Bystander Effects of Different Enzyme-Prodrug Systems for Cancer Gene Therapy Depend on Different Pathways for Intercellular Transfer of Toxic Metabolites, a Factor That Will Govern Clinical Choice of Appropriate Regimes

Transfer of suicide genes into tumor cells renders them sensitive to cytotoxic effects of specific prodrugs. We show here that both the herpes simplex virus thymidine kinase/ganciclovir (tk/GCV) and thymidine phophorylase/5′-deoxy-5-fluorouridine (tp/DFUR) suicide gene systems can induce cell death in tumor cells. Additionally in mixed cultures of cells with and without the suicide gene, death occurred in both cell types, indicative of a bystander effect. We demonstrate, in human and rodent cell lines, that the tk/GCV bystander effect requires gap junctional intercellular communication (GJIC). Where cultures lack GJIC, no bystander effect was observed. In communicating cultures, no correlation between level of GJIC and bystander effect was seen and this was due to high levels of tk activity. Additionally, we demonstrate that transfer of toxic metabolites from tk+ to tk¯ cells occurs within 2 hr of GCV application and, as no apoptosis could be detected until after this time, apoptosis is the result, not the cause, of the tk/GCV bystander effect. In the tp/DFUR system, a medium-mediated bystander effect, independent of GJIC and apoptosis, was observed. We demonstrated that combining tk/GCV and tp/DFUR suicide gene systems in culture was more effective than either therapy alone. We have investigated the bystander effects of the thymidine kinase/ganciclovir (tk/GCV) and thymidine phosphorylase/5′-deoxy-5-fluorouridine (tp/DFUR) enzyme/prodrug systems in vitro. We demonstrate that spread of toxicity to wild-type cells from tk-expressing cells requires functional gap junctions whereas transfer of toxicity in the tp/DFUR system occurs via the medium. Combining these enzyme/prodrug systems leads to an increased bystander effect.