Inhibition by Dexamethasone of Intracellular Binding of Phorbol Esters to Mouse Skin2

Abstract

The binding of phorbol esters to mouse epidermal subcellular fractions and the effect of tumor promoters on the protein complement of cytosol were investigated. When applied to mouse skin, ¹⁴C-labeled 12-0-tetradecanoylphorbol-13 acetate (TPA) and ³H-labeled 12-O-hexadecanoylphorbol-13 acetate (HPA) remained associated, after extensive dialysis, with epidermal chromatin, cytosol, microsomes, and mitochrondria. The specific activity of TPA and HPA binding to the subcellular fractions reached a peak by 1 hour after treatment. The powerful tumor promoter TPA had a greater affinity for the subcellular fractions than did the weaker promoter HPA. Simultaneous application of the anti promoting agent dexamethasone inhibited the binding to the cytosol and chromatin fractions from 1 to 24 hours after treatment but did not affect the other fractions. Label continued to be associated with cytosol protein after removal of unbound radioactivity by dialysis, gel filtration, and ultrafiltration. Polyacrylamide gel electrophoresis of the promoter- bound cytosol protein showed that most radioactivity was associated with the protein fraction previously reported to be a receptor for 3-methylcholanthrene. TPA and HPA binding to the cytosol receptor protein was inhibited between 3 and 12 hours after dexamethasone treatment. In vitro competition by a promoter or a non promoter for binding of radioactive TPA to the cytosol receptor protein correlated with promoting activity. However, dexamethasone did not compete in vitro for TPA binding to the cytosol receptor protein. When the protein complement of mouse epidermal cytosol was analyzed by electrophoresis on two different polyacrylamide gels, dexamethasone inhibited a slowly migrating fraction which was most enhanced between 12 and 24 hours after treatment. The findings suggested that both the chromatin and cytosol fractions were involved in the early action of skin tumor promoters.