Characterization and Assay of Progesterone Receptor in Human Mammary Carcinoma1

[CANCER RESEARCH 37, 464-471 , February 1977]

M. F. Pichon and E. Milgrom

Groupe de Recherches sur Ia Biochimie Endocrinienne et Ia Reproduction (INSERM U.135), FacultÃ©deMÃ©decine,Paris-Sud, 94270 BicÃ©tre,France

ever, only some patients are improved by such treatments. It SUMMARY would thus be of great practical importance to predict in advance which patient will respond and to nationalize the [3H]Pregn-4-ene-3,20-dione ([3Hjpmogestenane)-recepton choice of the surgical or pharmacological technique to be complexes from human mammary carcinoma were found to used. During the last 15 years, the detection and quantifica be stabilized in the presence of glycerol. The dissociation tion of estrogen receptors in mammary tumors have allowed rate constant was lowered and the equilibrium dissociation marked progress in this direction (11, 12, 16, 18, 21, 22, 34). constant was decreased (KD = 3 nM in the absence of However, the growth of mammary carcinoma may be can glycerol and 1.1 nM in the presence of 30% glycerol), traIled not only by estrogens but also by other hormones whereas no clean-cut effect on the association rate was including observed and no change occurred in the concentration andragens, glucacorticaids, pragestagens , and of binding sites. Gortisol was found to compete with prolactin (13). The study of specific receptors for these [3Hjpmagesterone only at concentrations higher than 1 @M. hormones should shed some bight on the problem of tumor This made it possible to distinguish [3H]pmogestemanebind hormonal dependence . The characterization and measu me ing to the receptor from binding to carticosteroid-binding ment of progesterone receptors have been hampered by the globulin. Synthetic progestins [6-chbaro-17-acetoxypregna lack of stability of the hormone-receptor complex as well as 4,6-diene-3,20-dione (chbonmadinane acetate), 17a-ethinyl, by the presence in the tissue extracts of CBG,2 which also 17- hydraxyestr-4-en-3-one (narethistenane), and 17,21- binds progesterone with high affinity. For these reasons d imethyl-1 9-nampmegna-4,9-diene-3,20-dione (R5020)] were and with 1 exception (31), this receptor has not been stud found to have a high affinity for the receptor, whereas ied until very recently, when a new synthetic compound, 5a-pmegnane-3,20-dione had an affinity about one-half that R5020, which has a low affinity for CBG and a high affinity of progesterone itself. 5f3-Pregnane-3,20-d ione, 17a-hy for the progesterone receptor, was described (26). How dnaxypnegn-4-ene-3,20-diane (estradial), 11fJ,21-dihydmaxy even, the use of the natural hormone, progesterone, fan the pregn-4-ene-3,20-diane (corticosterone), estra-1 3,5(10)- characterization and assay of its receptor may offer same tniene-3,1 7/3-dial, and 17/.3-hydraxyand rost-4-en-3-one advantages. It is mane readily available and a greaten num (testosterone) were weak inhibitors of [3H]proge@terane ben of laboratories would be able to practice the assay. In binding. Sedimentation on glycerol gradients showed addition, specificity problems may arise with the use of different patterns in different tumors; i.e., [3H]progestenone synthetic compounds. Far instance, it has recently been specific binding having the characteristics of receptor was shown that 11f3,21-dihydroxypregn-4-ene-3,20-dione (car found either in the 8 5 region, in the 4.5 5 region, or in ticosterone, the natural glucocorticoid) and dexamethasone both. Activated progesterone-receptor complex from hu (a synthetic glucocarticoid) are not bound identically in rat man mammary carcinoma cytosol was shown to bind to brain (7). Moreover, in human mammary carcinoma, evi human DNA. dence has been obtained that R5020 binds with relatively An assay of the receptor based on these binding proper high affinity to the glucacorticoid receptor (19, 32). This ties is described. This assay measures the total concentra has led us to establish a method for stabilizing proges tian of cytosal receptor since it makes possible the ex terone-mammary carcinoma receptor complexes and for change of endogenous hormone far excess added distinguishing them from pnogesterone-CBG complexes. It [3H]pragestenane. Of 55 biopsies examined by this method, was thus possible to set up an assay allowing the exchange 35 (64%) had a concentration of progesterone receptor of endagenous for added radioactive hormone. The total binding sites higher than 10 fmales/mg protein . Themewas a (hormone-bound and free) concentration of cytosol me positive correlation between the amounts of estrogen and ceptar could then be measured. This assay was used to progesterone receptors. examine 55 tumor biopsies.

