RESUMO
We previously reported that the retroviral vector expressing the herpes simplex virus-thymidine kinase gene under the control of 0.3-kb human alpha-fetoprotein (AFP) gene promoter (AF0.3) provided the cytotoxicity to ganciclovir (GCV) in high-AFP-producing human hepatoma cells but not in low-AFP-producing cells. Therefore, specific enhancement of AFP promoter activity is likely to be required to induce enough cytotoxicity in low-AFP-producing hepatoma cells. In this study, we constructed a hybrid promoter, [HRE]AF, in which a 0.4-kb fragment of human vascular endothelial growth factor 5'-flanking sequences containing hypoxia-responsive element (HRE) was fused to AF0.3 promoter. By means of the reporter gene transfection assay, hypoxia-inducible transcriptions that were mediated by [HRE]AF promoter were detected in low- and non-AFP-producing human hepatoma cells, but not in nonhepatoma cells. When the herpes simplex virus-thymidine kinase gene controlled by [HRE]AF promoter was transduced into hepatoma and nonhepatoma cells by a retroviral vector, the exposure to 1% O2 induced GCV cytotoxicity specifically in the hepatoma cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors consisting of the virus-infected hepatoma cells gradually disappeared by GCV administration. These results indicate that the hypoxia-inducible enhancer of the human vascular endothelial growth factor gene, which is directly linked to human AFP promoter, involves selective and enhanced tumoricidal activity in gene therapy for hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular/terapia , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Neoplasias Hepáticas/terapia , Regiões Promotoras Genéticas , alfa-Fetoproteínas/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Hipóxia Celular/genética , Fatores de Crescimento Endotelial/genética , Ganciclovir/toxicidade , Vetores Genéticos/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Linfocinas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Retroviridae/genética , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Ativação Transcricional , Transdução Genética , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , alfa-Fetoproteínas/biossínteseRESUMO
Cyclin D1, one of the G(1) cyclins, is frequently overexpressed in several types of carcinomas and is thought to play an important role in tumorigenesis and tumor progression including hepatocellular carcinoma. We constructed a retrovirus vector-carrying rat cyclin D1 cDNA in the reverse orientation, resulting in expression of antisense (AS) cyclin D1 mRNA. For efficient transduction of this recombinant retrovirus, two-step gene transfer was performed. The rat hepatoma cell line (dRLh84) was infected with this recombinant retrovirus after preinfection with adenovirus expressing the retrovirus receptor. In the rat hepatoma cells, AS cyclin D1 mRNA was expressed, inducing a decrease in the expression of endogenous cyclin D1 mRNA and an inhibition of cell growth. Moreover, two-step gene transfer of AS cyclin D1 into s.c. hepatoma xenografts resulted in inhibition of tumor growth and prolonged animal survival. In the virus-infected tumor xenografts, expression of cyclin D1 was immunohistochemically inhibited, and apoptosis of hepatoma cells was detected. These findings suggest that transduction of AS cyclin D1 is useful as an adjunct to standard treatments for hepatocellular carcinoma.