The proliferative response of low-density human cell cultures to tumor promoters and its relevance to carcinogenic mechanisms in vitro.

The effects of the tumor promoter -12-O-tetradecanoyl phorbol-13-acetate, its analogues, and other tumor promoters, on the proliferation of human skin fibroblasts (SF) have been investigated. We have previously shown (Kopelovich and Bias, 1979), that TPA caused a biphasic (upward concave) dose effect in the cloning efficiency assay of normal and mutant human fibroblastic cell strains. Here we report that the biphasic dose response pattern, consisting of an inhibitory phase and a stimulatory phase, was shared by the active analogues of TPA, e.g., phorbol-12,13-dibutyrate and phorbol-12,13-dibenzoate. This biphasic dose effect relationship, however, was not seen with phorbol-12,13-diacetate or the inactive analogues of TPA such as phorbol and 4-O-methyl-12-O-tetradecanoyl phorbol-13-acetate, nor was it seen with mezerein, teleocidin, or bile-acid derivatives of humans. An analysis of the cloning efficiency data by the median-effect equation (Chou and Talalay, 1981) showed that in low-density cultures both the inhibitory phase and the stimulatory phase of the dose-effect relationships of TPA, its analogues, mezerein, and teleocidin exhibited a linear median-effect plot and thus closely followed the basic mass-action principle. The median-effect plot of these data allowed quantitative determination of growth curve characteristic such as regression coefficient, slope (a measure of sigmoidicity), median-effect concentrations such as I50 for the inhibitory effect, and A50 for the stimulatory effect (i.e., the relative potency of the analogues) and the transition point of the biphasic phenomenon in the case of the phorbol esters. In addition, we have demonstrated a relationship between the dose response effect of TPA on the proliferation of various human cells and tumor progression in vitro.