FAS-MEDIATED APOPTOSIS IS INVOLVED IN THE ELIMINATION OF GENE-TRANSDUCED HEPATOCYTES WITH E1 E3-DELETED ADENOVIRAL VECTORS/

Gene-transduced hepatocytes with E1/E3-deleted adenoviral vectors are eliminated immediately and the expression of transduced genes disappea rs rapidly following the vector administration. In this report, we ana lysed the involvement of apoptotic cell death in the elimination of he patocytes infected with adenoviral vectors. An E1/E3-deleted adenovira l vector expressing Escherichia coli beta-galactosidase (LacZ) was inj ected via the portal vein into congenitally Fas-deficient mice (lpr), Fas ligand-deficient mice (gld) and their control mice, MRL and C3H. 5 -Bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal) staining of the l iver specimens showed that 80-100% of hepatocytes were LacZ positive a t 7 days after virus administration, suggesting that most of the hepat ocytes received the injected adenoviral vectors. In normal mice, the n umber of LacZ-positive cells decreased dramatically at 14 and 21 days after transduction and few positive cells were observed at day 28. bet a-Galactosidase activity, quantified by the O-nitrophenyl-beta-D-galac topyranoside assay, gave comparable results to X-gal staining. At days 14 or 21, many apoptotic hepatocytes and apoptotic infiltrating cells were detected with the terminal deoxyribonucleotidyl transferase-medi ated dUTP-digoxigenin nick end-labelling (TUNEL) in situ apoptosis det ection method. This observation suggested that the apoptotic process w as associated with the elimination of adenovirus-infected hepatocytes. To test the involvement of the Fas-Fas ligand interaction in this apo ptotic process, the period of transgene expression was measured in lpr and gld mice, which had received the same amount of AxCALacZ. X-Gal h istochemical analysis detected many LacZ-positive cells in lpr or gld mice liver even at 21 or 28 days after AxCALacZ injection. There were significant differences in the reduction rates of beta-galactosidase a ctivity of liver homogenates between lpr and MRL, or gld and C3H mice. Based on these observations, we conclude that the Fas-mediated apopto tic process is involved in the elimination of hepatocytes infected wit h E1/E3-deleted adenoviral vectors.