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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 Therapy volume 9, pages 1564–1575 (2002)Cite this article

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 (IC50 = 78 μM, 96-h incubation) via CD and to ganciclovir (GCV, IC50 = 1 μM, 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, 19F-NMR spectroscopy showed that incubation of cultured CDglyTK+ cells with 774 μM 5-FC for 16 h resulted in the following intracellular concentrations: 5-FC = 314 μM, 5-FU = 52 μM, cytotoxic fluoronucleotides = 163 μM; extracellular 5-FU reached only 6.4 μM. 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.

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Acknowledgements

The authors thank Dr Ivan Zuna (DKFZ) for performing statistical analyses, Gabi Müller (DKFZ) for supplying the AT-1/GFP cell line, Dr JF Gebert (University Clinic, Heidelberg, Germany) for supplying anti-cytosine deaminase antibody, and Dr RP Mason (University of Texas Southwestern Medical Center at Dallas) for the in vivo tumour pH measurements.

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Authors and Affiliations

  1. Clinical Cooperation Unit, Radiotherapeutical Oncology, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany

    H Corban-Wilhelm, G Becker, D Greulich & J Debus

  2. Central Spectroscopy Department, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany

    W E Hull

  3. Department of Medical Physics, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany

    U Bauder-Wüst

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  1. H Corban-Wilhelm
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Corban-Wilhelm, H., Hull, W., Becker, G. et al. 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 9, 1564–1575 (2002). https://doi.org/10.1038/sj.gt.3301834

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