INTRODUCTION MATERIALS AND METHODS Following the description by Beatson (4) of breast cancer regression after avaniectamy, various methods of endocrine Steroids and Buffer. 1,2,6,7-[3H]Progestemone (94 Gil treatment of this disease have been proposed (29). How mmale) was obtained from The Radiochemical Centre,

, This work has been supported by the InstitutNational de Ia Santd et de Ia 2 The abbreviations used are: CBG, corticosteroid-binding globulin; Recherche MÃ©dicaleand the DÃ©lÃ©gationGÃ©nÃ©caleaIa Recherche Scienti R5020, 17,21-dimethyl-1 9-norpregna-4,9-diene-3,2odione; progesterone, fique et Technique. pregn-4-ene-3,20-dione; cortisol, 11$,1 la,21 -trihydroxypregn-4-ene-3,20- Received August 3, 1976; accepted November 9, 1976. dione.

464 CANCER RESEARCH VOL. 37

Amersham, England. Its purity was periodically checked by Table 1 thin-layer chromatography an silica gel in the benzene:ethyl Assayof total cytosol progesteronereceptor in human mammary acetate (3:2, VA') system. 6,7-[3H]R5020 (51.4 Gi/mmole) carcinoma was a gift from Dr. J. P. Raynaud (Raussel Uclaf, Romain 1. Biopsy (@100mg) is kept in liquid nitrogen. yule, France). Non madioactiVe, chromatographically pure 2. Quick thawing (30 mm at 0Â°)andhomogenization in 6 volumes of Tnis buffer at 0Â°. steroids were obtained from Roussel Uclaf. The only buffer 3. Centnifugation (105,000 x g for 90 mm at 0Â°)yields cytosol. used was 10 mM Tnis: 1.5 mM EDTA: 0.5 mM dithiathreitol 4. Cytosol, 0.1 ml, is incubated in triplicate with: (a) 20 nM HGI, pH 7.4. Redistilled glycerol (Merck, Darmstadt, Ger [3H]progesterone and 1 j.tM unlabeled cortisol; (b) 20 nM many) was added in same experiments. [3H]progesterone, 1 @Munlabeled cortisob, and 2 @Munlabeled progesterone. Processing of the tumor. Human mammary carcinoma 5. After 2 hr at 0Â°,0.1 ml of Tnis buffer containing 60% glycerol is tissue was obtained by surgical biopsy. It was immediately added. The incubation is continued for 2 hr at 0Â°. dissected from fat, and necrotic tissues were excised. The 6. Dextran-coated charcoal suspension, 0.2 ml, in Tris buffer con specimens were cut into several pieces and were frozen in taming 30% glycerol is added. Mixing with a Vortex and agita liquid nitrogen where they were kept until used for the tion for 30 mm at 0Â°are performed. The suspension is centri fuged for 10 mm at 2000 x g. experiment (up to 2 months). The tissue was quickly thawed 7. Supernatant, 0.2 ml, is counted. Specific binding to progester at 0Â°,cut into small pieces, and homogenized with 6 vol one receptor = mean radioactivity bound in Incubations a â€” umes of buffer in an all-glass Patter-Elvehjem apparatus. mean radioactivity bound in Incubations b. The number of spe Cytosol was then obtained by centnifugation at 105,000 x g cific binding sites was calculated only when the differences between the total binding and nonspecific binding were found for 90 mm at 0Â°.The pellet was saved for DNA assay. to be statistically significant (P < 0.05, using Student's t test). Incubation of Cytosol with the Steroid. The steroid solu 8. Proteins are assayed in the cytosol and DNA in the 105,000 x g tian was introduced into a conical glass tube and evapo pellet. rated under air at roam temperature. The tube was cooled at 0Â°.Thecytasol was then added after dilution to the desired lize the receptor-hormone complex. Since glycerol has protein concentration if necessary. Incubation was per been shown to exert such a stabilizing effect on the rat formed at 0Â°with shaking in a Dubnoff incubator. uterus progesterone receptor (9), it was logical to study its Measurement of Protein-bound [3H]Progesterone. The effect on the human mammary binder. incubate was adjusted to 30% (v/v) glycerol. One volume of Cytosab from a tumor shown by previous experiments to a dextran (Dextran T 70; Pharmacia, Uppsala, Sweden, contain progesterone receptors (see Table 1) was incubated 0.05%):charcoal (Nonit A; Pralabo, Paris, France, 0.5%) until equilibrium was attained with various concentrations suspension in Tnis buffer containing 30% glycerol was of [3H]progesterone in either the presence or the absence of added. After being mixed with a Vortex apparatus, the 30% glycerol. Bound hormone was measured at equilib suspension was agitated far 30 mm at 0Â°.Itwas then centni nium, and Scatchard plots (28) were constructed (Chart 1). fuged at 2000 x g, and the radioactivity in the supernatant The equilibrium dissociation constant was KD= 3 nM in the was counted. The 30-mm period of contact with charcoal absence of glycerol and KD = 1.1 flM in its presence. Thus, a was shown by preliminary experiments to give, in 30% glyc 3.5-fold difference in affinity was observed , whereas the erol, minimal dissociation of the receptor-bound ham concentration of binding sites was identical in bath cases. mane but optimal dissociation of the nanspecificalby bound The change in the equilibrium dissociation constant could hormone. be due to modifications of either the association or the Nonspecific, nansaturable binding was measured in par dissociation rate constant. allel incubations with the addition of 2 j.tM unlabeled pro For technical reasons (time necessary to add glycerol, gesterane. It was subtracted from the total binding in order duration of contact with charcoal, high percentage of non to obtain the specific, saturable binding. specific binding after short incubations), it was difficult to Proteins were assayed by the technique of Lowry et a!. measure accurately the association rate constant. However, (20) with bovine serum albumin as standard protein. DNA no gross differences were observed between the rates of was assayed by the method of Burton (6). Radioactivity was association in the presence amin the absence of glycerol counted in a mixture of 3 ml ethanol and 10 ml tobuene (Chart 2). In bath cases equilibrium was attained in 60 mm. containing Omnifluor, 4 g/Iiter (NEN Chemicals GmbH, No alteration of the complexes was observed if the incuba Dneieichenhain, Germany). tion was continued for 4 hr whether glycerol was present or Assay of the estnadiob receptor was performed as de not. scnibed by the EORTC (European Organisation for Research Glycerol exerts a striking effect on the dissociation rate on Treatment of Cancer) group (8). The progesterone me constant (Chart 3). At 0Â°, in the absence of glycerol, k_1 = ceptor was assayed as described in Table 1. 0.041 min' (half-life, 17 mm); whereas in the presence of 30% glycerol, k_1= 0.0024 min1 (half-life, 290 mm). In both RESULTS cases the dissociation obeyed a 1st-order law, but the sta bility of the complexes was raised 17-fold under the influ Characterization of the Receptor ence of 30% glycerol. Distinction between [3H]Progesterone Binding to Recep Stabilization of Receptor-Steroid Complex by Glycerol. tor and Its Binding to CBG. A cytosol obtained from a To study the interaction of progesterone with its receptor in mammary carcinoma known to contain progesterone me human mammary carcinoma, it was first necessary to stabi ceptar (see Table 1) was incubated with [3H]progesterone

