Effect of phorbol ester tumor promoters on the expression of melanogenesis in B-16 melanoma cells.

Cells of the C3 clone of B-16 melanoma synthesize melanin only at confluence after which they senesce and can no longer be passaged. Addition to the cultures of 10(-8)--10(-7) M 12-O-tetradecanoylphorbol-13-acetate (TPA) shortly after plating delayed by about 2 days the onset of melanogenesis. TPA did not, however, affect the growth of the cells or the time at which they reached confluence. The ability of a series of phorbol esters to delay melanogenesis correlated with their tumor-promoting activity on mouse skin. The optimum time for addition of TPA was within the first 24 hr after plating; the inhibitory effect decreased when TPA was added at later points. alpha-melanocyte-stimulating hormone (5 x 10(-7) M) added to B-16 cultures 24 hr after plating slowed the growth of the cells and caused them to differentiate when still subconfluent. TPA also inhibited this alpha-melanocyte-stimulating hormone-induced melanogenesis. These results suggest that TPA inhibits a very early stage in a stepwise process that leads to the differentiation of these cultures. For reasons that are not apparent, the cells eventually escape from this inhibition. The B-16 melanoma cell culture system may be useful for studying the mechanism by which TPA and related tumor promoters affect cellular differentiation.