Transduction of cancer cells with herpes simplex virus thymidine kinase gene (HSVtk) followed by prodrug ganciclovir (GCV) treatment has been shown to induce apoptosis. In this study, four murine tumors including B16F10 melanoma, NG4TL4 sarcoma, H6 hepatoma and 1MEA 7R.1 hepatoma were found to vary in sensitivity to this gene therapy strategy in vitro but, at effective doses of GCV, the HSVtk-transduced cells of all four tumors showed similar kinetics of early rise in p53 protein levels, then cell cycle S-/G2-phase arrest and finally signs of apoptosis. Immunoblot analyses revealed that Pas (CD95/APO-1), Pas ligand (FasL) and two downstream mediators, RIP and caspase-3 (CPP32, YAMA, Apopain) were increased in GCV-treated HSVtk-transduced tumor cells after the cell cycle arrest and before apoptosis. Increased expression of Fast could also be observed in vivo in HSVtk-transduced tumors induced to regress by GCV treatment. Enzyme measurements using specific substrate showed that the caspase-3 activation followed kinetically the Fast expression. More than half of the HSVtk/GCV-induced cell death could be abrogated by addition to the cell culture medium of a specific antisense oligonucleotide to block Fast synthesis, a recombinant Pas/Fc chimeric protein to compete with Pas receptor for Fast binding, or cell-permeable specific tetrapeptide inhibitors of caspase-3 or caspase-8.
