Characteristics of Progesterone-Binding Components in Neoplastic Mammary Tissues of the Rat1

Home , Orosomucoid, Triamcinolone acetonide

[CANCER RESEARCH 36, 1886-1893, June 1976] Characteristics of Progesterone-binding Components in Neoplastic Mammary Tissues of the Rat1

James E. Goral2 and James L. Wittliff3

University of Rochester Cancer Center and Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

SUMMARY glucocorticoid-bmnding components of mammary tissues have been well characterized (8-10, 13, 18, 36, 37), little is Characteristics of [3H]progesterone-bmnding components known of the intracellular entities associated with proges were studied in cell-free preparations of two hormonally terone. responsive tumors: the R3230AG mammary adenocarci The detailed study of Lawson and Pearlman (20) first noma and 9,10-dimethyl-1 ,2-benzanthracene-induced mam suggested that the mammary gland of the pregnant rat mary tumor of the rat. Progesterone-binding macromole possessed the biological capacity to retain progesterone in cules from cytosols of both mammary neoplasms exhibited Vivo. Later studies demonstrated that the DMBA4-induced sedimentation coefficients of 3.5 to 4.0 S on sucrose mammary tumor of the rat (30, 39), as well as certain human gradients of either low or high ionic strength. From Scat mammary carcinomas (39), contained specific progester chard analyses of titration data, apparent dissociation con one-binding sites. Using organ cultures of mammary gland stants of 4 to 6 x 10@M were determined for ligand-binding from virgin mice, Mehta (26) reported that [3H]progesterone protein complexes from either tumor. Specific progester was associated with sites exhibiting high affinity. Atger et one-binding capacities varied considerably, ranging from al. (1) demonstrated that the cytoplasm of guinea pig mam 150 to 650 fmoles/mg of cytosol protein. Optimal binding of mary gland contained a progesterone-binding macromole [3H]progesterone was reached by 2 to 3 hr at 3Â°,pH7.4, and cule that was excluded by Sephadex G-25. However, be then decreased rapidly. Specificity studies indicated that cause of the low binding capacity, no further characteriza cortisol, corticosterone, and triamcinolone acetonide com tionstudieswereattempted. peted effectively for [3H]progesterone-binding. This sug In @dditionto normal mammary gland, several mammary gested that [3Hjprogesterone was bound largely to a macro tumors have been reported to be responsive to progester molecule distinct from transcortin, which does not bind one administration. Pharmacological doses of the steroid glucocorticoids containing 9a-fluoro groups. Aldosterone, hormone depressed the growth rate of the R3230AG mam as well as several androgens and estrogens, were weak mary adenocarcinoma (14) and increased the incidence of competitors of binding except at high concentrations. The DMBA-induced mammary tumors (15, 23). Since these car nature of the inhibition of progesterone-binding sites by cinomas responded differently to progesterone treatment, it triamcinolone acetonide and corticosterone was competi was suggested that these effects may be related to differ tive. Goncurrent titrations of [3Hjprogesterone and ences in the characteristics of progesterone-binding sites. [3H]triamcinolone acetonide-binding sites demonstrated This study determined some of the kinetic and molecular that their binding capacities were similar, considering the properties of specific [3H]progesterone-binding compo relative stabilities of the complexes. These results, which nents in these hormonally responsive mammary tumors of indicated that progesterone and glucocorticoids compete the rat. Additionally, in view of our previous observation for the same binding site, suggest that these hormones may that progesterone competed for glucocorticoid-binding influence mammary gland differentiation and development sites (9, 13), it was necessary to explore the binding of by a common mechanism. [3Hjprogesterone directly. A portion of the data reported here was presented elsewhere (12). I INTRODUCTION MATERIALS AND METHODS Mammary gland differentiation, development, and func tion are influenced by several steroid hormones, including Chemicals. All chemicals were reagent grade unless oth cortisol, estrogens, and progestogens (17, 19, 22, 26, 31, erwise specified. 