FEBRUARY1977 465

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Chart 1. Effect of glycerol on the binding equilibrium between [3H]progesteroneandreceptor.Cytosolwasincubatedwith varyingconcen Chart 3. Effect of glycerol on the rate of dissociation of [â€˜H]progesterone trations (0.5 to 50 nM) of [3H]progesterone in the presence (Curve a) or in the receptor complexes. Curve a, in the presence of 30% glycerol; Curve b, in the absence (Curve b) of 30% glycerol. All the incubates contained 1 @Munla absence of glycerol. Cytosol was incubated for 3 hr at 0Â°with 10 [email protected] beled cortisol to prevent binding of radioactive hormone to CBG (see (â€˜H]progesteroneand1 @.tMunlabeledcortisol in Tnisbuffer. At time 0 3 Chart 4). After 4 hr at 0Â°,1 volume of buffer containing either 30 or 60% volumes of buffer were added containing unlabeled progesterone and , in glycerol was added to obtain an equal concentration of glycerol (30%). some cases, glycerol. In all the incubates the final concentration of unlabeled Immediately afterward, a suspension of dextran-coated charcoal in 30% progesterone was 2.5 .tM and in one-half of the incubates glycerol was glycerol was added . Correction for the nonspecific binding was performed as presentat a concentrationof 30%.At varioustimes, abiquotsweretaken, to described above (see â€œMaterialsandMethodsâ€•).Cytosol contained 3.3 mg which 1 volume of Tnis buffer containing either 30 or 6O@'oglycerol was protein per ml. B, bound radioactivity; U, unbound radioactivity. added. A concentration of 30% glycerol was thus obtained in all the incu bates. Immediately afterward, a charcoal suspension in 30% glycerol was added. A correction was made for the nonsaturable, nonspecific binding. Protein concentration in the cytosol was 3 mg/mI. B, bound radioactivity.

