Differential effect of tumor promoters on phorbol-ester-receptor binding and membrane fluorescence anisotropy in C3H 10T 1/2 cells.

Abstract

The presence of high‐affinity membrane‐associated receptor sites for phorbol ester tumor promoters has been demonstrated with [3H]phorbol dibutyrate ([3H]phorbol Bt2) in the mouse embryo fibroblast cell line C3H 10 T1/2. Binding of [3H]phorbol Bt2 to intact cells occurs rapidly at 37°C and is also rapidly reversed upon addition of excess unlabeled phorbol Bt2. The dissociation can be resolved into two phases at 23°C. Scatchard analysis reveals a biphasic curve which is consistent with two classes of binding sites: a high‐affinity class with a dissociation constant of 16 nM and a total concentration of binding sites of 1.9 pmol/106 cells, and a low‐affinity class with a dissociation constant of 1000 nM and a total concentration of binding sites of 29.4 pmol/106 cells. Unlabeled phorbol Bt2, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (Myrphobol Ac), and the indole alkaloid tumor promoter teleocidin are potent inhibitors of [3H]phorbol Bt2 binding, producing 50% inhibition at 98 nM, 38 nM and 23nM, respectively. On the other hand, two compounds that lack tumor‐promoting activity, 4α‐phorbol‐12,13‐didecanoate (4α‐phorbol Dec2) and 4‐O‐methyl‐Myrphorbol Ac, only slightly inhibited phorbol Bt2 binding. The effects of these compounds on the physical state of the membrane of intact C3H 10T 1/2 cells was also assessed using two different fluorescence lipophilic probes. Phorbol Bt2. Myrphorbol Ac and teleocidin, but not 4α‐phorbol Dec2, induce significant decreases in the fluorescence anisotropy parameter obtained with both of the probes. However, the dose response curves for these effects are quite different from those obtained when the same compounds were tested for inhibition of [3H]phorbol Bt2 binding. This discrepancy suggests that although C3H 10T 1/2 cells contain an abundance of highaffinity receptor sites for phorbol ester and related tumor promoters, some of the membrane effects of these compounds may be mediated by direct binding of the tumor promoter to lipid domains in cell membrane.