1,2,6,7-[3H]Progesterone (96 to 105 Gi/ 40, 42). The initiation of these effects is thought to be mmole) was purchased from New England Nuclear Corp., mediated by specific steroid-binding components that mod Boston, Mass. , and 1,2,4-[3H]triamcinolone acetonide (9a- ulate intracellular events (16, 44). Although estrogen and fluoro-1 11j,16a ,17a ,21-tetrahydroxypregna-1 ,4-diene-3 ,20- dione-16,17-acetonide) (10.7 Gi/mmole) was obtained from

I This research was supported by USPHS Grant CA-12836 and CA-i 1198 Schwarz/Mann, Orangeburg, N. V. Unlabeled triamci from the National Cancer Institute. a Medical student Research Fellow supported by USPHS Grant T1-07-AM 01004-10. 4 The abbreviations used are: DMBA, 9,10-dimethyl-1 ,2-benzanthracene; 3 To whom requests for reprints should be addressed. l@,concentration of unlabeled steroid at which [3Hlprogesterone binding was Received November 11, 1975; accepted February 16, 1976. inhibited by 50%.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. Progesterone-binding Components in Mammary Tissue nolone acetonide and aldosterone were obtained from described previously was added to each vial and incubated Sigma Chemical Go. , St. Louis Mo. , and corticosterone, for 2 to 3 hr. Following the incubation period, cytosols were hydrocortisone, progesterone, and 17f3-estradiol were sup mixed with a pellet of dextran-coated charcoal, which was plied by Galbiochem, San Diego, Calif. Unlabeled dihydro prepared earlier by centrifuging 3 ml of the charcoal sus testosterone was purchased from Steraloids, Inc., Wilton, pension as suggested by Boylan and Wittliff (2). After an N. H. Tris-HGI (Trizma base) and DMBA were products of incubation of 10 mm, samples were centrifuged at 500 x g Sigma. Schwarz/Mann supplied the RNase-free sucrose, for 10 mm at 3Â°tosediment the charcoal. Aliquots (0.2 ml) of and Norit A was obtained from Matheson Coleman and Bell, the clear supernatant were removed and layered onto linear Norwood, Ohio. Omnifluor, from New England Nuclear 5 to 20% sucrose gradients prepared in Tris-HGI buffer, pH Corp., and Triton X-100, from Beckman Instruments, Inc., 7.4, containing 1.5 mM EDTA. Human serum albumin (4.6 S) Palo Alto, Calif. , were used in the preparation of a scintilla was used as a marker protein of known sedimentation ye tion cocktail. locity. Source and Maintenance of Animals. All animals were Calculations of Resufts. Computer programs described purchased from the Charles River Breeding Laboratories, earlier (3) for the Olivetti Programma 101 were used to Inc., Wilmington, Mass., and housed in the vivarium of the determine counting efficiencies and to convert radioactivity University of Rochester Medical Center. Female Fischer 344 data to steroid concentrations. Estimates of specific bind rats were used as hosts for the transplantable R3230AG ing by the dextran-coated charcoal assay were determined tumor, while the other mammary tumors studied were in as the difference between total and nonspecific binding. In duced by the carcinogen, DMBA, in young female Sprague the sucrose gradient procedure , steroid-binding compo Dawley rats. Transplantation of the R3230AC tumor and nents were identified by isotopic profiles. Specific binding induction of the DMBA-induced mammary tumor were per capacity was expressed as fmoles (10'@ mole) per mg of formed as described earlier (13). cytosol protein. Protein concentrations were estimated by Preparation of Cytosols. All procedures were performed the Geiger and Bessman (11) procedure when sulfhydryl at 0-3Â°unless otherwise noted. Mammary tumors were ex reagents were present, using bovine serum albumin as a cised quickly and placed in 10 mM Tris-HCI buffer, pH 7.4, standard. containing 