Radioimmunoassay for phorbol esters using rabbit antisera against phorbol succinate.

The phorbol nucleus was succinylated and then conjugated to bovine albumin using dicyclohexylcarbodiimide. Rabbits given injections of the conjugate developed antibodies which rose in titer progressively with repeated immunization. By the ninth bleeding, the binding of one antiserum, diluted 1:15,000, was saturated with about 10 nM [3H]phorbol-12,13-dibutyrate [( 3H]-PDBU) and had an average association constant, Ka, of 2.6 X 10(8) M-1. The serological specificity of the antisera was characterized by examining the inhibition of the [3H]PDBU-anti-phorbol succinate immune system by 18 phorbol-related compounds. The specificities of antibodies from two rabbits tested in detail were qualitatively similar. The rank order of inhibitory activity for certain phorbol-related compounds was PDBU [concentration of inhibitor required to give 50% inhibition of PDBU binding (IC50) = 7.6 nM] = phorbol-13-acetate [IC50 = 8.2 nM] greater than phorbol-12,13-dibenzoate greater than 4-beta-phorbol [IC50 = 124 nM] greater than or equal to phorbol-12,13-diacetate greater than or equal to phorbol-12-myristate-13-acetate [IC50 = 184 nM] greater than phorbol-13,20-diacetate greater than phorbol-12-acetate [IC50 = 2300 nM]. The following compounds showed no detectable serological activity: mezerein, 4-0-methylphorbol-12-myristate-13-acetate, ingenol, 4-alpha-phorbol, teleocidin B, and dihydroteleocidin B. These and other results indicated that the 4-beta-phorbol nucleus was required for serological activity, that esterification of the C-13 position with benzoate, acetate, or butyrate enhanced the immunoreactivity of 4-beta-phorbol, and that among the phorbol-related compounds examined there was no direct relationship between serological activity and biological potency as tumor promoters. Using the [3H]PDBU-anti-phorbol succinate immune system, we measured the concentrations of immunoreactive phorbol-related material in crude mixtures such as croton oil and performed pharmacokinetic studies in rats given PDBU s.c.

Specific receptors for phorbol esters in lymphoid cell populations: role in enhanced production of T-cell growth factor.

Phorbol ester tumor promoters act synergistically with concanavalin A to cause production of T-cell growth factor by normal human peripheral blood lymphocytes. A specific, saturable, binding component which may mediate the phorbol ester effect has been identified by using [20-3H]phorbol 12,13-dibutyrate in a whole-cell binding assay. Specific binding is maximal with 5 min at 37 or 23 degrees C but the level of bound ligand rapidly decreases to about 50% within 1 hr. At 4 degrees C, 2 hr are required to reach maximal binding, and the binding is stable for at least 20 hr. Binding is reversible at 37 and 4 degrees C with time courses similar to those for initial binding at the respective temperatures. Saturation of the specific binding occurs at a concentration (approximately 30 nM) consistent with that producing maximal T-cell growth factor activity. Scatchard analysis of the binding after 30 min at 37 degrees C demonstrates a lower Kd (9 nM) than that determined after 2 hr at 4 degrees C (22 nM). The median number of sites per cell for six donors was 2 X 10(5) (range, 1.3-4 X 10(5). Other tumor-promoting phorbol esters compete for [20-3H]phorbol 12,13-dibutyrate binding in approximate proportion to their activity in stimulating T-cell growth factor production. Phorbol, 4-alpha-phorbol didecanoate, dexamethasone, retinoic acid, butyric acid, and dimethyl sulfoxide do not compete for specific binding.

Binding of [3H]12-O-tetradecanoylphorbol-13-acetate to intact human peripheral blood lymphocytes.

Our studies indicate that tritiated 12-O-tetradecanoylphorbol-13-acetate ([3H]TPA) produced by the reduction of the C-20 aldehyde with sodium [3H]borohydride is recognized by the same cellular site as is unlabeled 12-O-tetradecanoylphorbol-13-acetate (TPA). None of the concentrations of TPA used in these studies had an effect on the cell number and viability of human peripheral blood lymphocytes (HPBL) when incubated up to 1 hr at temperatures of 37 and 4 degrees as compared to untreated controls. [3H]TPA was not significantly metabolized by these cells after 1 hr at 37 degrees. Examination of the binding of [3H]TPA with simultaneous examination of uptake of tritiated thymidine ([3H]dThd) in parallel cultures demonstrated a close correlation between the apparent binding constant (0.94 X 10(8) M-1) and the activation constant for TPA stimulation of [3H]-dThd incorporation (0.95 X 10(-8) M). Binding of [3H]TPA was examined in two experimental conditions in which TPA-induced mitogenesis was inhibited: (a) preincubation of HPBL at 37 degrees for 24 hr causes a decrease of [3H]dThd uptake of 50% and an apparent loss of binding sites for [3H]TPA; and (b) glucocorticoid inhibition of [3H]dThd uptake in HPBL by 50%, however, did not reduce [3H]TPA binding. Our data suggest that cellular receptors either at the membrane or in the cytoplasm exist for TPA in HPBL. Alterations in binding of TPA to these receptors may account for the decrease in mitogenic response in preincubation experiments.