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 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.
Department of Surgery II, Nagasaki University School of Medicine, Japan.
Y
Y
Kawashita
A
A
Ohtsuru
Y
Y
Kaneda
Y
Y
Nagayama
Y
Y
Kawazoe
S
S
Eguchi
H
H
Kuroda
H
H
Fujioka
M
M
Ito
T
T
Kanematsu
S
S
Yamashita
D000368:Aged; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000532:radiotherapy / Q000628:therapy*; D002478:Cells, Cultured; D003131:Combined Modality Therapy; D004268:DNA-Binding Proteins / Q000235:genetics*; D051766:Early Growth Response Protein 1; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000528:radiation effects; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D007763:Lac Operon; D008099:Liver / Q000166:cytology; D008113:Liver Neoplasms / Q000532:radiotherapy / Q000628:therapy*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger; D011836:Radiation Tolerance; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D013937:Thymidine Kinase; D015774:Ganciclovir