The binding of tamoxifen to human mammary carcinoma cytosol

Abstract

Interactions between tamoxifen and cytoplasmic binding sites from both ER+ and ER− human mammary carcinoma biopsies were investigated directly using [ 3H ] tamoxifen. Cytosol preparations were incubated for 16hr at 4°C with 1.25 nM [ 3H ] tamoxifen and increasing concentrations of unlabelled tamoxifen ( 1.5 nM-5 μM). Protein bound (B) and unbound (U) tamoxifen were separated by charcoal adsorption, and the affinity constants and binding site concentrations were calculated from measured values of B and U by a nonlinear, least squares regression analysis. A saturable, high affinity (K d = 6.0 ± 1.6 nM), tamoxifen binding site was present in ER+ but not ER− tumours. This site was present at 8.6 times the concentration of the high affinity oestradiol receptor site measured in the same tissue. Competition studies performed under similar assay conditions demonstrated that tamoxifen completely inhibited [ 3H ] oestradiol binding to its saturable binding site in ER+ tumours with a relative binding affinity 0.87 ±0.35% that of oestradiol. However, when [ 3H ] tamoxifen and cytosol were incubated with unlabelled oestradiol at concentrations as high as 10 μM there was no inhibition of [ 3H ] tamoxifen binding to its saturable binding site. It is concluded that under the present in vitro assay conditions tamoxifen is bound predominantly by a high affinity, saturable binding site in ER+ human mammary carcinoma cytosol that is distinct from the classical oestrogen receptor site.