MONITORING GENE-THERAPY WITH HERPES-SIMPLEX VIRUS THYMIDINE KINASE INHEPATOMA-CELLS - UPTAKE OF SPECIFIC SUBSTRATES

This study investigates the application of PET with specific substrate s for the assesment of enzyme activity after transfer of the herpes si mplex virus thymidine kinase (HSV-tk) gene. Methods: After transfectio n of a rat hepatoma cell line with a retroviral Vector containing the HSV-tk gene, different clones were established by G418 selection. Upta ke measurements were performed up to 48 hr in a TK-expressing cell lin e and in a control cell line using thymidine (TdR; measured under ther apy conditions), fluorodeoxycytidine (FdCyt) and ganciclovir (GCV). Ad ditionally, bystander experiments and inhibition/competition studies w ere done. Results: In TK-expressing cells GCV treatment caused an incr eased (up to 250%) TdR uptake in the acid-soluble fraction and a decre ase to 5.5% in the acid-insoluble fraction. The FdCyt uptake was highe r in the TK-expressing cells than in controls with a maximum after 4 h r (12-fold and 3-fold higher in the acid-insoluble and acid-soluble fr action). GCV accumulated up to 180-fold more in the acid-insoluble and 26-fold more in the acid-soluble fraction. GCV uptake occurred mainly by the nucleoside transport systems. Bystander experiments revealed a relation between growth inhibition or GCV uptake and the amount of TK -expressing cells. GCV uptake and growth inhibition were correlated wi th r = 0.96. Conclusion: Assessment of GCV accumulation may serve as a n indicator of the enzyme activity and of therapy outcome. TdR may be useful to measure therapy effects on DNA synthesis, whereas the potent ial of FdCyt has to be investigated in further studies.