induced suicide gene therapy has been used to successfully treat various cancers. TransfeX-mediated transfection of pCMV.Luc into HeLa cervical carcinoma and HSC-3, FaDu, and H357 oral squamous cell carcinoma (OSCC) cell lines in the presence of 10% serum has proved to be highly efficient, with low non-specific cytotoxicity. The plasmid pNGVL1-tk encoding HSV-tk under the control of the CMV promoter was delivered to the cells in vitro via the novel cationic liposomal reagent, TransfeX, followed by treatment with ganciclovir. METHODS: HeLa, HSC-3, FaDu, and H357 cells were seeded in 48-well culture plates one day prior to transfection, at a density of 1.5 × 105 cells per well in 1 ml of complete DMEM medium containing 10% serum (DME/10). Lipoplexes for transfection were prepared by mixing 1 µg pCMV.Luc or pNGVL1-tk per 100 µl of serum-free DMEM followed by the addition of 2 µl of TransfeX. Lipoplexes were added directly to cells grown in the presence of 10% serum. Twenty-four hours after transfection, 20 µg of ganciclovir was added per well and cells were incubated an additional 24 h. The Alamar blue assay was used to determine cell viability after 24, 48, and 120 h. Fresh medium and ganciclovir were added after each of these readings. RESULTS: After 24 h, 65% and 50% cytotoxicity were seen in HeLa cells and HSC-3 cells, respectively. FaDu and H357 cells were more resistant to treatment, showing 15% and 5% cytotoxicity, respectively, after 24 h. After 120 h, cytotoxicity remained the same in HeLa, and increased to 90% in HSC-3. In FaDu and H357, 65% and 30% cytotoxicity, respectively, were observed after 120 h. CONCLUSIONS: Suicide gene therapy is most effective against HSC-3 OSCC among the cells examined. Longer periods of incubation with ganciclovir or prolonged gene expression via enhanced episome vectors may be necessary to increase cytotoxicity in FaDu and H357 cells.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide