A Tag for Estrogen Receptor Binding Sites in Human Tissues

Home , Moxestrol

[CANCERRESEARCH38,3044-3050,September1978) 0008-5472/78/0038-0000$02.00 11fl-Methoxy-1 7-ethynyl-1 ,3,5(1O)-estratriene3 ,17fl-diol (Moxestrol), a Tag for Estrogen Receptor Binding Sites in Human Tissues

Jean-Pierre Raynaud,1 Pierre Marie Martin, Marie-Madeleine Bouton, and Tiiu Ojasoo Centrede RecherchesRoussel-Uciaf,93230Romainville,France(J-P. A., M-M. B., T. 0.1, and Institut J. Paoli et I. Calmettes,13273Marseille, France (p.M. M.J

ABSTRACT those that rely on the addition of excess radioinert steroid that binds selectively to the plasma protein (in order to A particularlysimple, sensitive, and inexpensivetech abolish the interference of the contaminant) or that rely on nique has been developedfor the routineassay of estro the displacement of labeled estradiol by estrogens that, gen receptorbindingsitesIn humantissues(endometrium unlike estradiol, do not bind hoSBP; and (b) those that allow and breast cancer specimens)with the use of a radiola the separation of the plasma protein from the receptor. beled potent estrogen, moxestrol[1lfl-methoxy-17-ethy Thus, attempts have been made ho abolish SBP in nyl-1,3,5(1O)-estratriene-3,17j3-dlolj.In these tissuesmox terference by adding either excess DHT or testosterone estrol binds specifically to the estrogen receptor with (10), which both bind to SBP. However, since estradiol which it forms a stable complexthat, at 25Â°,dissociates competes with DHT for SBP binding and DHT can compete twice as slowly as does the estradiol/receptor complex. with eslradiol for estrogen receptor binding, depending Unlikeestradiolitdoesnotbindwithhighaffinityto human upon the relative concentrations of specific plasma and plasmasex steroid-bindingprotein.In a correlationanal tissue binding sites, upon the relative affinities of DHT and ysis on 215 breast cancer specimens,ft was foundthat, eslradiol for these sites, and upon the chosen experimental for high bindingsite concentrations,determinationscar conditions (i.e., relative ligand concentrations, incubation ned out with moxestrolwere extremely well correlated temperature, and time), estrogen receptor sites are either with those performedwith estradiol (In the presence of under-oroverestimated. dihydrotestosteronein order to minimize interference Several teams have used excess estrogen antagonists fromestradiolbindingto sex steroid-bindingprotein).For such as CI 628 (Parke, Davis and Co., Detroit, Mich.) (3, 11) low binding site concentrations, determinations per or estrogens such as diehhylshilbestrol (16) ho measure formedwithestradioloverestimatedbindingsites in spite nonspecific binding, with the idea (7) that, if these com of the presenceof dihydrotestosterone.Thus,the use of pounds do not bind ho specific plasma estradiol binders, labeledmoxestrolinsteadof estradiolimprovesthe accu only tissue binding sites are assayed. This method has 2 racyof the measurementof specifictissuebinding.Satis drawbacks: (a) if the affinity of the antagonist for the factory conditions for a dextran-coated charcoal adsorp estrogen receptor is weak, as is often the case, very high tion exchangeassay have been developedin this paper; competitor concentrations are required; (b) competition by a maximumnumberof bindingsites are measured after a radioinert ligand for total specific labeled estradiol bind incubationwith2 to 10 n@radiollgandfor3 to 5 hr at 25Â°. ing is a less sensitive method of measurement than is the direct estimation of specific binding sites with a labeled INTRODUCTION ligand that does not bind to SBP; in the latter case, the ratio of specific to total binding is much higher. The assay of estrogen and progeshin receptors in the Currently available procedures for discriminating be cytoplasm of human mammary tumors is becoming a rou Iween specific receptor proteins and the SBP of serum are tine procedure for selection of patients for endocrine ther electrophoretic techniques [agar and polyacrylamide gel apy (12, 17). Several techniques have been developed for electrophoresis and isoelectric focusing (8, 27, 30, 32)J, gel the assay of the estrogen receptor (15), but these do not exclusion chromatography (26), and selective precipitation necessarily take into account the fact that estradiol, the of plasma or tissue proteins by ammonium or protamine radioligand in most common use, binds with relatively high sulfate (5). Electrophoretic procedures are easier, are much affinity to plasma SBP2 (31). This plasma protein is a quicker, and can analyze more samples simultaneously contaminant of many cytoplasmic preparations and can than can the more frequently used density gradient centrif constitute a major interference, particularly when the re ugation (29), but they nevertheless suffer from the not ceplor concentrations are very low. negligible disadvantage of being too expensive and time Available techniques to account for this interfering bind consuming for routine laboratory use in a clinical context. ing are either unsatisfactory or too cumbersome for routine In the progesterone receptor assay, the problems of use. They may be broadly classified into 2 categories: (a) interference by specific plasma binding to human cortico steroid-binding globulin were solved by replacing labeled progesterone by a highly potent labeled progestin, R 5020 (promegeshone, 17,21 -dimelhyl-1 9-nor-4,9-pregnadiene I To whom requests for reprints should be addressed.

2 The abbreviations used are: SBP, sex steroid-binding protein; DHT, 3,20-dione) which stabilizes the progeshin receptor by form dihydrotestosterone; DCC, dextran-coated charcoal; RBA, relative binding ing a slowly dissociating complex (19) and which binds with affinity. ReceivedSeptember14,1977;acceptedMay3, 1978. very low affinity to corticosteroid-binding globulin (6, 18).

3044 CANCER RESEARCH VOL. 38

In this paper, we propose the use of the highly potent eslro pulverizer and homogenized in 10 mM Tris-HCI (pH 7.4 at gen, moxestrol [A 2858, 11fJ-mehhoxy-17-ehhynyl-1,3,5(10)- 0Â°)/1 mM EDTA/12 mt@imonothioglycerol/10% glycerol estratriene-3,17f3-diol] (21) for the measurement of the buffer. Endometrium was homogenized (1/1 , w/v) in a estrogen receptor. Labeled moxeshrol has been used widely manual glass/glass homogenizer, and mammary tumors to detect this receptor in various structures of the central were homogenized (1/5, w/v) in a Polytron PT1O homoge nervous system of the very young rat (22, 23), since it does nizer (Setting 4; five 2-sec bursts at 5-sec cooling intervals). notbindtoestradiol-bindingprotein(20)and isatpresent Cytosol was prepared as above. being used to detect and assay estrogen receptors in DCC AdsorptionAssay. Plasmaand cylosolwere incu human mammary tumors, since it does not bind to SBP bated as described in the table and chart legends. All (24). II has the great advantage over labeled diethylshilbes cytosols were incubated with [6,7-3H]moxestrol (48 Ci/ trol, which also does not bind ho SBP, of being stable. An mmol). Cytosols A and B were incubated with [6,7- exchange assay similar to the one described for rat uterus 3HJestradiol (51 Ci/mmol); Cylosols C, and C2 were incu by Katzenellenbogen et a!. (13) has been developed for the baled with [2,4,6,7-3H]estradiol (91 Ci/mmol). measurement of the estrogen receptor in mammary tumors, For plasma and Cytosols A and B, a 100-@tIsample was with tritiated moxestrol as the radioligand. This assay is a stirred for 10 mm at 0â€”4Â°ina microtiter plate (Greiner simple, fast, yet sensitive method for an accurate evaluation plates, System Cooke M220-24 A) with 100 @.tIofa DCC of tissue binding sites only. suspension (0.625% Dextran 80,000/1 .25% charcoal Norit A) and then centrifuged for 10 mm at 800 x g at 0-4Â°.For MATERIALSAND METHODS Cytosols C1and C2, a 200-SI sample was stirred for 30 mm at 0-4Â°wihh500 .d of DCC suspension (0.05% Dextran/0.5% Test Compounds. 17f3-[6,7-3H]Estradiol(51Ci/mmol), charcoal Norit A/0.01% gelatin) and then centrifuged for 10 [6,7-3H]moxestrol (48 Ci/mmol), and [1-3H]DHT (24 Ci/ mm at1000 x g at0-4Â°. mmol) were synthesized at the Roussel Uclaf Research In each case the concentration of bound radioligand was Centre. 17f3-[2,4,6,7-3H]Eshradiol (91 Ci/mmol) was pur determined by measuring the radioactivity in an aliquot of chased from New England Nuclear (Boston, Mass.). The supernahant. Radioligand bound in the presence of a large purity of all radioligands (98%) was checked by thin-layer excess of radioinert hormone is assumed to be nonspecifi chromatography in the following solvent systems: benzene/ cally bound. The difference in supernatant radioactivity in ethyl acetate (7/3, v/v), methylene chloride/methanol (9/1, the presence and absence of radioinert hormone is identi v/v), and carbon letrachioride/acetone (7/3, v/v) for fied as the concentration of specifically bound hormone. [3H]moxestrol; benzene/ethyl acetate (1/1 , v/v) and