Regression of hepatocellular carcinoma in vitro and in vivo by radiosensitizing suicide gene therapy under the inducible and spatial control of radiation

To improve the efficacy and selectivity of gene therapy for hepatocellular carcinoma (HCC), we designed a strategy for suicide gene therapy in conjunc tion with radiation therapy using an HVJ-liposome vector system. The radio- inducible suicide gene was constructed by insertion of the early growth res ponse gene 1 (Egr-1) promoter upstream of the HSV-rk gene (EGF-tk), First, to test the tumor specificity of Egr-1, RT-PCR and immunohistochemistry wer e performed. The Egr-1 gene was highly expressed in HCC compared with norma l liver, where expression was barely detectable. Next, radiation-inducible activity of the Egr-1 promoter was examined in primary cultured normal hepa tocytes and human hepatoma cell lines Huh7, HepG2, and PLC/PRF/5 by lucifer ase assay as a reporter gene system. Egr-1 promoter activity was markedly i ncreased in hepatoma cell lines in a radiation dose-dependent manner, with maximum activation (15- to 28-fold) 12 hr after irradiation. In contrast, o nly a twofold increase in activation was noted in normal hepatocytes. An in vitro gene therapy experiment showed that EGR-tk-transduced hepatoma cells became highly sensitive to ganciclovir (GCV) after irradiation, but not wi thout irradiation, GCV with or without irradiation did not show any cytotox ic effects against control gene-transfected cells. In addition, a "radiosen sitization effect" was also demonstrated by combination therapy with the HS V-tk/GCV system and irradiation. To examine the efficacy of this EGR-tk/GCV gene therapy in vivo, xenografted liver tumors in nude mice were targeted using the HVJ-liposome vector system. EGR-tk-transfected tumors regressed s ignificantly after a combination therapy of irradiation and GCV in all mice (n = 8), and almost disappeared in 3 weeks without any side effects. In co mparison, tumors continued to grow in all mice (n = 8 in each group) treate d by transfer of EGR-tk followed by either irradiation without GCV or GCV w ithout irradiation. Our data indicate that HSV-tk gene therapy under the co ntrol of a radioinducible promoter is effective, and might be selective for hepatoma cells because of its inducible and radiosensitive capacity after radiation exposure as well as its tumor-specific activation.