Specific binding of phorbol ester tumor promoters

Publisher Website
Google Scholar
Abstract
[20-3H]Phorbol 12,13-dibutyrate bound to particulate preparations from chicken embryo fibroblasts in a specific, saturable, reversible fashion. Equilibrium binding occurred with a Kd of 25 nM; this value is very close to the 50% effective dose (ED50), 50 nM, previously determined for the biological response (induction of fibronectin loss) in growing chicken embryo fibroblasts. At saturation, 1.4 pmol of [20-3H]phorbol 12,13-dibutyrate was bound/mg of protein (approximately 7 .times. 104 molecules/cell). Binding was inhibited by phorbol 12-myristate 13-acetate (Ki = 2 nM), mezerein (Ki = 180 nM), phorbol 12,13-diacetate (Ki = 1.7 .mu.M), phorbol 12,13,20-triacetate (Ki = 39 .mu.M), and phorbol 13-acetate (Ki = 120 .mu.M). The measured Ki values are all within a factor of 3.5 of the ED50 values of these derivatives for inducing loss of fibronectin in intact cells. Binding was not inhibited by the inactive compounds phorbol (10 .mu.g/ml) and 4.alpha.-phorbol 12,13-didecanoate (10 .mu.g/ml) or by the inflammatory but nonpromoting phorbol-related diterpene esters resiniferatoxin (100 ng/ml) and 12-deoxyphorbol 13-isobutyrate 20-acetate (100 ng/ml). Biological responses to the phorbol esters in chicken embryo fibroblasts may be mediated by this binding activity and the binding activity corresponds to the phorbol ester target in mouse skin involved in tumor promotion. Binding was not inhibited by the nonphorbol promoters anthralin (1 .mu.M), phenol (1 mM), iodoacetic acid (1.7 .mu.M) and cantharidin (75 .mu.M) or by epidermal growth factor (100 ng/ml), dexamethasone acetate (2 .mu.M), retinoic acid (10 .mu.M) or prostaglandin E2 (.mu.M). These agents appear to act at a target distinct from that of the phorbol esters.