methyl Measurement of the Inhibition of [3H]Moxestroland ene chloride/methanol (9/1 , v/v) for [3H]estradiol. [3H]EstradiolBindingto Human Endometriumand Mam The following radioinert steroids were used: estradiol, mary Tumors. Aliquots of Cytosols A, B, and C2 were estriol, ehhynyl estradiol, diethylstilbeshrol, moxeshrol, RU incubated with 5 nM [3H]moxestrol or [3Hjestradiol and 16117 [11a-methoxy-1 7-ethynyl-1 ,3,5(10)-eshrahriene-3,17f3- various concentrations of unlabeled competitor (premixed) diol], tamoxifen (kindly supplied by ICI Ltd., Macclesfield, under different conditions of incubation temperature and England), progesterone, A 5020, testosterone, DHT, A 1881 time. After incubation, bound labeled steroid (B) was mea (17frhydroxy-1 7-mehhyl-estra-4,9,1 1-hrien-3-one), cortisol, sured by a DCC adsorption technique. The ratio B/B0, in dexamethasone (9a-fluoro-1 1f3,17,21-hrihydroxy-16a-methyl which B0represents the concentration of bound radioligand pregna-1 ,4-diene-3,20-dione), and aldosterone. in the absence of radioinert steroid, was plotted against the ExperimentalMateriaLHumanendometriumandprimary concentration of radioinert steroid added. The concentra breast cancer specimens from female patients were stored lion of radioinert steroid required for a 50% displacement in liquid nitrogen until use. They were analyzed by 3 teams of radioligand from its specific binding sites (after subtrac using the same basic technique (DCC adsorption analysis) lion of nonspecific binding) was determined. The ratio of with slight variations in experimental conditions. the 50% displacement concentration for radioligand and Cytosol Preparation.CytosolA was preparedfrom hu competitor gives the ABA of the competitor (14). man endometrium. The endometrium was powdered in the Measurementof DissociationRate Constants.Cytosois frozen state with a Thermovac tissue pulverizer and homog A and B were incubated with a saturating concentration of enized in 10 mM Tris-HCI (pH 7.4 at room hemperature)/0.25 [3H]estradiol or [3H]moxestrol and treated with DCC (1/10, M sucrose buffer (1/5, w/v) in a motor-driven glass/glass v/v; 6.25% Dextran 80,000/3.125% charcoal Norit A) to conical homogenizer. Cyhosol (supernatanl) was prepared remove excess free hormone. An excess of radioinert she by centrifuging homogenate at 105,000 x g for 45 mm at 0- roid was then added, and bound radioactivity was deter 4Â°ina 50 Ti rotor (Beckman L2 65B centrifuge). mined after different incubation times at 25Â°by a DCC Cytosol B was prepared from human mammary tumors. adsorption technique. Dissociation rates are given by the Frozen humors were thawed, crushed with an Ultraturrax slope of the line k_, t = â€”In(B/B0) in which B and B0 (four to five 5-sec bursts at 30-sec cooling intervals) and represent bound steroid at time t and time t = 0, respec homogenized in 10 mM Tris-HCI (pH 7.4 at room tempera lively. ture)/0.25 M sucrose buffer (1/7, w/v) in a motor-driven Density Gradient Centrifugation. Cytosols C, and C2were glass/glass homogenizer. Cytosol was prepared as above. incubated with 1 ho 30 np.i[3H]eshradiol in the presence of a Cytosols C1 and C2 were prepared from human endome 10-fold concentration of radioinert DHT or with an equiva trium and human mammary humors, respectively. They were lent amount of [3H]moxeshrol for 5 hr at 25Â°and then for 2 powdered in the frozen shale with a Thermovac tissue hr at 0Â°.The cytosol was treated with DCC to remove excess

