Retrovirus-mediated herpes simplex virus thymidine kinase gene transduction renders human thyroid carcinoma cell lines sensitive to ganciclovir and radiation in vitro and in vivo.

In an attempt to develop gene therapy for thyroid carcinomas, the present studies were undertaken to evaluate in vitro and in vivo therapeutic efficacy and toxicity of herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV) treatment, a widely used prodrug/suicide gene therapy, in human thyroid carcinoma cell lines, FRO and WRO cells, using a means of retrovirus-mediated gene transduction. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the HSV-tk gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) shifted from 250 to 0.5 mg/liter in FRO cells, and from 3,000 to 0.09 mg/liter in WRO cells with therapeutic indexes of 500 and 33,000, respectively. Treatment with 30 mg/liter GCV for 4 days led to complete cell death in HSV-tk tumor cells. Nontransduced cells mixed with transduced cells were also effectively killed by GCV (bystander effect). Low concentrations of GCV, which alone showed little cytotoxicity, enhanced radiation-induced cytotoxicity (radiosensitization). In vivo sc FRO-tk tumor models in nude mice also showed dose- and time-dependent tumor regression. The IC50 was less than 2 mg/kg, and treatment with 100 mg/kg GCV for 2 weeks completely eradicated all tumors. The bystander effect and radiosensitization were also obtained in vivo. These results suggest that the HSV-tk/GCV approach to human thyroid carcinoma cells appears to be very efficacious, with a wide therapeutic range, and exerts a bystander effect and radiosensitization both in vitro and in vivo. Thus, HSV-tk/GCV system, alone or in combination with radiotherapy, may be a promising suicide gene therapy for thyroid carcinomas.

Retrovirus-mediated gene therapy for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by human alpha-fetoprotein enhancer directly linked to its promoter.

We have previously reported that a retrovirus vector (LNAF0.3TK) carrying a herpes simplex virus thymidine kinase gene regulated only by the 0.3-kb human alpha-fetoprotein (AFP) promoter provides ganciclovir (GCV)-mediated cytotoxicity in high AFP-producing human hepatoma cells but not in low AFP-producing cells. In the present study, a retrovirus vector (LNAF0.3(E+)TK), in which herpes simplex virus thymidine kinase gene expression is under the control of a human AFP enhancer directly linked to its promoter, was constructed and compared with LNAF0.3(E+)TK. In the intermediate and low AFP-producing human hepatoma cells PLC/PRF/5 and huH1/cl.2, respectively, as well as in the high AFP-producing human hepatoma cells (HepG2), LNAF0.3(E+)TK sensitized these cells to GCV in vitro but did not affect cell growth in nonhepatoma cells (HeLa). In an animal model using athymic mice harboring PLC/PRF/5 cells, GCV treatment resulted in more pronounced growth inhibition in the LNAF0.3(E+)TK virus-infected cells than in the LNAF0.3(E+)TK virus-infected cells. These results indicate that the human AFP enhancer that is directly linked to its promoter involves selective and enhanced tumoricidal activity in gene therapy for hepatocellular carcinoma.

Enhanced expression of hepatitis B surface antigen by sodium butyrate in PLC/PRF/5 human hepatoma cells.

The effect of butyric acid, a natural fermentation product of colonic bacterial flora, on hepatitis B surface antigen (HBsAg) expression was investigated in HBsAg-positive PLC/PRF/5 human hepatoma cells. By Northern blot analysis, the levels of HBsAg mRNA increased dose-dependently using sodium butyrate (0-2 mmol/l). In transient chloramphenicol acetyltransferase plasmid transfection experiments, the HBsAg-preS2 promoter activity as well as the HBV enhancer 1 activity was stimulated by sodium butyrate, whereas the HBsAg-preS1 promoter activity was not. These results indicate that butyric acid functions as a physiological regulator of HBsAg expression through the portal blood flow and possibly contributes to increased expression ratio of preS2/S to preS1 polypeptides recognized in persistant HBV infection.

Differential regulation of glucose transporter-1 and -2 gene expression by hepatocyte growth factor in human hepatoblastoma cells.

Glucose transporters, GLUT-1 and GLUT-2, are key factors involved in facilitative glucose transport to hepatocytes. The aim of the present study was to clarify how hepatocyte growth factor (HGF) regulates expression of both genes in HepG2 human hepatoblastoma cells. HGF dose-dependently suppressed cell growth, but enhanced cellular glucose uptake together with increased expression of GLUT-1 protein. This increase resulted from elevation of its transcript. In contrast, no changes were found in expression of GLUT-2 protein or its transcript by HGF treatment. These results indicate that HGF stimulates glucose incorporation through the selective up-regulation of GLUT-1 gene expression in HepG2 cells.