1.5 mM EDTA, 250 mM sucrose, and 10 mM monothioglycerol. Tissues were minced on a Mcllwain tis sue chopper, stirred in the buffer to remove blood and milk RESULTS proteins, then blotted dry, and weighed. Using Duall ho mogenizers, mammary tissues were homogenized in buffer Time Courses of [3H]Progesterone Binding. Shown in using weight/volume ratios of 1/1 and 1/3 for R3230AC and Chart 1A, association of [3Hjprogesterone to specific sites in DMBA-induced mammary tumors, respectively. These ho cytosols from the R3230AG tumor occurred rapidly, reach mogenates were then centrifuged for 30 mm at 105,000 x g ing a maximum by -@2hr, which was followed by a decrease to prepare the cytosol fractions. in binding even in the presence of saturating concentra Dextran-coated Charcoal Procedure. This procedure is tions of labeled steroid. Specific binding capacity measured essentially an adaptation of the 3H-steroid-binding assays at 24 hr was only â€”25%ofthat determined at 2 hr. Similarly, that we reported earlier (10, 13). Constant volumes, 0.2 ml, binding of [3H]progesterone to specific sites in cytosols of the 105,000 x g supernatants were added to each of 0.5- from the DMBA-induced tumor was maximal by 2 to 3 hr dram glass shell vials (Fisher Scientific Go., Pittsburgh, (Chart 1B) at 3Â°,decreasing to undetectable levels by 24 hr. Pa.), containing [3H]progesterone alone, or in the presence Time courses of binding were comparable in 3 different of unlabeled progesterone, which had been evaporated specimens. As a result, an incubation period of 2 hr at 3Â° previously under nitrogen. In routine analyses a 100-fold was used routinely for experiments unless noted otherwise. excess of unlabeled progesterone was used. Triplicate re The rate constants of association at 3Â°were8.5 to 9.5 x 10@ actions were incubated for 2 to 3 hr at 4Â°unless noted M' min' and the t@of the complexes was 4 to 8 hr. These otherwise. Afterthe incubation period, 1 ml of a suspension values represent estimates, since true equilibrium was not of dextran-coated charcoal (10 mM Tris-HGI buffer, pH 7.4, established due to the rapid dissociation and/or degrada containing 1.5 mM EDTA, 0.25 M sucrose, 0.05% dextran, tion of steroid-binding protein complexes. These results are and 0.5 to 1.0% Norit A) was added. Following an additional in contrast with those seen using either [3H]triamcinolone incubation of 10 mm, the vials were centrifuged at 1500 rpm acetonide (13) or 17f3-[3H]estradiol (10). for 10 mm at 4Â°.Then an aliquot of each supernatant was Titrations of Specific Binding Sites with [3H]Progester removed and counted in toluene-based scintillation fluor one. Increasing concentrations of [3H]progesterone were containing Triton X-100 (4 g of Omnifluor in 300 ml of Triton incubated with cytosols from the R3230AG tumor (Chart 2), x-100 and 700 ml of toluene). and DMBA-induced tumors (Chart 3), each either in the Sucrose Gradient Centrifugatlon of Steroid-binding Pro presence or absence of unlabeled progesterone. Using the teins. The assay was similar to that previously described charcoal adsorption procedure, specific binding was calcu (10, 13). In each of several 0.5-dram shell vials, an appropri lated as the difference between total and nonspecific bind ate amount of [3H]progesterone (usually 40 to 50 nM) alone, ing. or in combination with an excess (usually 100-fold) of unla Saturation of specific binding sites in the cytosol from the beled progesterone, was evaporated under nitrogen just R3230AG tumor was observed at [3H]progesterone concen prior to the assay. An aliquot of cytosol (600 @d)preparedas trations approaching 150 nM (Chart 2A). Scatchard analysis

