Recent observations indicate that the
progesterone metabolite,
5alpha-pregnane-3,20-dione (5alphaP), which is produced at higher levels in tumorous breast tissue, promotes cell proliferation and detachment, whereas 3alpha-hydroxy-4-pregnen-20-one (
3alphaHP), which is produced at higher levels in nontumorous breast tissue, suppresses proliferation and detachment of MCF-7
breast cancer cells. The objective of the current study was to determine the presence and characteristics of binding sites for these endogenous putative
cancer-regulating
steroid hormones. Radiolabeled 5alphaP and
3alphaHP were used in radioligand binding assays on MCF-7 cell (membrane, cytosolic, and nuclear) fractions. Binding of [(3)H]5alphaP and [(3)H]
3alphaHP was observed only in the plasma membrane fraction, whereas
estradiol binding sites were confirmed in the cytosolic and nuclear fractions. The respective membrane binding sites exhibited specificity for the 5alphaP and
3alphaHP ligands with no appreciable displacement at 200- to 500-fold excess by other
steroids. The association rate constants were calculated as 0. 107/min and 0.0089/min and the dissociation rate constants were 0. 049 9 and 0.011 for 5alphaP and
3alphaHP, respectively. Saturation analyses indicated single classes of molecules with dissociation constants of 4.5 and 4.87 nM and receptor densities of 486 and 629 fmol/mg
protein, respectively, for 5alphaP and
3alphaHP. Exposure of MCF-7 cells to
estradiol for 1, 24, 48, and 72 h resulted in 2.3, 4. 2-, 2.99-, and 1.7-fold increases, respectively, in 5alphaP receptor density.
3alphaHP resulted in partial suppression of the
estradiol-mediated increase in 5alphaP receptor density. This is the first report of receptors for the
progesterone metabolites, 5alphaP and
3alphaHP, of their occurrence in
breast cancer cell membranes, and of the induction of 5alphaP receptors by
estradiol. The results provide further support for the potential importance of
progesterone metabolites in
breast cancer.