SEPTEMBER 1978 3045

free steroid, layered on a sucrose density gradient, and compete for [3HJDHT binding to SBP in human plasma. centrifuged as described in the chart legends (Beckman Estradiol exerts a marked competitive effect, about 30 times Spinco L265B centrifuge). The radioactivity of fractions lower than that of DHT. Ethynyl estradiol competes very collected from the hop of the tubes was measured. slightly and at very high concentrations only. Moxeslrol, EquilibriumDialysis.Dialysismembranes(UnionCarbide diethylstilbestrol, and tamoxifen do not compete at a 500- Corp., Chicago, Ill.) containing 0.5 ml of plasma (dilution 1/ fold excess. 50) from poshmenopausal women were introduced into 15 Equilibrium dialysis was used to evaluate nonspecific ml of 10 mM Tris-HCI (pH 7.4)/0.25 M sucrose buffer con binding. [3H]Moxestrol did not bind specifically to plasma taming 2.5 nM [3H]eslradiol or [3Hjmoxestrol and various from menopausal women and its nonspecific binding was concentrations (10 ho 2500 nM) of unlabeled steroid. After about 3 times lower than that of [3H]estradiol (Chart 2). magnetic stirring for 48 hr at 0Â°,the radioactivity of dupli cate 0.2-mi samples from inside and outside the bag was Bindingof Moxestrolto the TissueEstrogenReceptor determined.Resultswere representedin a proportion graph (1). ABA's have been deduced from standard competition curves (Chart 3) and recorded in Table 1. The receptor RESULTS identified in the cytoplasm of human mammary tumors is estrogen specific as indicated by the lack of competition Choice of Ligand to DistinguishIntracellular Receptors for [3Hjeshradiol binding by other classes of steroids (pro fromInterferingExtracellularSBP gestins, androgens, glucocorticoids, and mineralocorti coids). In a previous study (24), SBP levels were measured in The estrogen specificity of the moxestrol-labeled receptor plasma from 14 women with primary breast cancer. Levels was determined by competition. Eshradiol, ethynyl estradiol ranged from 30 to 110 pmol/ml plasma with a mean value and diethyistilbeshrol competed fairly markedly; the short of 48 Â±22 pmol/mI plasma and were well within the limits aching estrogen, estriol, had only a weak competitive effect. reported in the literature for healthy women (28). Even if The discrepancies observed in the ABA's for ethynyl serum contamination of cytosol samples is only about 0.1%, eshradiol and moxestrol under the various experimental the concentration of SBP in a given cytosol sample will be conditions can be explained by the differences in the as high as the concentration of intracellular tissue estrogen relative dissociation rates of these compounds compared receptors recorded for an average human mammary tumor hoestradiol under different incubation conditions. Different (50 fmol/ml cytosol). Thus, in any study of tissue estrogen RBA's are recorded according to the time and temperature receptors, it is preferable to use, instead of eshradiol, a of incubation (2). Thus, ligands that dissociate slowly from radioligand that does not bind to SBP. the receptor have a higher RBA after long rather than short Chart 1 shows the extent ho which various estrogens incubation times and may also have a higher ABA at high rather than low temperatures.

loG Sedimentation Coefficient of the Moxestrol/Receptor Complexin HumanEndometriumCytoplasm

Chart 4 illustrates 2 gradients obtained with cytosol from endometrium samples that had been freed of as much 75 plasma contamination as possible by thorough washing (4

0 limes) in Krebs-Ringer-phosphate buffer. On incubation of 0 logT 1 :@ @ 50 2 I- I A r@i -0.5 25

. -1.0@ . S â€¢ ib-@ i'0-8 lO@ l06 Competitor concentration (M) Chart2. Proportiongraphsof [3H)estradioland[3H)moxestrolbindingto Chart 1. Competitionfor [3H)DHTbindingin humanplasma.Aliquots of human plasma measured by equilibrium dialysis. In a proportion graph (1), 125 gd of human plasma (diluted to 1/20 in 10 mM Tris-HCI (pH 7.4)/0.25 N the logarithm of the fraction-bound steroid (log BIT) is plotted against the sucrose buffer) were incubated for 2 hr at 0Â°with5 [email protected][3H)DHTin the logarithm of the total steroid concentration (log 7). A line parallel to the presence of increasing concentrations of unlabeled steroids. Bound [3H)DHT abscissa indicates the presence of nonspecific binding only. A, [3H)estradiol; was measured by DCC adsorption. DES, diethylstilbestrol. ., [â€˜H)moxestrol.