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protein was calculated. The mean Â±S.E. from 6 separate determinations of the K@,was6.2 Â±0.9 x 108 M, and the average number of specific binding sites was 378 Â±70 fmoles/mg of cytosol protein. Specific progesterone-binding sites in the cell-free prepa rations of the DMBA-induced tumor saturated at approxi mately 150 nM [3H)progesterone (Chart 3.4). Shown in Chart 3B, Scatchard analysis (35) resulted in a K,.,of 4.3 x 108 M and the number of binding sites was 270 fmoles/mg of cytosol protein. A separate determination of the apparent dissociation constant gave a value of 3.9 x 10_8M, and the number of sites was 426 fmoles/mg of cytosol protein. Ligand Specificity of (3H]Progesterone-bindlng Sites. Various concentrations of unlabeled steroids were used in ligand competition experiments with [3H]progesterone to TIME (1w) determine the binding specificity. Gytosols from the Chart 1. Time courses of specific progesterone-binding by cytosol from R3230AG tumor and DMBA-induced tumors were prepared mammary carcinomas of the rat. A. Cytosol was prepared from R3230AC tumors and incubated at 3Â°with36 flM [3Hjprogesterone in the presence and as described previously. Table 1 presents the I50values for absence of 4 x i0-@ M unlabeled progesterone. At various times, triplicate several unlabeled competitors. Triamcinolone acetonide, determinations were performed by the dextran-coated charcoal procedure. over a concentration range of 10_8to 10@ M, competed as Specific binding (as shown) was determined as the difference between the mean value of total binding and that of binding in the presence of unlabeled well as unlabeled progesterone for [3H]progesterone-bind steroid; variability was less than 5% for each set of determinations. B. ing sites in cytosol. Since triamcinolone acetonide does not Aliquots of cytosol that were prepared from DMBA-induced mammary tumor associated readily with transcortin (7), it was concluded that homogenized in Tris buffer, pH 7.4, were incubated at 3Â°with 38 flM [3H)progesterone in the presence and absence of 6 x i0@ M progesterone. the progesterone-binding sites described were distinct from those of the serum protein. Naturally occurring glucocorti coids such as corticosterone and hydrocortisone also in hibited [3H]progesterone binding significantly at physiologi cal concentrations. Aldosterone, 17/3-estradiol, and dihydro testosterone were weak competitors except at high concen trations in cell-free reactions. Similar results were observed with binding components from the DMBA-induced tumors. The I5@corresponded well with the theoretical value. From the inhibition data in Table 1 it was possible to calculate the

PROGCSTERONI ( nM ) SOUND (nM) Chart 2. Titration of progesterone-binding sites in the cytosol from the R3230AC mammary adenocarcinoma. A . Constant volumes of cytosol were incubated with increasing concentrations of [3Hjprogesterone at 3Â°for3 hr in the presence ( 0) and absence (â€¢)of2 x i0@ N unlabeled progesterone. Each point represents the mean of triplicate determinations made by the dextran-coated charcoal procedure. Specific binding (@) was estimated as the difference between total binding and binding in the presence of unla baled progesterone. B. Scatchard analysis of titration data in A gave a K,, value of 6.1 x 10-' M and a binding capacity of 663 fmoles/mg of cytosol protein. PROGESTERONE (nM) Chart 3. Titration of progesterone-binding sites in the cytosol from the (35) of these data yielded a straight line, indicating a single DMBA-induced mammary tumor. A. Constant volumes of cytosol were incu class of binding sites with high affinity (Chart 2B). For this bated for 3 hr at 3Â°withincreasing concentrations of [3H]progesterone in the presence ( 0) and absence (â€¢)of2 x 10@N unlabeled progesterone. Mess particular preparation of the R3230AG tumor, an apparent urements of binding were made by the dextran-coated charcoal procedure. K,, of 6.1 x lO-@ M was obtained for the complex of proges Eachpoint represents the mean of 3 determinations. â€¢,specificbinding. B. Scatchard analysis of titration data for progesterone-binding (shown in A) terone and binding component. From the intercept on the indicated a Kdvalue of 4.3 x 10_aM and a binding capacity of 270 fmoles/mg abscissa, a binding capacity of 663 fmoles/mg of cytosol ofcytosolprotein.

