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Characterization and Assay of Progesterone Receptor in Human Mammary Carcinoma1

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Characterization and Assay of Progesterone Receptor in Human Mammary Carcinoma1 [CANCER RESEARCH 37, 464-471 , February 1977] Characterization and Assay of Progesterone Receptor in Human Mammary Carcinoma1 M. F. Pichon and E. Milgrom Groupe de Recherches sur Ia Biochimie Endocrinienne et Ia Reproduction (INSERM U.135), FacultédeMédecine,Paris-Sud, 94270 Bicétre,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 Médicaleand the DélégationGénécaleaIa 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 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. Progesterone Receptor in Mammary Carcinoma 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 0°)andhomogenization in 6 volumes of Tnis buffer at 0°. steroids were obtained from Roussel Uclaf. The only buffer 3. Centnifugation (105,000 x g for 90 mm at 0°)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 0°,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 0°. 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 0°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 0°,cut into small pieces, and homogenized with 6 vol one receptor = mean radioactivity bound in Incubations a — 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 0°.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 0°.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 0°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 0°.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.
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