Specific binding of phorbol ester tumor promoters

[20- 3 H]Phorbol 12,13-dibutyrate bound to particulate preparations from chicken embryo fibroblasts in a specific, saturable, reversible fashion. Equilibrium binding occurred with a K d of 25 nM; this value is very close to the 50% effective dose (ED 50 ), 50 nM, previously determined for the biological response (induction of fibronectin loss) in growing chicken embryo fibroblasts. At saturation, 1.4 pmol of [20- 3 H]phorbol 12,13-dibutyrate was bound per mg of protein (approximately 7 × 10 4 molecules per cell). Binding was inhibited by phorbol 12-myristate 13-acetate ( K i = 2 nM), mezerein ( K i = 180 nM), phorbol 12,13-dibenzoate ( K i = 180 nM), phorbol 12,13-diacetate ( K i = 1.7 μM), phorbol 12,13,20-triacetate ( K i = 39 μM), and phorbol 13-acetate ( K i = 120 μM). The measured K i values are all within a factor of 3.5 of the ED 50 values of these derivatives for inducing loss of fibronectin in intact cells. Binding was not inhibited by the inactive compounds phorbol (10 μg/ml) and 4α-phorbol 12,13-didecanoate (10 μg/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). These data suggest that biological responses to the phorbol esters in chicken embryo fibroblasts are mediated by this binding activity and that 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 μM), phenol (1 mM), iodoacetic acid (1.7 μM), and cantharidin (75 μM), or by epidermal growth factor (100 ng/ml), dexamethasone acetate (2 μM), retinoic acid (10 μM), or prostaglandin E 2 (1 μM). These agents thus appear to act at a target distinct from that of the phorbol esters.