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1Relative Table were 4.5 x 10-' and 7.0 x l0-@M, respectively, as calculated for(3HJprogesterone-bindingdissociation constants of variousligands from the equation: tumorDissociationCompetitivesites in the R3230ACmammary Slope (I) = slope (P) [1 + [/]/K] (M)Progesterone steroid l@,a(M) constant 10@Triamcinolone 8.2 x 10_a 6.2 x where I is inhibitor and P is progesterone (21). io-'Corticosterone acetonide 2.6 x io-' 2.3 x Concurrent Titration of Glucocorticoid and Progester 10@@Hydrocortisone 1.1 x i0@ 6.6 x io-'Aldosterone 1.0 x 10@ 8.7 x one-binding Sites. Evidence that certain glucocorticoids 10@@17/3-Estradiol 7.1 x 10@ 6.2 x inhibited progesterone binding competitively suggested 10@Dihydrotestosterone 1.4 x 10@ 1.2 x that these steroids bound to common sites on the cytoplas 5.2x 10@ 3.1x 10@ mic components. Titration of specific glucocorticoid and a Concentration of [3H]progesterone, 40 to 60 nM. progesterone-binding sites was performed on the same cy b Mean value of 6 determinations by Scatchard analysis of titra tosol from either the R3230AG or DMBA-induced tumors. tion data. From Scatchard analysis of the titration data for the R3230AGtumor presented in Chart 5, it was determined that relative dissociation constants for each unlabeled ligand the number of glucocorticoid-binding sites was 420 fmoles/ binding complex using the following relationship suggested mg of cytosol protein, while the progesterone-binding ca by Rodbard (32): pacity was 282 fmoles/mg of protein. In a separate experi ment, values of 441 and 498 fmoles/mg of protein were iÃ§.= K1,[C]50 obtained for the number of binding sites of [â€˜H]progester [P]50 one and [3H]triamcinolone acetonide, respectively. As was expected, the number of progesterone-binding sites was where iÃ§.is the dissociation constant of the competitor less than that of glucocorticoid-binding sites, based on the binding site complexes, K,, is the dissociation constant of relative stabilities and affinities of these ligand-binding [3H]progesterone-binding site complexes determined by protein complexes at 3Â°.Further studies using cytosol from Scatchard analysis of titration data, [C]@,is the concentra the DMBA-induced tumor confirmed the observation that tion of unlabeled competitor that inhibited the binding of the relative binding capacities of progesterone and tn labeled ligand by 50%, and [P]@,is the concentration of amcinolone acetonide were similar (data not presented). In unlabeled progesterone that inhibited the binding of this experiment the number of progesterone-binding sites [3Hlprogesterone by 50%. The relative dissociation con-' stants estimated by this relationship (Table 1) indicated that triamcinolone acetonide, corticosterone, and hydrocorti sone had similar affinities as that of progesterone for bind ing sites. Progesterone-binding sites showed significantly lower affinity for aldosterone, as well as for the androgen and estrogen tested. Nature of the Inhibition of [3H]Progesterone-binding by Triamcinolone Acetonide and Corticosterone. From ligand specificity studies it was suggested that inhibition of pro gesterone-binding sites by naturally occurring and syn thetic glucocorticoids was of a competitive nature. For fur ther delineation of this observation, cytosol preparations from the R3230AC tumor were titrated with various concen trations of [3H]progesterone in either the presence or ab sence of limiting concentrations of one of the following: unlabeled progesterone, triamcinolone acetonide, or corti costerone. From the titration data, double reciprocal plots (21) were constructed to evaluate the nature of the inhibi tion by these steroids. Unlabeled progesterone, at several limiting concentrations, demonstrated competitive inhibi tion for [3H]progesterone-binding sites (data not shown). Both triamcinolone acetonide and corticosterone also ex @ hibited inhibition of a competitive nature for specific pro [ 3HJPROGESTERONE-FREE (nM) FREE (102 flM') gesterone-binding sites (Chart 4). From analysis of double Chart 4. Inhibition of progesterone-binding