Specific binding of phorbol esters to Friend erythroleukemia cells — general properties, down regulation and relationship to cell differentiation

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Abstract
Specific and saturable binding sites for [20-3H]phorbol 12,13-dibutyrate ([3H]PDBu) were demonstrated in intact Friend erythroleukemia cells (FELC), in which inducible erythroid differentiation is reversibly inhibited by phorbol esters. The binding of [3H]PDBu to intact cells was maximal within only 15 min of incubation at 37°C, after which there was a gradual decrease; binding at 4°C however, was a slow process, requiring > 180 min for maximal binding. A Scatchard analysis showed that the dissociation constant for binding of [3H]PDBu is 8.3 nM; at saturation, ∼1.75 × 105 molecules of [3H]PDBu are bound per cell. The binding of [3H]PDBu is blocked by 12-O-tetradecanoyl phorbol-13-acetate, phorbol 12,13-didecanoate, mezerein, 4-O-methyl-12-O-tetradecanoyl phorbol-13-acetate and resiniferatoxin, but not by phorbol or 4α-phorbol 12,13-didecanoate. There was, in general, a good correlation between the potency of these agents in inhibiting [3H]PDBu binding and their activity in promoting tumors on mouse skin. Inducers of differentiation, such as hexamethylene bisacetamide, dimethyl sulfoxide and butyric acid, as well as inhibitors of cell differentiation, dexamethasone and local anesthetics, did not significantly block the binding of [3H]PDBu to intact FELC. When FELC were induced to differentiate with 4 mM hexamethylene bisacetamide (∼80% of cells were benzidine-positive), a slight decrease (10–20%) in the number of binding sites at saturation was seen, but the dissociation constant was not changed. When the cells were precultured with non-radioactive phorbol esters, a significant decrease in [3H]PDBu binding was observed, suggesting a homologous down regulation of phorbol ester receptors. Scatchard analysis indicated that the decrease in [3H]PDBu binding was due to a decrease in the number of binding sites and not to a change in affinity. Such specific phorbol ester binding sites might mediate a number of biochemical and biological effects of phorbol esters on FELC.