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

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 degree C, nonspecific binding of the highly lipophilic [3H]TPA to membranes can be reduced to approximately 10% of the total binding. A comparative study of homogenates of several organs with this test revealed specific [3H]TPA binding/microgram DNA in the order brain much greater than lung approximately equal to spleen approximately equal to liver approximately equal to kidney approximately equal to thymus approximately equal to ovaries. Specific binding sites were also detected in 600 x g supernatant fractions of homogenates from epidermis, forestomach, glandular stomach and small intestine. Competition experiments showed displacement of [3H]TPA by TPA greater than phorbol-12,13-didecanoate greater than 12-deoxyphorbol-13-tetradecanoate greater than phorbol-12,13-dibutyrate (PDBu) greater than phorbol-12,13-diacetate greater than 4-O-methyl-TPA greater than 4 alpha-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 approximately 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.