sites in cytosol from the reciprocal plots a Kmfor progesterone of 4.5 x 10@ M was R3230AC adenocarcinoma by glucocorticoids. A constant volume of cytosol obtained that agreed well with a K,@valueof 4.3 x 10_' M, prepared from tumors was incubated at 3Â°for2 hr with increasing concentra tions of [3Hlprogesterone either alone ( 0) or in the presence of 1.5 x 10@M determined by Scatchard analysis of the data. From the data unlabeled progesterone (â€¢),1.5 x i0@ P.4triamcinolone acetonide (I), or 1.5 presented in Chart 4, a Kmfor progesterone of 8.4 x 10'@M x i0@ Mcorticosterone (0). Binding capacities shown in A were measured by the dextran-coated charcoal procedure. A double reciprocal plot of the was determined compared to a K@valueof 9.3 x 10W'M. The specific binding data from A is presented in B. Each point represents the K, values for triamcinolone acetonide and corticosterone mean of 3 determinations.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. J. E. Goral and J. L. Wittliff observed earlier from time-course experiments. As seen in Charts 6 and 7, either unlabeled progesterone or triamcino lone acetonide partially inhibited the binding of 3Hligand to the 3.5 to 4.0 S components. Since transcortin, which also sediments in this region (18), does not bind tniamcinolone a 0 acetonide readily (7, 34), it was concluded that the majority E a. of [3H]progesterone was associated with a distinct cellular â€˜-9 component. Bound steroid under the 3.5 to 4.0 S peak, which was not inhibited by either unlabeled progesterone or tniamcinolone acetonide, was thought to result from serum a z proteins (e.g. , albumin and a,-acid glycoprotein) contami nating the mammary tumors. Presumably, as [3H]progester one dissociated from specific cellular components, it was a z 4 associated with contaminating proteins sedimenting at 0 -a â€”4S,which bound it less specifically. I 1, DISCUSSION

Although the biochemical properties of estrogen- and glucocorticoid-binding components in the mammary gland and mammary tumors have been described (8-10, 13, 18, Chart 5. Titration of glucocorticoid and progesterone-binding sites in 36-38, 44), relatively little is known about progesterone cytosol from the R3230AC tumor. Constant volumes of cytosol were incu binding components in these tissues. Studies in vivo by bated with various concentrations of either [3H]progesterone or Lawson and Pearlman (20) demonstrated localization of [3Hjtriamcinolone acetonide each either alone or in the presence of their respective unlabeled ligand at 10@ M (A). Specific binding of progesterone [3H]progesterone and its metabolites in the mammary gland (I) and triamcinolone acetonide (S) was determined as the difference be of the pregnant rat at levels 2 to 3 times that of plasma. tween total and nonspecific binding measured by the dextran-coated char However, isolation of the binding components was not re coal procedure. Each point represents the mean of 3 determinations. From Scatchard analysis of the titration data in B , the dissociation constants were ported. Similarly, Mobbs (30) has found low levels of selec estimated as 3 x 10-' N forthe progesterone-binding complexes and 7 x i0' tive uptake in vivo of [â€˜4G]progesteronein DMBA-induced N for triamcinolone acetonide-binding complexes. Binding capacities were 282and420fmoles/mgofproteinforprogesteroneandtriamcinoloneaceto mammary carcinomas. In these studies hormone-respon nide, respectively. sive DMBA-induced mammary tumors of the rat did not concentrate progesterone or its metabolites to the same was 270 fmoles/mg of protein while that of the tniamcino extent as did labeled estradiol. In hormone-unresponsive lone acetonide binding sites was 400 fmoles/mg of cytosol protein. Molecular Characteristics of [3H]Progesterone-blndlng Components. The sedimentation properties of [3H]pro gesterone-binding macromolecules were found to be unlike those observed for [3H]tniamcinolone acetonide binding proteins (13). Gytoplasmic binding components were labeled in vitro with [3H]progesterone either alone or in the presence of unlabeled tniamcinolone acetonide or