DOWN-REGULATION OF TYROSINASE MESSENGER-RNA LEVELS IN MELANOMA-CELLS BY TUMOR PROMOTERS AND BY INSULIN

Mouse melanoma cells in culture respond to melanocyte-stimulating hormone (MSH) or to cyclic AMP analogues by demonstrating an increase in tyrosinase activity. In this study the effect of the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), on the hormonal induction of tyrosinase was examined. TPA was found to lower basal levels of tyrosinase activity in melanoma cells and to reduce tyrosinase levels in cells treated with either MSH (10-7 M), dibutyryl cAMP (10-4 M), isobutylmethylxanthine (IBMX, 10-4 M), or with the potent MSH analogue, [Nle4,d-phe7]-α-MSH. The phorbol ester, phorbol 12,13-dibutyrate was also effective in lowering tyrosinase activity levels, while 4α-phorbol 12,13-didecanoate, which does not bind protein kinase C, was ineffective. In order to determine how TPA may reduce tyrosinase activity in melanoma cells, the levels of tyrosinase mRNA in untreated or TPA-treated cells were determined by Northern blot analysis. A marked down-regulation of constitutive levels of tyrosinase mRNA was observed in cells treated with the tumor promoter. Tyrosinase mRNA levels in cultures exposed to TPA for 48 h were only 7% of control levels. Tyrosinase mRNA levels in cells treated with both MSH and TPA were also lower than in cells treated with MSH alone. Previous studies from this laboratory have shown that insulin both lowers basal tyrosinase activity in melanoma cells and antagonizes the MSH stimulation of the enzyme. We have now determined that this inhibition is also due to reduced levels of tyrosinase mRNA. These results implicate a role for protein kinase C in the phorbol ester and insulin-mediated down-regulation of tyrosinase gene activity in melanoma cells and suggest the possible presence of both inhibitory and stimulatory DNA control elements in the tyrosinase gene which may control transcriptional rates. © 1990.