Gene therapy of hepatocellular carcinoma in vitro and in vivo in nude miceby adenoviral transfer of the Escherichia coli purine nucleoside phosphorylase gene

Expression of viral or bacterial enzymes in tumor cells to convert nontoxic prodrugs into highly toxic metabolites is an attractive gene-therapeutic a pproach for the treatment of hepatocellular carcinoma (HCC). The Escherichi a coli purine nucleoside phosphorylase (PNP) converts purine analogs into f reely diffusible metabolites, which are highly toxic to dividing and nondiv iding cells. We investigated the antitumor effects of PNP in the human HCC cell lines, HepG2, Hep3B, and HuH-7, and performed a comparison with herpes simplex thymidine kinase (TK), The genes for PNP, TK, and enhanced green f luorescent protein (EGFP) were delivered to HCC cells by identical adenovir al vectors. Fludarabine and ganciclovir (GCV) served as prodrugs for PNP an d TK, respectively. Expression of PNP highly sensitized HCC cells to fludar abine treatment. Fludarabine concentrations between 0.5 and 1 mu g/mL kille d 100% of the cells expressing PNP with no detectable toxicity in control c ells expressing EGFP, Expression of PNP in as few as 10% of HCC cells induc ed efficient killing of most bystander cells. Expression of TK followed by GCV treatment produced a potent growth inhibition but failed to kill all TK -expressing HCC cells. More importantly, the TK system exhibited a lower de gree of bystander effect. Adenoviral delivery of PNP followed by fludarabin e administration prevented subcutaneous and intrahepatic tumor formation in nude mice and was also effective for the treatment of established tumors. These results demonstrate the potential of the PNP/fludarabine system for t he treatment of HCC.