Differing patterns of cytotoxicity of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in various human cell strains.

Inhibition of colony formation by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) over a wide range of concentrations (10(-4)-10(2) ng/ml) was examined in two normal human diploid fibroblast strains and eight cell lines derived from various human tumors. Three dose-response patterns were observed: (i) no killing at any dose, which is characteristic of rodent cells; (ii) increasing cytotoxicity with TPA doses of 0.1 ng/ml or greater; and (iii) a biphasic response with maximal cytotoxicity at 1.0 ng/ml, and minimal effects at much lower or higher concentrations. The latter response group included both normal and tumor cell strains. When normal cells were incubated concurrently with superoxide dismutase or CuDIPS, survival was enhanced in a dose-dependent manner. Specific binding of [3H]PDBu to cells from each of the three response categories was studied to determine whether the cells might contain two classes of specific phorbol ester receptors. Scatchard plots yielded straight lines, consistent with one class of binding sites. The possible significance of this cytotoxic effect of TPA in human cells at dose levels usually considered typical for specific phorbol ester responses is discussed.

Kinetics and subcellular localization of specific [3H]phorbol 12, 13-dibutyrate binding by mouse brain.

The specific binding of [3H]phorbol 12,13-dibutyrate ([3H]-PDBU) to particulate preparations from mouse brain has been further characterized. Kinetic analysis, using a filtration assay to measure binding, yielded a second-order rate constant at 23 degrees of 3.75 X 10(7) M-1 min-1 and a first-order dissociation rate constant of 0.21 min-1. The Kd of 5.6 nM calculated from the kinetic data agreed well with the value determined previously in equilibrium binding studies. The Kd for [3H]PDBU binding varied only slightly with temperature. From its temperature dependence, [3H]PDBU binding appeared to be associated with a small increase in enthalpy (delta H degrees = +0.4 kcal/mol) and a large increase in entropy (delta S degrees = +38 e.u.). Such values are characteristic for hydrophobic interactions. The dissociation rate constant for binding, in contrast to the Kd, varied dramatically with temperature. The half-time for release ranged from 1.75 min at 30 degrees to 62 min at 4 degrees. The Kd for binding was Ca2+ sensitive; chelation of Ca2+ by ethyleneglycolbis(beta-aminoethyl ether)N,N'-tetraacetic acid increased the Kd 2.4-fold. Upon subcellular fractionation, the specific [3H]PDBU binding activity was exclusively particulate; no binding to cytosol was detectable. Binding clearly did not correlate with nuclear or mitochondrial markers. On the other hand, a broader distribution of binding activity was seen on sucrose density gradients than for either Na+-K+-adenosine triphosphatase activity or binding of quinuclidinyl benzilate (a muscarinic cholinergic antagonist). The localization of specific [3H]PDBU binding to the plasma membrane therefore remains uncertain.