3046 CANCER RESEARCHVOL. 38

Table 1 a Competition for (3HJestradiol and (3HJmoxestrol binding in cytosol 100 S S from human endometrium and from human mammary tumors S Standardcompetition curves were obtained by incubating repli Dexamethasone 2 cortls@ caterHimoxestroland aliquots of cytosol with 5 flM rH]estradiol or Progesterone (prernixed)underincreasingconcentrations of unlabeledcompetitor g C-) R 5020 wasmeasureddifferent incubation conditions. Bound radioactivity ofligand by DCC adsorption. The ratio of the concentration 0 75. displaceradioligandho the concentration of competitor required to ofestradiol binding by 50% (ABA) was determined. The ABA wastakentobe100.Mammary tumors Endometrium [3HjEstradiol[3H]moxestrolCytosol [3H]Moxestrol hr,hr,25Â°B, 5 Cytosol C,, 5 Cytosol A, 2 0Â°Estradiol hr,25Â° 100Ethynylestradiol 100 100

75Moxestrol 110 100 1: 25Diethylstilbestrol 100 100 80Estriol 6AU16117 6 4Tamoxifen 0.15Progesterone <0.1A <0.1 0.1 <0.1A18815020 <0.1 0.1 <0.1DHT 0.15Testosterone <0.1 <0.1 <0.1Cortisol

<0.1Dexamethasone <0.1 <0.1 <0.1Aldosterone <0.1 <0.1

<0.1

iO@ Competitor concentration (M)

Chart 3. Competition for [â€˜H)moxestrolbinding to cytosol from human endometrium(a) and from human mammarytumors (b) CytosolA (5 mg proteinperml)wasincubatedfor 2 hr at 0Â°(a),andCytosolC,wasIncubated for 5 hr at 25Â°(b) with 5 flN [â€˜H)moxestrolin the presence of increasing concentrations of unlabeled steroids. Bound radioligand was measured by DCCadsorption. this cytosol with [3H]eshradiol in the presence of DHT, 2 complexes were formed that sedimented in the 45 and 85 Chart 4. Density gradient centrifugatlon of [@H)estradioIand [3H)moxestrol regions respectively. Only the 8S radioactivity peak was binding to cytosol from human endometrium freed from plasma contamina tion. Cytosol C, (7 mg protein per ml) was layered (150 @tI)ona 5 to 25% suppressible by excess radioinert estradiol (Chart 4a). The linear sucrose gradient which was centrifuged at 322,000 x g (50,000 rpm in same cytosol incubated with [3H]moxestrol in the absence an SW 60 rotor) for 16 hr at 0-4Â°.a, binding of 10 n@i[â€˜H)estradiol(91Cu of DHT gave a major 85 radioactivity peak nearly totally mmol) plus 100 nN DHT in the absence (A) or presence (@) of 1000 flM estradiol. b, binding of 10 n@i[@H)moxestrol(48 Ci/mmol) in the absence (â€¢) suppressible by excess radioinert moxeshrol (Chart 4b) and or presence (0) of 1000 nM moxestrol. [â€˜@C)OvaIbumin([â€œC]Ov)sedimented a very small unsuppressible 45 peak. at 3.6S (4).