pro gesterone, and sedimented on linear 5 to 20% sucrose gradients of low and high ionic strength. On gradients of either low or high ionic strength, the progesterone-binding complexes from the cytosol of the R3230AC tumor sedi mented at 3.5 to 4.0 S (Ghart 6). Likewise, progesterone binding components in the cytosol of the DMBA-induced tumor sedimented at 3.5 to 4.0 S on sucrose gradients either without or with 0.4 M KGl (Chart 7). In contrast, [3H]tniamcinolone acetonide-binding components in these tissues sedimented at 7 to 8 S on sucrose gradients of low ionic strength and at 4 to 5 S on gradients containing 0.4 M KGI (13). The specific progesterone-binding capacity of both the FRACTION NUMBER DMBA-induced and the R3230AG tumors observed at 14 to Chart 6. Sedimentation properties of progesterone-binding components 16 hr, using the sucrose gradient procedure, was lower than labeled in vitro from R3230AC mammary tumors. Cytosol was incubated with 49 flM [3H]progesterone either alone (â€¢)or in the presence of 5 x 10_a hi that observed at 2 to 3 hr, estimated with the dextran-coated unlabeled progesterone ( 0) or 5 x 10a N triamcinolone acetonide (0) for 2 charcoal method. The difference in binding apparently was hr at 3Â°.Aliquots of the reaction were centrifuged on linear 5 to 20% sucrose due to the lability of the steroid-binding protein complexes gradients of low ionic strength (A) or in the presence of 0.4 hi KCI (B).

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tumors, progesterone appeared to increase the concentra tion of estradiol or its metabolites, although the retention of estradiol itself was unchanged. A major problem in determination of specific progester

one-binding components in mammary tissues has been .5 contamination of preparations with serum proteins such as corticosteroid-binding globulin (43), or a,-acid glycopro â€˜2 tein, which bind with a high affinity (33). a,-Acid glycopro a tein (orosomucoid) has a single binding site for progester z

one with binding affinities of 6.7 to 10.4 x 10@M' and 2 sediments at â€”2.9S (33). However, corticosteroid-binding z globulin has a higher affinity for progesterone, i.e., KA @-3x 0 108 M' (43). Thus, distinguishing specific intracellular 0 [3H]progesterone-binding components from corticosteroid 0 binding globulin was accomplished by using unlabeled x triamcinolone acetonide as a competitor, since it does not a, bind to the serum protein. In serum of the pregnant guinea pig, a distinct progester one-binding component has been reported (6). Burton et al. (5) designated this protein as progesterone-binding globu TOP TOP lin because of its migration as an a-globulin. Levels of this FRACTION NUMBER binding component were found to rise 100-fold during preg Chart 7. Sedimentation properties of progesterone-binding components labeled in vitro from DMBA-induced mammary tumors. Cytosol was incu nancy (6). Further, isolation and characterization of proges bated with 49 flM [3Hjprogesterone either alone (â€¢)orin the presence of 5 x terone-binding globulin indicated binding sites with high 10_aN unlabeled progesterone (0) or 5 x 10 hi triamcinolone acetonide affinity, KA â€”3x 10@M', and sedimentation coefficients, (0), for 3 hr at 3Â°.Aliquots of the reaction were centrifuged on linear 5 to 20% 4.7 S (4). Since there was no evidence of this protein in rat sucrose gradients of low ionic strength (A) or the presence of 0.4 hi KCI(B). serum, it was not considered a contamination problem in the characterization of intracellular progesterone and bind with binding sites in cell-free preparations of mammary ing components of the mammary tumors reported here. tumors was maximal by 2 to 3 hr, followed by rapid dissocia Specific cellular components binding progesterone in tion and/or degradation. This contrasts with the earlier DMBA-induced tumors was first described by Terenius (39). report of Terenius (39), in which a 16-hr incubation was Using tumors undifferentiated with regard to hormone re used to estimate progesterone-binding capacity in cytosols sponsiveness, he found that these tumors bound from