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.
Corban-Wilhelm
H
H
Clinical Cooperation Unit, Radiotherapeutical Oncology, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Hull
W E
WE
Becker
G
G
Bauder-Wüst
U
U
Greulich
D
D
Debus
J
J
D000818:Animals; D000963:Antimetabolites / Q000493:pharmacokinetics / Q000494:pharmacology; D024201:Bystander Effect; D002470:Cell Survival / Q000187:drug effects; D043525:Cytosine Deaminase; D004195:Disease Models, Animal; D018572:Disease-Free Survival; D005437:Flucytosine / Q000493:pharmacokinetics / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D009700:Nucleoside Deaminases / Q000235:genetics; D011355:Prodrugs / Q000493:pharmacokinetics; D011471:Prostatic Neoplasms / Q000473:pathology / Q000628:therapy*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D000963:Antimetabolites; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D005437:Flucytosine; D013937:Thymidine Kinase; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil