Involvement of Fas (CD95/APO-1) and Fas ligand in apoptosis induced by ganciclovir treatment of tumor cells transduced with herpes simplex virus thymidine kinase

Transduction of cancer cells with herpes simplex virus thymidine kinase gen e (HSVtk) followed by prodrug ganciclovir (GCV) treatment has been shown to induce apoptosis. In this study, four murine tumors including B16F10 melan oma, 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 dos es of GCV, the HSVtk-transduced cells of all four tumors showed similar kin etics of early rise in p53 protein levels, then cell cycle S-/G2-phase arre st and finally signs of apoptosis. Immunoblot analyses revealed that Pas (C D95/APO-1), Pas ligand (FasL) and two downstream mediators, RIP and caspase -3 (CPP32, YAMA, Apopain) were increased in GCV-treated HSVtk-transduced tu mor cells after the cell cycle arrest and before apoptosis. Increased expre ssion of Fast could also be observed in vivo in HSVtk-transduced tumors ind uced to regress by GCV treatment. Enzyme measurements using specific substr ate showed that the caspase-3 activation followed kinetically the Fast expr ession. More than half of the HSVtk/GCV-induced cell death could be abrogat ed by addition to the cell culture medium of a specific antisense oligonucl eotide to block Fast synthesis, a recombinant Pas/Fc chimeric protein to co mpete with Pas receptor for Fast binding, or cell-permeable specific tetrap eptide inhibitors of caspase-3 or caspase-8.