Detection of hepatitis B virus genome in hepatocellular carcinoma from both hepatitis B surface antigen- and antibody to hepatitis C virus-negative patients.

Twenty-two patients who tested seronegative for both hepatitis B virus (HBV) and hepatitis C virus (HCV) markers and undefined pathogenesis of hepatocellular carcinoma (HCC) were studied using polymerase chain reaction (PCR). Among these patients, 5 (23%) were positive for HBV-DNA in serum by PCR, but no patient was positive for HCV-RNA by reverse transcription PCR. Liver tissue specimens were available for analyzing HBV genome by PCR in 17 patients, and HBV sequences were detected in 10 (59%) patients. These results indicate that HBV is commonly implicated in serologically undefined pathogenesis of HCC in Japan.

Retrovirus-mediated gene therapy for human hepatocellular carcinoma transplanted in athymic mice.

Gene therapy using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of the 0.3-kb human alpha-fetoprotein (AFP) gene promoter (LNAF0.3TK virus) in combination with ganciclovir (GCV) treatment was performed in athymic mice harboring AFP-producing HuH-7 human hepatoma cells. GCV treatment resulted in pronounced growth inhibition of the virus-infected HuH-7 xenograft in mice, but did not affect growth of the parental xenograft. These results indicate that the AFP gene promoter sequence allows enough therapeutic gene expression to induce the GCV-mediated cytotoxicity in vivo in AFP-producing human hepatoma cells.

Utilization of variant-type of human alpha-fetoprotein promoter in gene therapy targeting for hepatocellular carcinoma.

We previously reported that the retroviral vector (LNAFW0.3TK) expressing the herpes simplex thymidine kinase (HSVtk) gene under the control of the 0.3 kb human alpha-fetoprotein (AFP) promoter provided the ganciclovir (GCV)-mediated cytotoxicity in the high AFP-producing (HuH-7) but not in the low AFP-producing (huH-1/cl.2) human hepatoma cells. In the present study, we constructed the retroviral vector (LNAFM0.3TK) in which the HSVtk gene expression is regulated by the variant-type of the 0.3 kb human AFP promoter with a G-to-A substitution at nucleotide -119, a point mutation responsible for hereditary persistence of human AFP and the vector was applied to three human hepatoma cell lines HuH-7, huH-1/cl.2 and intermediate AFP-producing cells (PLC/PRF/5). By the reporter gene transfection assay, the activity of the variant-type of the promoter was much higher than that of the wild-type of the promoter in both HuH-7 and huH-1/cl.2 cells. Consistent with this, LNAFM0.3TK infection could sensitize huH-1/cl.2 cells, as well as HuH-7 and PLC/PRF/5 cells to GCV, but did not affect cell growth of nonhepatoma cells (HeLa). In addition, the bystander effect was achieved more efficiently by LNAFM0.3TK infection than LNAFW0.3TK infection in HuH-7 cells. These results suggest that the variant-type of the human AFP promoter ensures the therapeutic gene expression in gene therapy particularly for the low AFP-producing hepatoma cells.

Retrovirus-mediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by alpha-fetoprotein enhancer/promoter.

In the present study, to achieve more selective and efficient therapeutic gene expression in hepatoma cells, we compared the therapeutic efficacies of the retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene by the alpha-fetoprotein (AFP) enhancer/promoter in the forward (LNAFE0.3TK) and reverse (LN[AFE0.3TK]R) orientation to the vector long terminal repeats. By Northern blotting, the level of the HSV-tk mRNA in LN[AFE0.3TK]R-infected HepG2 human hepatoma cells was much higher than that in LNAFE0.3TK-infected cells. Consistent with this, LN[AFE0.3TK]R infection into HepG2 cells caused a greater cytotoxicity by ganciclovir exposure together with a stronger bystander effect than LNAFE0.3TK infection. In an animal model, intratumorous injection of LN[AFE0.3TK]R with ganciclovir treatment resulted in pronounced growth inhibition of HepG2 tumor. Thus, the reversely oriented therapeutic gene expression under the control of AFP enhancer/promoter is a possible candidate for the retrovirus-mediated gene therapy for hepatocellular carcinoma.