Aphidicolin induces 6-thioguanine resistant mutants in human diploid fibroblasts

Cytotoxic and mutagenic effects of aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta, were studied in human diploid VH-10 fibroblasts. The cells were treated (2 or 4h) with APC at concentration ranges of 10-40 microM. The effect of APC on cell survival after 4 h treatment was significantly higher than after 2 h treatment. The mutagenicity of APC was investigated at the HPRT locus, and the frequency of HPRT mutants was estimated by selection in medium containing 6-thioguanine (6-TG). Treatment of fibroblast cells with 20 microM of APC for 2 or 4 h resulted approximately in 5 or 10 times increase of 6-TG resistant mutant frequencies, respectively, compared to untreated control cells.The cell cycle analyses performed during the expression time (9-12 days) have shown that after 2 and 4h treatment with APC the cells were blocked in G2 phase during the majority of the expression period, compared to control cells. Four days after the treatment, the amount of cells in G2 phase increased about two-fold (28.6-31.8% compared to 13.5% in the untreated cells). The mode of cell death during the expression time was via necrosis, rather than apoptosis, which was demonstrated by fluorescein-diacetate (FDA)-staining and terminal dUTP nick end labeling (TUNEL)-method.