Specific binding of the tumor promoter TPA in various mouse organs as measured by a 'cold acetone-filter assay'

Abstract

Rapid, specific, saturable and partially reversible binding of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate ([³H]TPA) to a particulate fraction of mouse brain can be demonstrated by means of a ‘cold acetone-filter assay’; by washing with cold acetone at -20°C, nonspecific binding of the highly lipophilk [³H]TPA to membranes can be reduced to ∼10% of the total binding. A comparative study of homogenates of several organs with this test revealed specific [³H]TPA binding/ μg DNA in the order brain > lung ≅ spleen ≅ Over ≅ kidney ≅ thymus ≅ ovaries. Specific binding sites were also detected hi 600 × g supernatant fractions of homogenates from epidermis, forestomach, glandular stomach and small intestine. Competition experiments showed displacement of [³H]TPA by TPA > phorbol-12,13-didecanoate > 12-deoxyphorbol-13-tetradecano-ate > phorbol-12,13-dibutyrate (PDBu) > phorbol-12,13-diacetate > 4-O-methyl-TPA > 4α-phorbol-12,13-didecanoate in the brain particulate fraction. Specific [³H]TPA binding is sensitive to heat, periodate and dithiothreitol, but is relatively insensitive to urea or to 1–5% solutions of several common detergents. It is estimated that the present binding test is ∼500 times more sensitive than the widely-accepted [³H]PDBu assay; perhaps more important, the present method employs TPA, which is extremely effective both as a tumor promoter in vivo and as a pleiotropic effector of cells in vivo and in vitro.