Phenotypic expression of carcinogen-initiated epidermal cells to tumor cells by 12-O-tetradecanoylphorbol-13-acetate in a manner dependent on 3T3 fibroblast-derived humoral factor(s).

Primary cultured newborn mouse epidermal cells cultured in the low Ca2+ (0.02 mM) medium showed typical basal cell morphology and proliferated as a monolayer. A stepwise increase in medium Ca2+ concentration induced terminal differentiation of epidermal cells. In the case of epidermal cells obtained from newborn mice transplacentally initiated with 7,12-dimethylbenz[a]anthracene (DMBA) or epidermal cells initiated in vitro by DMBA, a small number of rapidly growing cellular foci with epidermal morphology appeared and proliferated when the medium Ca2+ concentration was raised. Without increasing Ca2+ concentration, such foci never appeared. However, the Ca2+ concentration of the extracellular milieu of basal epidermal cells is known to be very low in in vivo epidermis. Under the low Ca2+ conditions, 12-O-tetradecanoylphorbol-13-acetate (TPA), a most potent skin tumor promoter, never induced rapidly growing cellular foci. When the initiated epidermal cells were co-cultured with 3T3 fibroblasts but without direct cell-to-cell contact, TPA induced rapidly growing cellular foci even under the low Ca2+ condition. Without initiation, such cellular foci hardly appeared. 3T3 fibroblasts induced only a very small number of cellular foci in the absence of TPA. Co-culture with mouse peritoneal macrophages was not effective in inducing such cellular foci, indicating that the effect is 3T3 fibroblast specific. The conditioned medium of 3T3 fibroblasts was also capable of inducing such cellular foci. Three of these rapidly growing cellular foci were cloned and designated as WYF-30, WYF-31 and WYF-32 respectively. All of these three cell lines grew rapidly in the normal (1.8 mM) Ca2+ medium, indicating that these cell lines were resistant to Ca(2+)-induced differentiation. In the low Ca2+ medium, the growth of these three cell lines was stimulated by TPA. All three cell lines formed colonies in soft agar. The number of colonies formed under the normal Ca2+ condition was larger than that formed under the low Ca2+ condition. Under the low Ca2+ condition, the colony formation of each cell line was augmented by TPA. All the cell lines formed tumors in nude mice. These results indicate that TPA induces phenotypic expression of dormant initiated cells to tumor cells in a manner dependent on 3T3 fibroblast-derived humoral factor(s).