Characterization of a human placental factor which inhibits specific binding of phorbol esters to cultured cells

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Abstract
Phorbol ester receptors have been demonstrated in a variety of cells and tissues using [3H]phorbol-12,13-dibutyrate (PDBu) as a ligand. In a search for possible endogenous ligand(s) for the receptor, the human placenta was used as a source. A factor that can inhibit the binding of [3H]PDBu on different types of cells was purified (133-fold) from an extract of human placenta. This factor, PEBIF (phorbol ester binding inhibitory factor), is sensitive to pepsin and resistant to trypsin treatment. It is heat- and acid (pH3)-stable and can be precipitated by 80% ethanol with no loss of activity. PEBIF inhibits binding whether it is added before or after incubation of [3H]PDBu with human amniotic membrane cells (FL). Inhibition occurs at both 37.degree. C and 4.degree. C and is rapid and reversible; it does not require intact cells, since it also occurs with membrane fractions. PEBIF does not act like a binding protein for PDBu, and the kinetics of the inhibition on FL cells is non-competitive. Inhibition was also observed in rat liver cells (IAR 6) and Friend erythroleukemia cells (FELC) [mouse]. Differentiation of FELC induced by hexamethylene bisacetamide can be inhibited by 12-O-tetradecanoyl-phorbol-13-acetate(TPA) only if TPA-sensitive cells (TS 19-101) are used; no inhibition is observed with TPA-resistant cells (TR 19-9). The same is true of PEBIF. These 2 clones have about the same number of receptors, with no change of affinity; and the extent of inhibition of PDBu binding by PEBIF was similar in the 2 clones. Like TPA, PEBIF can increase 2-deoxyglucose uptake in mouse fibroblasts (BALB/3T3 cells). This physiological factor may play a role in the regulation of cell differentiation and/or in the modulation of carcinogenesis.