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

Gene Ther. 2002 Dec;9(23):1564-75. doi: 10.1038/sj.gt.3301834.

Abstract

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.

MeSH terms

  • Animals
  • Antimetabolites / pharmacokinetics
  • Antimetabolites / pharmacology
  • Bystander Effect*
  • Cell Survival / drug effects
  • Cytosine Deaminase
  • Disease Models, Animal
  • Disease-Free Survival
  • Flucytosine / pharmacokinetics
  • Flucytosine / pharmacology
  • Fluorouracil / pharmacology
  • Genetic Therapy / methods*
  • Magnetic Resonance Spectroscopy
  • Male
  • Nucleoside Deaminases / genetics
  • Prodrugs / pharmacokinetics
  • Prostatic Neoplasms / pathology
  • Prostatic Neoplasms / therapy*
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Thymidine Kinase / genetics
  • Transfection
  • Tumor Cells, Cultured

Substances

  • Antimetabolites
  • Prodrugs
  • Recombinant Fusion Proteins
  • Flucytosine
  • Thymidine Kinase
  • Nucleoside Deaminases
  • Cytosine Deaminase
  • Fluorouracil