| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 87 === Abstract
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 tumor cell lines including B16F10 melanoma, NG4TL4 sarcoma, H6 hepatoma and 1MEA 7R.1 hepatoma were found to vary in their 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 arrest in S-/G2-phase and finally signs of apoptosis. Immunoblot analyses revealed that Fas (CD95/APO-1), Fas ligand (FasL) and two downstream mediators, RIP and CPP32/Caspase-3 (YAMA, Apopain) were increased in GCV-treated HSVtk-transduced tumor cells after the cell cycle arrest and before apoptosis. Increased expression of FasL could also be observed in vivo in HSVtk-transduced tumors induced to regress following GCV treatment. Enzymatic measurements using specific substrate showed that the CPP32/Caspase-3 activation followed kinetically the FasL expression. To further demonstrate the involvement of Fas/FasL pathway in this GCV-induced apoptosis events, most of the HSVtk/GCV-induced cell death could be abrogated by addition to the cell culture medium of a specific antisense oligonucleotide to block FasL synthesis, a recombinant Fas/Fc chimeric protein to compete with Fas receptor for FasL binding, or cell-permeable specific tetrapeptide inhibitors of CPP32/Caspase-3 or FLICE/Caspase-8.
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