Binding Sites of Droloxifene in the Cytosol of 7,12‐Dimethylbenz[α]anthracene‐induced Rat Mammary Tumor Cells

The binding sites, other than the estrogen receptor (ER), of the antiestrogens droloxifene (DROL, (E)‐α‐[p[2‐(dimethylamino)ethoxy]‐phenyl]‐α'‐ethyl‐3‐stilbenol) and tamoxifen (TAM), and estradiol‐17β (E(2)) in the cytosol of 7,12‐dimethylbenz[α]anthracene‐induced rat mammary ER‐positive tumor cells were studied using a high‐performance liquid chromatography (HPLC) gel filtration assay. The cytosol was incubated with (3)H‐labeled drug with or without unlabeled drug, and separated by HPLC gel filtration. (3)H‐E(2) produced two major peaks of radioactivity at fractions No. 40 and No. 70. The peak at fraction No. 70 was identified as the ER in an ER‐enzyme‐immuno assay. This peak was dose‐dependently inhibited by unlabeled DROL or TAM, DROL being a more potent inhibitor than TAM. The peak at fraction No. 40 was also inhibited by co‐incubation with unlabeled DROL or TAM. (3)H‐DROL or (3)H‐TAM provided only one peak at fraction No. 43. This peak was thought to be an antiestrogen binding site (AEBS), because it was inhibited by unlabeled antiestrogen hut not by E(2). The results suggest that the antiestrogens DROL and TAM have a higher affinity for the AEBS than for the ER in the absence of E(2), while in the presence of E(2) both have an affinity for the ER and inhibit E(2) binding to the ER.