dpmbound

Chart 2. Effect of glycerol on the rate of association of [3H]progesterone to receptor.Curvea, associationin the presenceof 30%glycerol;Curveb, association in the absence of glycerol. Cytosol was added at time 0 to 1 volume of buffer containing 5 n@ [3H]progesterone and 1 @.tMunlabeled cortisol. One-half of the incubations contained 30% glycerol, and one-half were performed in the absence of glycerol. Hormone receptor association was allowed to proceed at 0Â°forvarious times. At the end of the incubation, 1 volume of buffer containing 60 or 30% glycerol was added in order to obtain in all cases a final concentration of 30% glycerol. Charcoal suspension in [Ml UnIaÃ³@II.d hormone 30% glycerol was immediately added. Time of contact with charcoal was 30 Chart 4. Differences in affinity for cortisol and progesterone of mammary mm. Correction was performed for the nonspecific, nonsaturable binding. carcinoma cytosol (A) and plasma (B). A. Mammary carcinoma cytosol was Protein concentration in the cytosob was 3 mg/mI. The maximal binding is prepared in Tnis buffer containing 30% glycerol. It was incubated for 4 hr at 0Â° different in Curve a and Curve b due to the differences in equilibrium with 20 nM [3H]progesterone alone or with increasing concentrations of association constant observed in the presence and in the absence of glycerol unlabeled cortisol (Curve a) or progesterone (Curve b) (50 nM to 5 @&M). (seeChart 1). Bound radioactivity was measured. Protein concentration was 3.7 mg/mb. B. Plasma was diluted with Tnis buffer and glycerol (final concentration, 30%) to the same protein concentration as in mammary cytosol. Incubation and and increasing amounts of unlabeled cartisal (Chart 4A, bound radioactivity measurements were performed as in A. Curve a). A small decrease in binding of radioactivity was observed with 50 nM cartisol. There was not further campe cortisal was due to a chase of radioactivity from GBG and titian when the cortisol concentration was increased from that competition for receptor binding of [3H]progestenone 50 nM to 1 @M.At concentrations higher than 1 tiM, a net was observed only with amounts of cortisol oven 1 @M. decrease of bound radioactivity was seen. With unlabeled This interpretation was further strengthened when a simi progesterone most of the [3H]progesterone binding was bar experiment was performed. with plasma (Chart 4B). It abolished at 1 ,iM (Chart 4A, Curve b). was then observed that [3H]pmagestemanewas chased from It is thus probable that the decrease obtained with 50 nM GBG by 50 nM unlabeled cortisol (Chart 4B, Curve a). Na