SEPTEMBER 1978 3047

Table2 C Dissociation rates ofcytosolestrogen moxestrol and estradiol from human â€˜6) 25Â°Cytosol receptor at 25Â°prior A was incubated with 25 nM radioligand for 1 hr at 0) wasincubatedto addition of 2500 nM radioinert steroid. Cytosol B U, of5000 with 2.5 n M radioligand for 24 hr at 0Â°prior to addition .@ nM radioinert steriod.Endometriurn tumorCytosol Mammary Bk_, A Cytosol

U, t,12(10 t,,@ k_, (hr)Moxestrol 5sec â€˜) (hr) (10 â€˜sec â€˜) @0 C 3.2Estradiol 4 4.8 6 .0 .2 11 1.7 13 1.5 .@

a S â€¢NS 200

150

1100 Moxestrol binding sites (fmoles/mg protein) Chart 6. Correlation between estrogen binding sites assayed with 0 [3H)estradiol (in the presence of DHT) and with [â€˜H)moxestrolalonein cytosol @ 50 NS from human mammary tumors. 0, 100 tumors from pre- and postmenopau A sal patients of whom approximately 30% had undergone radiotherapy. Cytosol B (250 pJ)was incubated for 20 hr at 0Â°witheither 5 n@[3H)estradiol plus 100 nM DHT or 5 ni.i [3Hjmoxestrol in the presence or absence of 2500 i 2 3 4 5 Hours flM radioinert hormone. â€¢,1 15 tumors from postmenopausal patients only. CytosolC, (200 @.tl)wasincubatedfor 16hr at 0Â°witheither 2 nM[3H)-estra b S diol plus 20 nM DHT or 2 n@ [â€˜H)moxestrolin the presence or absence of I 2000 nN radioinert ligand. Binding sites were assayed by a DCC adsorption 5.NS technique. Cytosols from Tumors 58, 61, and 91 were submitted to density I gradient centrifugation (see Chart 7). assayed as a function of incubation time at 25Â°with a saturating radioligand concentration (10 nM). A maximum I number of binding sites were measured within 3 hr whether NS with [3H]eshradiol (plus 100 nM DHT) or with [3H]moxeslrol. This number remained constant until at least 5 hr incuba lion. Choice of RadioligandConcentration(Chart Sb). For concentration(nM) saturation of 150 fmol of estrogen-binding sites per mg Chart 5. Binding site concentration in cytosol from human mammary cytosol protein, a concentration of 2 nM [3H]moxeslrol was tumors measured as a function of incubation time (a) and radioligand required. Above this concentration the binding curves ob concentration (b). In a. Cytosol B (5 mg protein per ml) was incubated with 10 nN estradiol and 100 nN DHT for 2 hr at 0Â°andthen treated with DCC to tamed in the absence and presence of radioinert ligand remove free steroid. Aliquots of this cytosol were then incubated for various were parallel; in other words, specific binding was no time periods at 25Â°with 10 nN radioligand in the absence or presence of longer dependent on radioligand concentration. Nonspe 2500 [email protected] steroid. In b, Cytosol C@(2.5 mg protein per ml) was incubated with [3H]moxestrol for 5 hr at 25Â°in the absence or presence of cific binding was low. radioinert moxestrol. Bound radioactivity was measured by DCC adsorption. Satisfactory conditions for an exchange assay of estro ., [3H)moxestrol; 0, [3H)moxestrol plus radioinert moxaetrol; A, gen-binding sites in the cytoplasm of human mammary [3Hjaetradiol, t@,[3H)estradiol plus radloinert estradiol; 5, specific binding; NS,nonspecificbinding. tumors would thus appear to be incubation of cytosol with 2 to 10 nM [3H]moxestrol for 3 to 5 hr at 25Â°. Developmentof an ExchangeAssay of Estrogen-binding Siteswith Moxestrol Assay of Estrogen-bindingSitesin the Cytoplasmof Hu man MammaryTumors Choice of IncubationTemperature. Both estradioland moxestrol dissociate very slowly from the estrogen receptor Binding sites were assayed as described in the legend of at 0Â°(2),but when the temperature is increased to 25Â°,the Chart 6 either with [3Hjmoxestrol alone or with [3H]eslradiol dissociation rate of estradiol exceeds that of moxestrol. At in the presence of 20 or 100 nM DHT to minimize interfer 25Â°moxestrol dissociated 2 ho 3 times more slowly than did ence by plasma binding. Values for [3H]estradiol (plus DHT) estradiol in cytosols from both human endomelrium and binding were plotted against those for [3Hjmoxeshrol bind mammary humors (Table 2). ing and were exceptionally well correlated especially for Choiceof IncubationTime(ChartSe).Bindingsiteswere high binding site concentrations. For low binding site

