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 microg/mL). No signs of toxicity were found in control cells expressing EGFP even at high prodrug concentrations (20 microg/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 CYP4B1/4-1M 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.