466 CANCER RESEARCHVOL. 37

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. Progesterone Receptor in Mammary Carcinoma further decrease of bound radioactivity was seen even at 5 have bound the hormone and have been activated, are able @M.The inhibitory effect of progesterone was markedly to interact with nuclear acceptors. The binding of various smaller, a finding that is in agreement with the known receptors to DNA has been studied with the use of many difference in affinity of both steroids far human GBG at 0Â° techniques including chromatography on Sephanase 2B (1, (33). 23). DNA is excluded from this gel, whereas neceptar-ste Steroid Specificity of Progesterone Receptor (Chart 5). raid complexes and free hormone are included. Activated The ability of various unlabeled steroids to compete with [3H]progestemone-mammary carcinoma receptor complexes [3H]pragesterone was studied . The concentrations giving did bind to human DNA (Chart 7, Curve a). Conversely, 50% inhibition of [3H]pragestenone binding were compared with that of progesterone (= 100). Synthetic progestative compounds had a high affinity: R5020, 127%; 17a-ethinyl, 17-hydroxyestr-4-en-3-one (horethisterone), 83%; 6-chbora 17-acetoxypregna-4,6-diene-3,20-dione (chbormadinane), 72%. 5a-Pregnane-3,20-dione was also a patent inhibitor (48%). 20a-Hydraxypregn-4-ene-3,20-dione had a lower affinity (26%). 5f3-Pregnane-3,20- diane (4%), 17a-hyd roxy pregn-4-ene-3,20-dione (17 hydraxypmogestenone) (3%), I 1fJ,21-dihyd roxypregn-4-ene-3,20-dione (corticosterone) (2.5%), 17f3- hydroxyandrost-4-en - 3- one (testosterone) (0.6%), and estra-1 ,3,5(10)-tniene-3,1 7f3-diol (estradiob) (0.4%) were very weakly bound. Density Gradient Sedimentation (Chart 6). From the me suIts of the experiments reported above, progesterone me Chart 6. Density gradient sedimentation of progesterone receptor. Cyto ceptomscould be characterized as macromolecules binding sol containing 10% glycerol was incubated for 2 hr at 0Â°with: (a) 10 nM to [3H]progesterone with high affinity (this may be shown by [3H]progesterone; (b) 10 nM [3H]progesterone Ã· 1 @Munlabeledcortisol; (c) 10 nM [3H]progesterone + 1 j@Munlabeled cortisol + 1 @Munlabeledproges competition with excess unlabeled progesterone), whereas terone; (d) 10 nM [3H]R5020. Incubate, 0.2 ml, was centrifuged for 9 (A) or 10 the distinction from GBG could be made by the absence of (B)hr at 49,000rpmin a Sw50.1rotorthrougha 20to 40Â°@oglycerolgradient. competition with excess unlabeled cortisal. In different tu A andB represent2 differenttumor specimens.CytosolsA andB contained 10 and 15.7 mg protein per ml, respectively. Specific activity of radioactive mar biopsies, macromolecules with these characteristics R5020 has been normalized to 94 Ci/mmole (identical to that of were found either in the 8 5 on in the 4.5 S regions or in [3H]progesterone). bath. Two representative patterns are shown. In Chart 6A an 8 S peak is observed, whereas in Chart 6B only a shoulder is present in this region, the major binder having a sedimenta tian coefficient of 4.5 5. Albumin and GBG migrate also in the 4 5 region, but the binding protein is neither of them since it has a low capacity (competition by unlabeled pro gesterane) and no affinity for cortisol. It may be observed that the sedimentation pattern of bound [3H]R5020 is strictly supenimposable on that of [3H]pmogestemone. Binding to DNA. Steroid hormone receptors, after they

Chart 7. Interaction with DNA of [3H]progesterone-receptor complexes (a) and [3Hjprogesterone-CBG complexes (b). In Curve a, cytosol was prepared in Tnis buffer containing 20% glycerol (protein concentration, 9 mg/mI). It was incubated for 2 hr at 0Â°with 10 nM [aH]progesterone. It was then activated for 1 hr at 15Â°andcooled for 15 mm at [email protected] with 1 volume of human DNA (Azornm 10.5) was performed for 30 mm at 0Â°.0.4ml of the incubate was chromatographed on a column of Sepharose 2B (0.6 x 25 cm) in Tnisbuffer containing20%glycerol. Fractions(0.5 ml) werecollected,of Chart5. Steroidspecificityof progesteronereceptor.Cytosolcontaining which 0.2 ml was counted for radioactivity. b was identical to a except that 30% glycerol was incubated for 2 hr at 0Â°with 3 nM [3H]progesterone and diluted female plasma (protein concentration 9 mg/mI) was used. DNA was increasing concentrations of unlabeled steroids. Bound radioactivity was prepared from human placenta according to the technique of Bernardi and measured with the dextran-coated charcoal technique. 100 = binding in the Stutz (30) including chromatography on hydroxylapatite as the last step (5). absence of competitor. Unlabeled steroids: A, progesterone; B, R5020; C, In the absence of DNA no radioactivity was present in the void volume of the 17a-ethinyl,1 7-hydroxyestr-4-en-3-one (horethisterone); D , 5a-pregnane column. On Sepharose 2B the separation between receptor-steroid com 3,20-dione; E, 5$-pregnane-3,20-dione; F, 17fl-hydroxyandrost-4-en-3-one plexes not bound to DNA or CBG-steroid complexes on one hand and free (testosterone); G, estra-1 .3,5(10)tniene-3,17p-diol (estradiol). hormoneon the other hand is not complete.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. M. F. Pichon and E. Milgrom [3H]progestemone bound to GBG did not interact with DNA enol), and the concentration of unlabeled cortisal has been (Chart 7, Curve b). selected to chase totally the radioactive hormone from GBG without affecting the binding to the receptor. Parallel incuba Assay of the Receptor tions in the presence of excess unlabeled progesterone allow one to connect fan the nonspecific binding. Altenna The method of assay is summarized in Table 1. It involves tively, it is possible to perform incubatians with various an incubation in 2 successive periods. An initial incubation concentrations of hormone and to use Scatchard plots. of 2 hr in the absence of glycerol allows fan the exchange However, no special information is obtained from the slope between the endagenaus hormone of the patient and the of the Scatchard plot, since the affinity constants of all radioactive hormone added for the assay (the half-life of the receptors tested were similar. Thus, only the concentration progesterone-receptor complex in the absence of glycerol of binding sites is of interest. In such a situation a better is 17 mm). The 2nd 2-hr period in the presence of glycerol statistical precision is achieved by performing many nepli results in a higher binding of the steroid to the receptor, due cates of binding assay at a hormonal concentration that to decreased equilibrium dissociation constant, and stabi saturates the receptor. lizes progesterone-receptor complexes (at 20 nM Results of Assay. Fifty-five biopsies were studied (Tables [3H]pmogestenoneconcentration, equilibrium is actually at 2 and 3), including 38 primary tumors, 11 local recurrences, tamed in less than 1 hm).The concentration of radioactive 3 lymph nodes, and 3 metastases. None of the patients has hormone has been chosen to saturate the receptor (18-fold received previous hormonal treatment on radiotherapy. higher than the K1)= 1.1 flM in the presence of 30% glyc Of the 55 biopsies tested (each taken from a different

