Cytosine deaminase versus thymidine kinase: a comparison of the antitumor activity.

The efficacy of single and combination suicide gene therapy was evaluated using a Herpes simplex virus thymidine kinase/ganciclovir system and Escherichia coli cytosine deaminase/5-fluorocytosine system on the rat prostate tumor cell line R3327 AT-1. The wild-type R3327 AT-1 cell line was transfected with a bifunctional fusion gene CDglyTK, which had the advantage that the resulting R3327 AT-1/CDglyTK cell line has the same amount of cytosine deaminase and thymidine kinase molecules. The percentage of viable R3327 AT-1/CDglyTK cells after 96 h incubation with 0.1 micro g/ml ganciclovir or 10 micro g/ml 5-fluorocytosine were 85% and 52% of controls, respectively. The cell viability when both suicide genes systems were activated was 43%. For in vivo analysis, Copenhagen rats were injected subcutaneously with R3327 AT-1 or R3327 AT-1/CDglyTK cells and treated with 30 mg/kg ganciclovir, 500 mg/kg 5-fluorocytosine, or both prodrugs together. A survival of 83% with the thymidine kinase/ganciclovir and 57% with the CD/5-FC could be observed. Only co-administration of thymidine kinase- and cytosine deaminase-specific prodrugs resulted in a 100% recurrence-free survival of the Copenhagen rats with a Dunning R3327 AT-1/CDglyTK prostate tumor and showed an additive cytotoxic effect. Calculation of the degree of activation and the potential of activation can be used to predict the success of a suicide gene therapy. In our case, the cytosine deaminase/5-fluorocytosine system had a low degree of activation (value 40), which is also found in the low response to 5- fluorocytosine in vivo (57% tumor free).

Cytosine deaminase and thymidine kinase gene therapy in a Dunning rat prostate tumour model: absence of bystander effects and characterisation of 5-fluorocytosine metabolism with 19F-NMR spectroscopy.

The rat prostate tumour cell line R3327 AT-1 was transfected with a gene coding for a fusion protein comprised of cytosine deaminase (CD from E. coli) and thymidine kinase (TK from Herpes simplex virus, HSV-1). The resulting AT-1/CDglyTK cell line was sensitive to the prodrug 5-fluorocytosine (IC(50) = 78 microM, 96-h incubation) via CD and to ganciclovir (GCV, IC(50) = 1 microM, 96 h) via TK. Subcutaneous tumours generated from 100% CDglyTK(+) cells responded well to 5-FC therapy (500 mg/kg, i.p., 14 daily treatments, four out of seven animals in remission) and to GCV therapy (30 mg/kg, i.p., 14 daily treatments, five of six animals in remission). However, experiments with mixtures of CDglyTK(+) and CDglyTK(-) cells showed low levels of connexins (intercellular gap junctions) and no bystander effect for nontransfected cells using either 5-FC or GCV therapy. Furthermore, (19)F-NMR spectroscopy showed that incubation of cultured CDglyTK(+) cells with 774 microM 5-FC for 16 h resulted in the following intracellular concentrations: 5-FC = 314 microM, 5-FU = 52 microM, cytotoxic fluoronucleotides = 163 microM; extracellular 5-FU reached only 6.4 microM. Thus, in this model system intracellular trapping of 5-FU (slow export) contributes to the failure of the CD/5-FC bystander effect via an extracellular route.