Improvement in the efficiency of the in vitro transformation assay method using BALB/3T3 A31-1-1 cells

In order to improve the in vitro transformation assay using BALB/3T3 cells, which is routinely conducted with minimal essential medium (MEM) containing 10% fetal calf serum (FCS), we examined the effect of a new medium after the cells had been treated with carcinogens. Preliminary experiments suggested that the use of T medium (modified DME/F-12) supplemented with insulin, transferrin, ethanolamine and sodium selenite plus 2% FCS resulted in a high transformation frequency. The present study confirmed that this medium was very efficient at inducing transformation foci. Transformation frequency was highest when 12-O-tetradecanoylphorbol-13-acetate (TPA) was started from 1 week after the carcinogen treatment. According to the new protocol using this medium, the transformation frequency was five times higher and transformation foci appeared much earlier than with the protocol using usual MEM plus 10% FCS medium. In addition, we tested several typical carcinogens and a promoter in order to confirm the applicability of the new protocol to the two-stage transformation assay. N-Methyl-N'-nitro-N-nitro-soguanidine (MNNG) and benzo[a]pyrene, but not noncarcinogenic benzo[e]pyrene, induced transformation foci. In the metabolic activation system dimethylnitrosamine gave a highly positive transformation. Okadaic acid, a non-TPA-type tumor promoter, enhanced transformation of MNNG-initiated cells. These studies demonstrate that the use of the proposed medium drastically improves transformation frequency in the two-stage in vitro transformation assay.