Table 2 Concentration of estrogen and progesterone receptors in primary tumors Progester Estrogen one receptor receptor (fmoles/ (fmoles/ mg pro- mg pro Patient Age Hormonal status tein) tein) 1 54 Postmenopausab 0 0 2 62 Postmenopausal 0 0 @ 3 64 Postmenopausal 0 0 4 72 Postmenopausal 0 0 5 77 Postmenopausal 83 178 6 65 Postmenopausal 95 16 7 67 Postmenopausal 107 14 8Â° 55 Postmenopausal 114 0 9 49 Postmenopausal 121 31 10 66 Postmenopausal 139 31 11 73 Postmenopausal 211 112 12 58 Postmenopausal 212 278 13 60 Postmenopausal 225 90 14 74 Postmenopausal 242 46 15 68 Postmenopausal 251 0 16 72 Postmenapausal 269 123 17 70 Postmenopausal 322 197 18 65 Postmenopausal 334 0 19 77 Postmenopausab 367 33 20 73 Postmenopausal 393 62 21 67 Postmenopausal 395 113 22 68 Postmenopausal 451 180 23 77 Postmenopausal 478 29 24 77 Pastmenopausal 544 200 25 61 Postmenopausal 751 33 26 84 Postmenopausal 1241 131 27 40 Ovaniectomized (10 ms.) 0 0 28 44 Ovaniectomized (3 yr) 0 0 29 61 Ovariectomized(11 yr) 192 228 30 â€˜47 Ovariectomized (3 yr) 259 285 31 41 Normal cycle, luteal phase 0 0 32 46 Normal cycle, luteal phase 0 0 33 47 Normal cycle, luteal phase 46 159 34 41 Normal cycle, luteal phase 53 25 35 46 Normal cycle, follicular phase 91 1484 36 37 Normal cycle, follicular phase 191 25 37 48 Normal cycle, follicular phase 204 0 38 35 Normal cycle, follicular phase 293 462

a in menopause for 3 years. All the other patients were in menopause for over 5 years.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. Progesterone Receptor in Mammary Carcinoma Table 3 Concentration of estrogen and progesterone receptors in recurrences and metastasesProges

teroneEstrogenreceptorreceptor(fmoles/(fmoles/mgmg

pro PatientAgeHormonal statusproteintein)Local

recurrences3963Postmenopausal29504071Postmenopausal3804164Postmenopausal1901064283Postmenopausal2143064380Postmenopausal34104458Postmenopausal348424562Postmenopausal970594648Ovariectomized

yr)256354735Normal (2 determined0134836Normal cycle, phase nat phase401384939Normal cycle, luteal determined870Metastases50'54Postmenopausal62451cycle, phase not

a58bPostmenopausal96052@42Ovaniectomized yr)503553c43Ovariectomized (7 yr)76054@42Normal (2 determined0055d44Normal cycle, phase not cycle, luteabphase100

a Lymph node metastases. b In menopause for 5 years. All the other patients were in menopause for over 5 years. e Brain metastases. d Bone metastases.

