BINDING OF [H-3]12-O-TETRADECANOYLPHORBOL-13-ACETATE TO INTACT HUMAN PERIPHERAL-BLOOD LYMPHOCYTES

Abstract

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) [a carcinogen]. 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 h at temperatures of 37 and 4.degree. C as compared to untreated controls. [3H]TPA was not significantly metabolized by these cells after 1 h at 37.degree. C. 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 .times. 108 M-1) and the activation constant for TPA stimulation of [3H]-dThd incorporation (0.95 .times. 10-8 M). Binding of [3H]TPA was examined in 2 experimental conditions in which TPA-induced mitogenesis was inhibited: preincubation of HPBL at 37.degree. C for 24 h causes a decrease of [3H]dThd uptake of 50% and an apparent loss of binding sites for [3H]TPA; and glucocorticoid inhibition of [3H]dThd uptake in HPBL [human peripheral blood lymphocytes] by 50%, however, did not reduce [3H]TPA binding. Apparently cellular receptors 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.