Leonhard Mohr

Ethanol inhibits hepatocyte proliferation in insulin receptor substrate 1 transgenic mice

BACKGROUND & AIMS Long-term ethanol consumption is known to impair the ability of the liver to regenerate, but the molecular mechanisms are poorly understood. Multiple growth factors promote hepatocyte proliferation, some of which involve the insulin receptor substrate 1 (IRS-1)-mediated signal transduction pathway. To explore effects of ethanol on the IRS-1 signal liver growth in vivo, studies in transgenic mice overexpressing IRS-1 in the liver were performed because these mice show constitutive activation of the downstream signal transduction pathways leading to enhanced hepatocyte proliferation. METHODS Tyrosyl phosphorylation of IRS-1 and subsequent protein-protein interactions were examined in liver lysates from animals fed ethanol or control diet. Activity of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) was assessed by specific enzymatic assays. Hepatocyte proliferation was measured by incorporation of [3H]thymidine into liver DNA. RESULTS Tyrosyl phosphorylation of IRS-1, association of IRS-1 with PI3K, and activation of downstream PI3K and MAPK pathways were greatly reduced as a result of long-term ethanol consumption. Ethanol virtually abolished the enhanced hepatocyte DNA synthesis induced by expression of the IRS-1 transgene. CONCLUSIONS Altered transmission of growth signals through the IRS-1-mediated signal transduction cascade may represent a molecular mechanism of how ethanol inhibits hepatocyte proliferation.

Rabbit cytochrome P450 4B1: A novel prodrug activating gene for pharmacogene therapy of hepatocellular carcinoma.

Gene therapy using vector-mediated transfer of prodrug activating genes is a promising treatment approach for malignant tumors. As demonstrated recently, the novel prodrug activating gene coding for rabbit cytochrome P450 4B1 (CYP4B1) is able to induce tumor cell death at low micromolar concentrations in glioblastoma cells after treatment with the prodrug 4-ipomeanol (4-IM) in vitro and in vivo. The rabbit CYP4B1 converts this prodrug and other furane analogs and aromatic amines, such as 2-aminoanthracene, to highly toxic alkylating metabolites, whereas the human isoenzyme exhibits only minimal enzymatic activity. In the present study, the cDNA encoding rabbit CYP4B1 was used for pharmacogene therapy of hepatocellular carcinoma (HCC). Cell clones derived from the human HCC cell lines Hep3B, HuH-7, and HepG2 and stably expressing the chimeric protein CYP4B1-EGFP (the CYP4B1 coding sequence fused to the enhanced green fluorescent protein (EGFP) gene) were selected. HCC clones expressing EGFP served as controls. 4-IM rapidly induced tumor cell death in CYP4B1-EGFP-expressing clones at low concentrations (a 50% lethal dose of between 0.5 and 2 μg/mL). No signs of toxicity were found in control cells expressing EGFP even at high prodrug concentrations (20 μg/mL). Cell death occurred by apoptosis and was independent of functional p53. A pronounced direct bystander effect was observed in Hep3B cells, whereas bystander HepG2 and HuH-7 cells were highly resistant to toxic 4-IM metabolites. These results demonstrate that the CYP4B1/4-IM system efficiently and rapidly induces cell death in HCC cells, and that a cell line-specific mechanism may exist that limits the extent of the bystander effect of this novel prodrug activating system.

Nucleic acid-based antiviral and gene therapy of chronic hepatitis B infection

Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. There is a need to develop new antiviral approaches for the treatment of this disease. We have explored various nucleic acid‐based strategies designed to inhibit HBV replication including: the use of antisense RNA and DNA constructs, DNA‐based immunization techniques to stimulate broad‐based cellular immune responses with particular emphasis on the generation of cytotoxic lymphocyte (CTL) activity to viral structural proteins, hammerhead ribozymes to cleave HBV pregenomic RNA in vitro and dominant negative HBV core mutant proteins as inhibitors of nucleocapsid formation within cells. In order to optimize these antiviral effects, various novel expression vectors have been developed to deliver such DNA constructs to cells. For example, adenoviral vectors carrying genes that encode for dominant negative proteins have been employed to transfect hepatocytes in vitro and in vivo. In addition, plasmid vectors have been produced to promote expression of HBV structural genes following injection into muscle cells as a means to stimulate the hosts cellular and humoral immune response in the context of histocompatibility antigen (HLA) class I and II antigen presentation. These experimental approaches may have important implications for the generation of efficient antiviral effects during chronic HBV infection.