patient), 20 (36%) contained no detectable progesterone described the possible use of the natural hormone, with the receptor or had a small concentration of this receptor (<10 provisions that the complex is stabilized by glycerol and fmoles/mg cytosol protein). that a proper concentration of unlabeled cortisol inhibits There was a clear relation between the concentrations of binding to CBG without decreasing the binding to the me estrogen and progesterone receptors. Of the 11 tumors cepton. Cortisol also prevents [3Hjprogesterone from bind containing less than 10 fmoles estrogen receptor pen mg ing to the glucocorticaid receptor. [3H]Progestenone is protein, only 1 contained more than 10 fmoles progesteane readily available to all laboratories, may be obtained with a receptor per mg protein. Of the 20 tumors with a medium specific activity of â€˜â€”100Ci/mmole,and has the advantage content in estrogen receptor (11 to 200 fmales), 14 (70%) of the specificity of the natural hormone (see â€œIntroduc contained over 10 fmoles progesterone receptor per mg tionâ€•).Under these conditions (presence of 30% glycerol), protein. Of the 24 tumors with a very high estrogen receptor the affinity of R5020 for the receptor was only very slightly content (oven 200 fmoles), 20 (83%) contained oven 10 higher than that of progesterone and the sedimentation fmoles progesterone receptor per mg protein. profiles obtained with both compounds were similar. Olden patients had a slightly higher proportion of tumors The number of positive results of the assay described in containing more than 10 fmoles receptor per mg protein: 11 this paper is higher than that reported by Horwitz and Mc to 20 (55%) in the group below 50; and 24 of 35 (69%) in the Guime(15) (20 positive results in 50 patients), and the results group aver 50 years. are of the same order as those reported by Raynaud et a!. Concentrations of oven 10 fmoles/mg protein were ob (27) (34 of 59). Both groups used R5020, and it is not known served in 24 of 35 (69%) postmenapausal patients, 4 of 7 whether only free receptor was measured or whether ex castrated patients, and 7 of 13 cycling patients. change of possible endagenous hormone was performed. The criteria of positivity were also different. Horwitz and McGuime (14) did not set a lower limit of concentration, DISCUSSION whereas Raynaud et a!. (27) compared tumors on a weight basis. In this study an arbitrary concentration of 10 fmoles/ The precise determination of the progesterone receptor mg protein was used as a cut-off paint to compare groups of in human mammary carcinoma has been made difficult by patients. the instability of the hormone-receptor complex and by the The existence of 2 forms of the receptor (8 and 4.5 S) is presence of CBG, which binds progesterone with high aftin very similar to that described for target organs of various ity. To salve these problems, the use of R5020, a synthetic animals (17, 24). It is not known whether their ratio is under progestin, has been proposed (15, 27). In this paper we have hormonal control as has been shown for guinea pig mecep

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. M. F. Pichan and E. Milgrom tar (25). It would also be interesting to investigate these 2 REFERENCES forms far possible different functional capabilities in, for instance, their binding to DNA. 1. Baxter,J. 0., Rousseau, G. G., Benson, M. L., Garcea, A. L., lto,J., and Tomkins, G. M. Role of DNA and Specific Cytoplasmic Receptors in The mechanism of the effect of glycerol on progesterone Glucocorticoid Action. Proc. NatI. Acad. Sci. U. S., 69: 1892-1896, 1972. binding to receptor is not clearly understood. The decrease 2. Baxter, J., and Tomkins, G. M. Glucocorticoid Hormone Receptors. In G. RaspÃ©,(ed), Advances in the Biosciences, Vol. 7, pp. 331-347. New in dissociation rate constant (17-fold) was greater than the York: Pergamon Press, 1971. increase in equilibrium association constant (3.5-fold). 3. Bayard,F.,Louvet,J. P.,Monrozies,M.,Boulard,A.,andPontonnier,G. 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M. F. Pichon and E. Milgrom

Cancer Res 1977;37:464-471.

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