3048 CANCERRESEARCHVOL. 38

20 30 Fractions Chart 7. Density gradient centrifugation of [â€˜H)estradioland[â€˜H]moxestrolbinding to cytosol from human mammary tumors. Cytosol C, from Tumors 61 (left), 58 (center), and 91 (right) (7, 4, and 4 mg protein per ml, respectively), which had not been previously treated with DCC, was incubated with either 30 n@i [3H)estradiol (91 Ci/mmol) plus 300 nN DHT or 30 nii (3H]moxestrol (48 Ci/mmol) in the absence or presence of 1000 ni@radioinert steroid. A, [3H)estradiol; L@,[3H)estradiol plus radioinert estradiol; â€¢,[3H]moxestrol; 0, [3Hjmoxestrol plus radioinert moxestrol. This was layered (200 s.d) on a 5 to 25% linear sucrose gradient which was centrifuged at 369,000 x g (60,000 rpm in an SW 60 rotor) for 18 hr at 0Â°.[â€˜C)Ovalbumin([â€˜C)Ov)sedimented at 3.6S (4). concentrations, the points diverged away from the correla in the assay of specific estrogen receptor sites is to use, lion curve toward the estradiol axis, suggesting that, in instead of estradiol, a radiolabeled synthetic estrogen that spite of the presence of 20 or even 100 nM DHT in the assay does not bind ho SBP. A highly suitable molecule for this medium, part of estradiol binding to SBP might have been purpose would seem ho be moxestrol, a highly potent measured.Incasesinwhich 100 nM, and notonly20 nM, estrogen both in animals (21) and in humans (9, 25) that, DHT had been added, the points were closer to the theoret unlike estradiol, does not bind specifically to human ical correlation curve, but it would seem that even a 100 nM plasma, whether this binding is measured indirectly by DHT concentration is not sufficient to eliminate the influ displacement of labeled DHT or directly by equilibrium ence of plasma contamination when very few estrogen dialysis. Furthermore, the nonspecific binding of moxestrol receptor binding sites are present. is about 3 times lower than that of estradiol. The cytosol of the numbered tumors was analyzed in Like estradiol (15), moxestrol gives rise, in sucrose den sucrose density gradients. In humors with high estradiol sihygradient patterns, to specific 8S and some displaceable and moxestrol-binding site concentrations (Tumors 58 and 45 binding but, unlike estradiol, none of this 45 displacea 61), either 85 radioactivity peaks alone (Chart 7a, Tumor 61) ble binding can be accounted for by SBP. Thus, assays of or 85 peaks with 45 peaks (Chart 7b, Tumor 58) were estrogen-binding sites in the cytoplasm of 215 human detected. In tumors with high estradiol-binding site concen mammary humors with estradiol in the presence of DHT to trahions but very low moxeslrol-binding sites, sucrose den minimize interference by SBP and concomitantly with mox sity gradients revealed the presence of a 45 displaceable estrol alone showed that in receptor-rich preparations estradiol radioactivity peak but no moxeshrol radioactivity (>100 fmol/mg protein), estradiol-plus-DHT values and peak (Chart 7c, Tumor 91). These results suggest that moxestrol values were extremely well correlated. On the borderline tumors that are considered receptor positive ho other hand, in receptor-poor preparations, estradiol-plus the natural hormone could in fact be merely contaminated DHT tended ho give falsely high binding measurements as a by plasma. result of contaminating SBP binding and in spite of the presence of DHT. Density gradient cenhrifugahion of several DISCUSSION humors with high estradiol-plus-DHT values and low mox estrol values (of which a few are illustrated in this paper) One of the simplest methods of eliminating interference revealed unsahurable binding only, in the 45 region. by contaminating plasma binding to proteins such as SBP In our correlation analysis, moxestrol was used under the

