Tumor promoter 12-O-tetradecanoylphorbol-13-acetate receptors in normal human transitional epithelial cells

Abstract

As a prelude to study the promotion with TPA of in vitro transformation of human urothelial cells (HUC) in culture, we characterized tumor promoter TPA receptors in primary cultures of HUC. [ ³ H]TPA bound specifically to intact living HUC; maximum specific binding was attained in ∼30 min at 37°C. [ ³ H]TPA bound to HUC in a saturable and competitive manner. Scatchard analysis of specific binding to intact cells displayed a single slope corresponding to an equilibrium dissociation constant (Kd) of 0.56 nM; at saturation TPA-binding capacity was 2.37 pmol/10 ⁶ HUC (1.43 × 10 ⁶ sites per cell). [ ³ H]TPA bound specifically and with high affinity to the particulate fractions of HUC; binding was both saturable and reversible. Saturation of the specific binding of [3H]TPA occurred at ∼1 nM at 4°C. Scatchard analysis of specific binding to the particulate fraction displayed a single slope corresponding to a Kd of 1.08 nM; at saturation TPA-binding capacity was 2.05 pmol/mg protein (750 000 molecules per HUC). [ ³ H]TPA binding was inhibited by the biologically active phorbol ester, phorbol didecanoate, whereas inactive phorbol did not compete for TPA binding. Binding was not affected by sodium saccharin, epidermal growth factor, retinoic acid or dexamethasone. [ ³ H]TPA bound specifically to the HUC cytosolic fraction but only in the presence of calcium and phosphatidylserine. Calcium-activated and phospholipid-sensitive protein kinase activity was detected in HUC fractions. These results indicate the presence of high-affinity specific receptors for TPA in HUC.