Immunotherapy of Murine Hepatocellular Carcinoma by α-Fetoprotein DNA Vaccination Combined with Adenovirus-Mediated Chemokine and Cytokine Expression

Most hepatocellular carcinomas (HCCs) express oncofetal alpha-fetoprotein (AFP). We and others have demonstrated efficient tumor control mediated by cellular immune responses in mice bearing subcutaneous tumors derived from the AFP-expressing murine HCC cell line Hepa 1-6 by DNA vaccination against AFP. In the present study, we examined AFP DNA vaccination in the AFP-expressing primary murine HCC model BW7756. In this model AFP DNA vaccination resulted in only minimal lymphocytic infiltration and failed to control tumor growth. To augment the AFP-specific cellular immune response, intratumoral expression of chemokine IP-10 (interferon-inducible protein-10) and the proinflammatory cytokine interleukin (IL)-12 by adenoviral vectors (AdmIL-12 and AdmIP-10) was analyzed. Intratumoral injection of AdmIL-12 and AdmIP-10 resulted in transient tumor regressions, without prolongation of animal survival. By contrast, AFP DNA vaccination followed by intratumoral injection of AdmIL-12 and AdmIP-10 resulted in tumor regression in all animals and in prolongation of animal survival; in 25% of animals the tumors became undetectable. This study demonstrates for the first time that activation of effector cells against a tumor antigen induced by the combination of DNA vaccination and intratumoral chemokine and cytokine expression is superior to the respective treatment strategies alone. This effect may be mediated by attraction of activated effector cells to the tumor tissue.

ENHANCED GENE DELIVERY AND EXPRESSION IN HUMAN HEPATOCELLULAR CARCINOMA CELLS BY CATIONIC IMMUNOLIPOSOMES

AbstractA targeted vector allowing enhanced gene transfer to human hepatocellular carcinoma (HCC1) cells in vitro was developed using cationic liposomes covalently conjugated with the mAb AF-20. This high affinity antibody recognizes a rapidly internalized 180 kDa cell surface glycoprotein which is abundantly expressed on the surface of human HCC and other cancer cells. Quantitative binding analysis of liposomes with target cells by flow cytometry showed specific association of mAb-targeted liposomes with human HCC cells. Using mAb-targeted cationic liposomes containing 20% DOTAP, in the presence or absence of serum, gene expression in HuH-7 cells was enhanced up to 40-fold as compared to liposomes conjugated with an isotype-matched non-relevant control antibody. Transfection specificity was not observed in a control cell line that does not express the antigen recognized by mAb AF-20. This study demonstrates that cationic liposome formulations can be targeted with monoclonal antibodies (mAbs) to enhance s...

Adenovirus-mediated gene transfer to orthotopic hepatocellular carcinomas in athymic nude mice

Gene therapy may become an option for the treatment of malignant tumors such as hepatocellular carcinoma (HCC), once safe and efficient vector systems have been established. Due to their stability in vivo , recombinant adenoviral vectors are promising vectors for gene delivery to HCC. To study the characteristics of gene delivery into HCCs by recombinant adenoviral vectors in vivo , we established an in situ HCC model in the livers of athymic nude mice by intrahepatic injection of human HCC cells. Recombinant adenovirus vectors expressing β-galactosidase (Ad2CMVβgal) were injected via the tail vein of mice bearing HCC or directly into intrahepatic tumors. Levels of β-galactosidase expression in tumor tissue and surrounding normal liver were analyzed by histochemistry or for quantification by a chemiluminescence assay in tissue homogenates. Following tail vein injection, high levels of β-galactosidase expression were found in the liver, but virtually no gene expression could be detected in the tumor tissue. In contrast, after direct injection of Ad2CMVβgal into intrahepatic HCCs, high levels of β-galactosidase expression were detected in the tumor tissue. However, single transduced hepatocytes scattered throughout the normal liver could also be identified. These results indicate that barriers such as the endothelial lining of the tumor vasculature impair the efficiency of adenoviral vectors for gene delivery into HCCs by intravenous administration, which can be overcome by direct injection into the tumor tissue. However, due to the observed transduction of disseminated hepatocytes following intratumoral administration, additional HCC-specific targeting to further enhance the safety of adenoviral vectors may be required. Cancer Gene Therapy (2001) 8, 573–579

