Inhibition of Terminal Differentiation of Hamster Epidermal Cells in Culture by the Phorbol Ester 12-O-Tetradecanoylphorbol-13-Acetate¹

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the terminal differentiation of hamster epidermal cells in culture was studied. Epidermal cells were isolated from 1-day-old Syrian hamsters by separating the epidermis from the dermis by cold trypsin treatment. A large number of cells were isolated by this procedure without contamination with dermal fibroblasts. When grown in culture, the epidermal cells divided rapidly, stratified, and differentiated as measured by elaboration of abundant keratin-like amorphous material, red staining with rhodanile blue (which is characteristic of cornifying epithelium), and formation of cornified envelopes. These structures were measured by electron microscopy and quantitation of detergent-insoluble cell ghosts. TPA markedly inhibited this differentiation of the hamster epidermal cells in culture. When grown in the presence of TPA (5 to 1000 ng/ml) for three or more days, the epidermal cell monolayers failed to stain positively with rhodanile blue, and the cell stratification and production of keratin-like material was reduced. The differentiation of the epidermal cells was quantitated by measuring the percentage of cells with cornified envelopes; TPA reduced by up to 70% the number of these terminally differentiated cells. Phorbol didecanoate also inhibited the differentiation of hamster epidermal cells in culture, while phorbol was inactive. The effect of TPA was reversible. When TPA was removed from the media, the cells rapidly differentiated to the same extent as did untreated cells. TPA also stimulated DNA synthesis of the epidermal cells, especially after 10 days in culture when the vast number of cells in control cultures had ceased DNA synthesis. These results are discussed in view of the fact that TPA has not been demonstrated to promote epidermal carcinogenesis in Syrian hamsters.