Regression of Hepatocellular Carcinoma in Vitro and in Vivo by Radiosensitizing Suicide Gene Therapy under the Inducible and Spatial Control of Radiation

Abstract

To improve the efficacy and selectivity of gene therapy for hepatocellular carcinoma (HCC), we designed a strategy for suicide gene therapy in conjunction with radiation therapy using an HVJ-liposome vector system. The radio-inducible suicide gene was constructed by insertion of the early growth response gene 1 (Egr-1) promoter upstream of the HSV-tk gene (EGF-tk). First, to test the tumor specificity of Egr-1, RT-PCR and immunohistochemistry were performed. The Egr-1 gene was highly expressed in HCC compared with normal liver, where expression was barely detectable. Next, radiation-inducible activity of the Egr-1 promoter was examined in primary cultured normal hepatocytes and human hepatoma cell lines Huh7, HepG2, and PLC/PRF/5 by luciferase assay as a reporter gene system. Egr-1 promoter activity was markedly increased in hepatoma cell lines in a radiation dose-dependent manner, with maximum activation (15- to 28-fold) 12 hr after irradiation. In contrast, only 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 without irradiation. GCV with or without irradiation did not show any cytotoxic effects against control gene-transfected cells. In addition, a "radiosensitization effect" was also demonstrated by combination therapy with the HSV-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 significantly after a combination therapy of irradiation and GCV in all mice (n = 8), and almost disappeared in 3 weeks without any side effects. In comparison, tumors continued to grow in all mice (n = 8 in each group) treated by transfer of EGR-tk followed by either irradiation without GCV or GCV without irradiation. Our data indicate that HSV-tk gene therapy under the control 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.

Activation of MAPK signaling pathway is essential for Id-1 induced serum independent prostate cancer cell growth

“…The cDNA was synthesized using the SuperScript TM First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). The cDNAs were then amplified by PCR with Egr-1 specific primers (upstream: 5'-TGA CCG CAG AGT CTT TTC CT-3', downstream: 5'-TGG GTT GGT CAT GCT CAC TA-3') (Kawashita et al, 1999). The PCR conditions were as follows: an initial denaturation at 958C was followed by 30 cycles of PCR (948C for 1 min, 558C for 1 min and 728C for 2 min), and a final extension at 728C for 5 min.…”

Radiation to control gene expression

“…1,2 Other groups have used the Egr-1 promoter with the gene for HSVtk which produced enhanced tumor cell killing in the presence of the prodrug ganciclovir following radiation treatment. 3,4 Ionizing radiation activates transcription of the Egr-1 gene and the CArG [CC(A+T rich) 6 GG] domain of the promoter which are important for this response. 5 A synthetic promoter has been described based on four copies of the CArG element of the Egr-1 promoter serving as the enhancer for the CMV promoter.…”

Radioresponsive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy for malignant brain tumors

“…14,15 An alternate approach for the improvement of the specificity of gene therapy involves the use of radiosensitive promoters to activate gene expression in the radiation field in a selective manner. [15][16][17][18][19] The use of a radiationinducible promoter permits the expression of a therapeutic gene to be confined within a specific irradiated volume for a chosen period of time. This tight spatial and temporal control of therapeutic gene expression potentially increases the efficiency and safety of gene therapy.…”