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Comparison of different methods to assess the cytotoxic effects of cytosine deaminase and thymidine kinase gene therapy

Cancer Gene Therapy volume 11, pages 208–214 (2004)Cite this article

Abstract

Dunning R3327 AT-1 rat prostate tumor cells were transfected with a double-fusion suicide gene (CDglyTK) that coded for the cytosine deaminase from E. coli and the thymidine kinase (TK) from HSV-1. The resulting cell line AT-1/CDglyTK was incubated with 10 and 20 μg/ml 5-FC or 0.25 μg/ml GCV, or both 5-FC and GCV 96 hours before harvest. The MTS assay detected cell viabilities of 50±5 and 25±5% after 5-FC treatment, and 50±5% after GCV treatment. The dye exclusion and the colony-forming assay confirmed the data of the MTS assay with GCV (47±5 and 32±5%), but presented different results for the 5-FC incubation. We detected 100±1 and 85±5% viable cells after 10 μg/ml 5-FC, and 97±1 and 85±5% after 20 μg/ml 5-FC treatment, respectively. S-phase arrest in both suicide gene systems was noticeable and a significant increase in cell granularity was observed after incubation with GCV or GCV & 5-FC. This study demonstrates that 5-FC and the metabolized 5-FU act not only as genotoxic reagents, but also as RNA-directed agent, because of the recovery of the cells. On the other hand, a significant S-phase block could be observed after 24 hours incubation with GCV. This short time is enough to incorporate the genotoxic GCV metabolites in the nascent DNA to impair the cell cycle.

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Authors and Affiliations

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

    Heike Corban-Wilhelm, Gabriele Becker, Ditmar Greulich, Klaus Braun & Jürgen Debus

  2. Institute of Pathology, University of Heidelberg, Germany

    Volker Ehemann

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  1. Heike Corban-Wilhelm
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Correspondence to Heike Corban-Wilhelm.

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Corban-Wilhelm, H., Ehemann, V., Becker, G. et al. Comparison of different methods to assess the cytotoxic effects of cytosine deaminase and thymidine kinase gene therapy. Cancer Gene Ther 11, 208–214 (2004). https://doi.org/10.1038/sj.cgt.7700667

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