A STUDY OF THE EFFECTS OF PHORBOL 12-MYRISTATE-13-ACETATE ON CELL-DIFFERENTIATION OF PURE HUMAN MELANOCYTES INVITRO

Pure human melanocyte cultures were established in a serum-free medium containing epidermal growth factor (10 ng/ml), hydrocortisone (10(-7) M), insulin (5-mu-g/ml), transferrin (10-mu-g/ml), cholera toxin (2 ng/ml), isobutylmethyl xanthine (10(-4) M) and bovine pituitary extract (30-mu-g/ml). To investigate the biological effects of PMA on melanocytes in vitro, the cells were incubated in media containing various concentration of PMA (including 0 nM, 85 nM and 170 nM), and grown continuously for 12 days without passage. The cells were observed for changes in cell morphology, cell cycle, cytoskeleton and HLA-DR expression. In addition, the effect of PMA on tyrosinase activity was also evaluated. The results revealed that the higher the PMA concentration, the higher the percentage of large irregularly shaped melanocytes. These large melanocytes were three to ten times the size of small bipolar or multipolar cells. A higher concentration of PMA was also associated with a higher percentage of melanocytes in the S and G2-M phases of the cell cycle and with a higher percentage of melanocytes as tetraploid and octaploid karyotypes. The cytoskeleton (vimentin) in the large irregularly shaped cells appeared disaggregated as compared with that in the usual dendritic cells with a compact distribution. HLA-DR was found to be expressed on some melanocytes growing in media containing PMA, appearing both in large dendritic cells and large irregularly shaped cells. None of the cells expressed HLA-DR when cultured in the abscence of PMA. PMA at 85 nM, but not at 170 nM, significantly stimulated tyrosinase activity as compared with the controls (0 nM PMA). It thus seems appropriate to study the carcinogenic or other properties of melanocytes in systems that do not contain PMA, such as the serum-free medium proposed by us.