Aphidicolin induces 6-thioguanine resistant mutants in human diploid fibroblasts

Abstract

Cytotoxic and mutagenic effects of aphidicolin (APC), an inhibitor of DNA polymerases α and δ, were studied in human diploid VH-10 fibroblasts. The cells were treated (2 or 4 h) with APC at concentration ranges of 10–40 μM. 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 μM 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 4 h treatment with APC the cells were blocked in G 2 phase during the majority of the expression period, compared to control cells. Four days after the treatment, the amount of cells in G 2 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.