title: Comparison of different methods to assess the cytotoxic effects of cytosine deaminase and thymidine kinase gene therapy.
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 microg/ml 5-FC or 0.25 microg/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 microg/ml 5-FC, and 97+/-1 and 85+/-5% after 20 microg/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.
authors.affiliation: Clinical Cooperation Unit Radiotherapeutical Oncology, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany. h.corban-wilhelm@dkfz.de
authors.forename: Heike
authors.initials: H
authors.lastname: Corban-Wilhelm
authors.forename: Volker
authors.initials: V
authors.lastname: Ehemann
authors.forename: Gabriele
authors.initials: G
authors.lastname: Becker
authors.forename: Ditmar
authors.initials: D
authors.lastname: Greulich
authors.forename: Klaus
authors.initials: K
authors.lastname: Braun
authors.forename: Jürgen
authors.initials: J
authors.lastname: Debus
mesh_terms: D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002470:Cell Survival; D043525:Cytosine Deaminase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005437:Flucytosine / Q000378:metabolism / Q000633:toxicity; D015774:Ganciclovir / Q000378:metabolism / Q000633:toxicity; D018014:Gene Transfer Techniques; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods*; D018259:Herpesvirus 1, Human / Q000235:genetics; D008297:Male; D011471:Prostatic Neoplasms / Q000473:pathology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods; D014410:Tumor Stem Cell Assay
chemical_list: D011993:Recombinant Fusion Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; D005437:Flucytosine; D013937:Thymidine Kinase; D043525:Cytosine Deaminase; D015774:Ganciclovir