Use of an orthogonal design method to study two-stage chemical carcinogenesis in BALB/3T3 cells

An orthogonal design method was used to study two-stage chemical carcinogenesis in BALB/3T3 cells. Four factors were studied: different concentrations of the initiator N-methyl-N-nitro-N'-nitrosoguanidine (MNNG); different concentrations of the promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA); different concentrations of fetal calf serum (FCS) and different times at which TPA was added after cell initiation. From the results of three experiments designed by Table L9(3(4)), L8(2(7)) and L16(4(5)), 0.3 microgram/ml MNNG was the highest possible initiating concentration and 0.25 microgram/ml TPA was the minimum effective concentration for promoting activity. There is synergy between MNNG and TPA, the optimum combination in sequential treatment being 0.3 microgram/ml MNNG and 0.25 microgram/ml TPA. The 10% FCS standard concentration was the optimal one; however, below 5% few foci appeared. The time at which TPA was added had little effect on cloning efficiency and transformation frequency. So the use of this orthogonal design in cell culture has many advantages: several factors can be tested simultaneously; it is easy to find the optimal protocol conditions and the dose-response relationship is stable, which enables the reproducibility to be improved. In addition, the different tables proposed may help to reveal unexpected problems.