Intracellular fate of non-viral vectors in oral cancer cells
Nejat Düzgüneş: 0000-0001-6159-1391
87th General Session of the International Association for Dental Research (IADR) and 38th Annual Meeting of the American Association for Dental Research (AADR)
April 1-4, 2009
Date of Presentation
Journal of Dental Research
Journal Volume Number
88 (Special issue A)
Introduction: One problem in the use of cationic lipid-DNA complexes (“lipoplexes”) for gene therapy of Oral Squamous Cell Carcinoma (OSCC) is the variability in transfection of different OSCC cells. Objective: Test the hypothesis that the variability in transfection arises from the differential uptake of lipoplexes, using fluorescent transfection reagents. Methods: KB, HSC-3 and H-357 human OSCC cells were seeded in 48-well culture plates , and used at approx. 85% confluence. The plasmid pCMV.Luc expressing luciferase was complexed with optimal volumes (2µl/well) of Fluo-Metafectene. Transfection activity was assessed by luciferase expression assayed 48 h after transfection, using the Promega Luciferase Assay System and a luminometer. Fluorescence was quantitated 48 h after transfection using a Perkin-Elmer Luminescence Spectrometer, and observed in a Nikon-Diaphot microscope right after transfection or 48 h afterwards. Results: Representative luciferase activities in HSC-3, KB and H-357 cells were 100100, 73607 and 147 Relative Light Units/ml. Fluorescent lipoplexes were taken up by HSC-3 and KB cells to a greater extent than by H-357 cells, as determined by microscopy and fluorometry. Conclusion: While the uptake of fluorescent lipoplexes correlates considerably with transfection activity, there appears to be a barrier to gene transfer in H-357 cells, since a significant amount of lipoplexes (albeit lower than that in HSC-3 and KB cells) are taken up by these cells. In cell lines that are difficult to transfect, the efficacy-limiting step in gene transfer to OSCC cells appears to be lipoplex processing beyond initial uptake. These additional steps may include destabilization of the endosomal membrane, escape of the DNA into the cytoplasm and transport of DNA into the nucleus. We are exploring the use of fluorescence markers for lysosomes and nuclei to better define the intracellular localization of lipoplexes. Supported by an AADR Student Research Fellowship (C.L.-D.).
Intracellular fate of non-viral vectors in oral cancer cells.
Paper presented at 87th General Session of the International Association for Dental Research (IADR) and 38th Annual Meeting of the American Association for Dental Research (AADR) in Miami, FL.