Immunological approach to hepatocellular carcinoma

Summary. A library of monoclonal antibodies (MoAbs) has been produced against a human hepatocellular carcinoma (HCC) cell line designated FOCUS in order to study the antigenic properties of transformed hepatocytes. Several monoclonal antibodies (MoAbs) were initially selected for study since they bound to antigens which were overexpressed in HCC tissues compared with the adjacent uninvolved normal liver counterpart; in addition, these MoAbs revealed low level antigen expression on other normal human tissues. Subsequently, HCC cell lines were metabolically labelled and the antigens further characterized by immunoprecipitation and Western blot analysis. If the MoAb recognized a primary linear epitope on a protein, cloning was performed using a ΛGT11 cDNA expression library prepared from the FOCUS HCC cell line. These studies characterized the HCC associated antigen(s) at the molecular level. This review illustrates the value of such an experimental approach to search for and identify HCC associated antigens and emphasizes the biological properties of novel proteins may be defined and characterized by these techniques. More important, our investigations have described unique proteins that may not only be important in the pathogenesis of HCC but also demonstrates how such antigen‐antibody systems may be used to develop strategies for immuno‐targetting and gene therapy of HCC.

TRANSGENIC OVEREXPRESSION OF INSULIN RECEPTOR SUBSTRATE 1 IN HEPATOCYTES ENHANCES HEPATOCELLULAR PROLIFERATION IN YOUNG MICE ONLY

Aim:  The insulin receptor substrate‐1 (IRS‐1) is a multisite docking protein which plays a central role in the signal transduction of growth factors such as insulin and insulin‐like growth factors (IGF‐1 and IGF‐2). It is found to be frequently overexpressed in human hepatocellular carcinoma (HCC).

Mechanisms of cell death induced by the suicide genes encoding purine nucleoside phosphorylase and thymidine kinase in human hepatocellular carcinoma cells in vitro

For gene therapy of hepatocellular carcinoma (HCC), the Escherichia coli purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system may be more useful than the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system as a result of a stronger bystander effect. To analyze the molecular mechanisms involved in PNP/fludarabine-mediated cell death in human HCC cells in comparison with HSV-tk/GCV, we transduced human HCC cells of the cell lines, HepG2 and Hep3B, with PNP or HSV-tk using adenoviral vectors, followed by prodrug incubation. Both systems predominantly induced apoptosis in HepG2 and Hep3B cells. PNP/fludarabine induced strong p53 accumulation and a more rapid onset of apoptosis in p53-positive HepG2 cells as compared with p53-negative Hep3B cells, but efficiency of tumor cell killing was similar in both cell lines. In contrast, HSV-tk/GCV-induced apoptosis was reduced in p53-negative Hep3B cells as compared with p53-positive HepG2 cells. HSV-tk/GCV, but not PNP/fludarabine, caused up-regulation of Fas in p53-positive HepG2 cells and of Fas ligand (FasL) in both HCC cell lines. These results demonstrate cell line-specific differences in response to treatment with PNP/fludarabine and HSV-tk/GCV, respectively, and indicate that PNP/fludarabine may be superior to HSV-tk/GCV for the treatment of human HCC because of its independence from p53 and the Fas/FasL system.

Immunotherapy directed against alpha-fetoprotein results in autoimmune liver disease during liver regeneration

BACKGROUND & AIMS Priming immune responses against alpha-fetoprotein (AFP) highly expressed in the majority of hepatocellular carcinomas results in significant antitumoral T-cell responses. Liver regeneration in humans and mice, however, is also associated with increased AFP expression. Therefore, we evaluated the risk of AFP-directed immunotherapeutic approaches to induce autoimmunity against the regenerating liver. METHODS Mice were immunized with DNA encoding mouse AFP. For induction of liver regeneration, partial hepatectomy was performed and mice were monitored by serial histopathologic examinations and measurements of serum ALT activities (U/L), and by determination of the kinetics of AFP-specific T-cell responses. RESULTS Livers of AFP immune mice without partial hepatectomy were characterized by minor lymphocytic infiltrations without transaminase elevations. By contrast, a significant hepatocyte damage was observed in regenerating liver that correlated well with the number of AFP-specific CD8(+) T cells, the activity of liver regeneration, and the level of AFP synthesis. Autoimmune liver damage was mediated by CD4(+) T cell-dependent CD8(+) cytotoxic T lymphocytes. CONCLUSIONS These results show that priming of T-cell responses against shared tumor-specific self antigens may be accompanied by induction of autoimmunity dependent on the level of expression of the self antigen and have important implications for the development of antitumoral vaccines targeted against antigens that are not strictly tumor-specific.