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

被引:69
作者
Kawashita, Y
Ohtsuru, A
Kaneda, Y
Nagayama, Y
Kawazoe, Y
Eguchi, S
Kuroda, H
Fujioka, H
Ito, M
Kanematsu, T
Yamashita, S
机构
[1] Nagasaki Univ, Sch Med, Atom Bomb Dis Inst, Dept Nat Med, Nagasaki 8528523, Japan
[2] Nagasaki Univ, Sch Med, Dept Surg 2, Nagasaki 8528523, Japan
[3] Osaka Univ, Sch Med, Div Gene Therapy Sci, Osaka 5650871, Japan
[4] Nagasaki Univ, Sch Med, Dept Pharmacol 1, Nagasaki 8528523, Japan
[5] Nagasaki Univ, Sch Med, Dept Pathol, Nagasaki 8528523, Japan
关键词
D O I
10.1089/10430349950017842
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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-rk 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.
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页码:1509 / 1519
页数:11
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