SEPTEMBER1978 3049

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. J-P. Raynaud et a!. same routine conditions as was estradiol, namely, DCC 10. HShnel, R., Ratajczak, T., and Twaddle, E. Problems in Quantitation and saturation analysis after incubation of cytosol with radioli Isolation of Estrogen Receptors. In: Abstracts, Fifth International Con grass of Endocrinology, Hamburg, July 1976, p. 54, 1976. gand for about 20 hr at 0Â°.These conditions could be 11. Jensen, E. V., Block, G. E., Smith, S., Kyser, K., and DeSombre, E. A. improved by performing the assay at 25Â°.Al25Â°themoxes Estrogen Receptorsand Breast Cancer Responseto Adrenalectomy. NatI. Cancer Inst. Monograph, 34: 55-70, 1971. trol/receptor complex in cytoplasm from human endome 12. Jensen, E. V., Smith, S., and DeSombre, E. A. Hormone Dependency in trium and mammary tumors dissociates twice as slowly as Breast Cancer. J. Steroid Biochem., 7: 911-917, 1976. does the eslradiol/receptor complex, leading to effective 13. Katzenellenbogen, J. A., Johnson, H. J., Jr., and Carlson, K. E. Studies on the Uterine Cytoplasmic Estrogen Binding Protein. Thermal Stability replacement of the endogenous hormone by synthetic ra and Ligand Dissociation Rate. An Assay of Empty and Filled Sites by dioligand in an exchange assay. It would seem that a Exchange. Biochemistry, 12: 4092â€”4099,1973. maximum number of binding sites are measured after 3 hr 14. Korenman, S. G. Relation between Estrogen Inhibitory Activity and Binding to Cytosol of Rabbit and Human Uterus. Endocrinology, 87: incubation at 25Â°with a saturating moxeshrol concentration 1119-1123,1970. (2 ho 10 nM). The choice of radioligand concentration 15. McGuire, W. L., Carbone, P. P., and Vollmer, E. P. (eds.). Estrogen Receptorsin HumanBreastCancer.NewYork: RavenPress,1975. depends upon whether the sample under study is a recep 16. McGuire, W. I., De Ia Garza, M., and Chamness, G. C. Evaluation of br-rich or receptor-poor specimen; ideally, a 2-concentra Estrogen Receptor Assays in Human Breast Cancer Tissue. Cancer Rae., lion assay would yield the most information. If the radioli 37:637-639,1977. 17. McGuire, W. L., Horwitz, K. B., Pearson, 0. H., and Segaloff, A. Current gand concentration is too low, binding sites are undereshi Status of Estrogen and ProgesteroneReceptors in Breast Cancer. mated since they are not saturated; if it is too high, the Cancer, 39:2934-2947,1977. interference of nonspecific binding is no longer negligible, 18. Philibert, D., and Raynaud, J. P. Binding of Progesterone and A 5020, a Highly Potent Progestin, to Human Endometrium and Myometrium. since the ratio of specific ho nonspecific binding is low. Contraception, 10: 457-466, 1974. As confirmed in a recent paper (16), DCC saturation 19. Philibert, D., and Raynaud, J. P. Cytoplasmic Progestin Receptors in analysis is a simple accurate method for receptor assay. Mouse Uterus. In: W. L. McGuire, J. P. Raynaud, and E. E. Baulieu (eds.),ProgesteroneReceptorsin Normaland NeoplasticTissues,pp. The use of moxeshrol in such an assay would further 227-243. New York: Raven Press, 1977. improveaccuracy. 20. Raynaud, J. P. Influence of Rat Estradiol Binding Plasma Protein (EBP) on Uterotrophic Activity. Steroids, 21: 249-258, 1973. 21. Raynaud, J. P., Bouton, M. M., Gallet-Bourquin, D., Philibert, D., ACKNOWLEDGMENTS Tournemine, C. , and Azadian-Boulanger, G. Comparative Study of EstrogenAction.Mol. Pharmacol.,9:520-533,1973. We would like to thank Dr. J. P. Gautray (Centre Hospitalier Intercom 22. Raynaud, J. P., and Moguilewsky, M. OntogenÃ©sedes Rdcepteurs des munalde CrÃ«teil)forsupplyinghumanendometriumand Dr. H. Magdelenat OestrogÃ¨neschezle Rat.In: A. Soulairac,J. P.Gautray,J. P. Rousseau, (Fondation Curie-Institut du Radium) for supplying mammary tumors. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. 11β-Methoxy-17-ethynyl-1,3,5(10)-estratriene-3,17β-diol (Moxestrol), a Tag for Estrogen Receptor Binding Sites in Human Tissues

Jean-Pierre Raynaud, Pierre Marie Martin, Marie-Madeleine Bouton, et al.

Cancer Res 1978;38:3044-3050.

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