Abstract
Oestrogen receptor protein (ER) was detected in 9 of 11 samples of malignant breast tissue and 8 of 9 samples of normal breast tissue. Levels of cytosolic ER (ERc) in malignant breast were 21-1102 fmol mg-1 soluble protein (Kd 1.8 X 10(-9)-3.1 X 10(-8) mol l-1) and those of nucleosolic ER (ERn), 13-526 fmol mg-1 soluble protein (Kd 2.1 X 10(-9)-1.4 X 10(-8) mol l-1). In normal breast tissue ERc levels were 33-640 fmol mg-1 soluble protein (Kd 1.3 X 10(-10)-3.2 X 10(-9) mol l-1), ERn was detected in only 2 samples, 8 and 87 fmol mg-1 soluble protein with Kd 3.2 X 10(-9) and 1.4 X 10(-9) l mol-1 respectively. 17 alpha-ethinyl-13 beta-ethyl-17 beta-hydroxy-4,15-gonadiene-3-one (gestodene), a new synthetic progestogen displaced 3H-oestradiol (3H-E2) from both ERc and ERn in malignant tissue but not in normal breast, or these receptors from endometrial tissue. In competition studies gestodene was approximately 3 times more effective in displacing 3H-E2 from ERc and ERn in malignant breast tissue than the natural ligand. Quantitation of ER by gestodene were ERc, 12-1134 fmol gestodene bound mg-1 soluble protein (Kd 1 X 10(-9)-8.1 X 10(-9) mol l-1); ERn, 17-531 fmol gestodene bound mg-1 soluble protein (Kd 1.6 X 10(-9)-1.1 X 10(-8) mol l-1). L-13-ethyl-17 alpha-ethinyl, 17 beta-hydroxy-gonen-3-one (levonorgestrel) showed no binding to ER in malignant breast, normal breast or endometrial tissue. In circulation both gestodene and levonorgestrel displaced E2 from sex hormone binding globulin more than any of the androgens tested. These results suggest that gestodene is a progestogen with oestrogenic and/or antioestrogenic properties and provide strong evidence for differences in ER from malignant and normal breast tissue.
Highlights
Quantitation of ER by gestodene were ERr, 12-1134 fmol gestodene bound mg- soluble protein (Kd 1 x 10-9-8.1 x 10-9 mol l-1); ER., 17-531 fmol gestodene bound mg ' soluble protein (Kd 1.6 x O1-9.1 x 10 8moll-')
The assay for ER, and ER. were carried out on all samples of malignant, normal and endometrial tissues exactly as the ER assay described above except that a constant amount of 3H-gestodene (6,500c.p.m.) and varying amounts (0-16pmol) of radioinert gestodene were employed as the binding ligands
Competition studies showed that gestodene displaced 3H-E2 from ER, and ER, in malignant fmol E2 bound mg 1 soluble protein fmol gestodene bound mgsoluble protein
Summary
3H-gestodene (specific activity 2.15 TBq mmol- 1), 3Hlevonorgestrel (specific activity 1.44 TBq mmol - 1) and the corresponding radioinert compounds were a gift from Schering Chemicals (UK) Ltd. 3Hoestra-1,3,5(10)-triene-3,17fl-diol (Oestradiol, E2) Aliquots (0.4ml) of these were incubated (cytosols for 2 h and nucleosols for 18 h) with a constant amount of 3H-E2 (6,000 c.p.m.) and varying amounts (0-16 pmol) of either gestodene or levonorgestrel, and using varying amounts (018.4pmol) of radioinert E2. Were carried out on all samples of malignant, normal and endometrial tissues exactly as the ER assay described above except that a constant amount of 3H-gestodene (6,500c.p.m.) and varying amounts (0-16pmol) of radioinert gestodene were employed as the binding ligands. Aliquots (0.4 ml) of the above dilution were incubated as described for the two-tier column SHBG assay (Iqbal & Johnson, 1977) with constant amounts (25,000 c.p.m.) of either 3H-DHT, 3H-testosterone, 3H-E2, 3H-5a-androstane-3a(or 3ft, 17#-diol and varying amounts (0-180 pmol) of their respective radioinert moieties. Displacement of 3H-progesterone and 3H-cortisol was studied by using varying concentrations (0-180 pmol) of their respective radioinert ligands and in a parallel series of experiments .displacement of 3H-progesterone and 3H-cortisol was studied by using varying amounts of radioinert gestodene or levonorgestrel (0165 pmol)
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