DMBA-induced mammary tumors of the rat. Our esti [3H]progesterone in a specific fashion and at low levels, 11 mates of binding capacity made by the dextran-coated char to 42 fmoles/mg of cytosol protein. His data also provided coal procedure were 10-fold higher than those of Terenius, the first evidence of specific progesterone-binding sites in suggesting that the values he reported may be underesti human mammary carcinoma. Using Sephadex G-25 chro mates due to ligand dissociation. In our study considerable matography, Atger et al. (1) demonstrated specific proges variation in the level of progesterone-binding capacity was terone-binding components in cytoplasm of mammary noted in cytosols from both R3230AC and DMBA-induced gland from guinea pig. Further characterization was mammary tumors although, in general, the quantities were hindered by the low levels of the binding components. similar. Dissociation constants were in the range of 4 to 6 x @ Using organ cultures in vitro of mammary gland from virgin 1 M for progesterone-binding complexes in cytosols from mice, Mehta (26) demonstrated the presence of a cytoplas both the R3230AG and DMBA-induced tumors. Mehta (26) mic component in these tissues that bound [3H]progester reported slightly higher dissociation constants for proges one specifically. Binding saturated at 10@ M and a dis terone-binding complexes; however, he used whole mam sociation constant of 1.5 x 10@ M were determined from maryexplants in organ culture compared to cell-free prepa Scatchard analysis of binding data. rations used in this study. In this paper we report a more detailed characterization of Ligand specificity of binding showed significant competi [3H]progesterone-binding components and indicate that the tion by progesterone, as well as by several naturally occur biochemical properties of these entities in cytosols from the ring glucocorticoids, and by tniamcinolone acetonide. R3230AC adenocarcinoma and DMBA-induced tumors of These data indicated that the intracellular progesterone the rat are similar. Our data indicate that the molecular binding components of mammary tissues were distinct from basis of the difference in progesterone responsiveness by those of serum, namely transcortin, which does not bind these tumors is unrelated to the properties of specific pro triamcinolone acetonide specifically (7, 34). Neither andro gesterone-binding components in cytosol. Rather, it is sug gens nor estrogens were competitors for progesterone gested that the variation in hormonal response is due to a binding sites except at high concentrations. Similar ligand difference(s) in intracellular events beyond the initial bind specificities have been described for progesterone-binding ing. components in the uterus (20, 24). However, other investi A number of differences between this study and earlier gators have characterized another progesterone-binding reports are noteworthy. Association of [3H]progesterone component that did not bind glucocorticoids such as corti

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. J. E. Goral and J. L. Wittliff sol (25), suggesting the existence of 2 distinct progester of these hormones on mammary tumor growth and regres one-binding components. sion are under investigation. The competition of progesterone-binding sites by triamci nolone acetonide was of a competitive nature, suggesting ACKNOWLEDGMENTS that these hormones compete for the same sites. Addition ally, concurrent measurements of the number of glucocorti The authors wish to thank W. Swanson and M. Ludwig Swanson of the Animal Tumor Research Facility of the University of Rochster Cancer Center, coid-and progesterone-binding sites indicated that they who transplanted the R3230AC tumor line and performed the intubations of were of the same order. [3HjProgesterone-binding capaci DMBA. The assistance of Barbara Beatty was most appreciated. We also would like to express our gratitude to Dr. R. Hilf, Dr. R. Mehta, and Dr. E. ties were consistently lower than those of [3H]tniamcinolone Stotz for their comments and critical reading of the manuscript. acetonide, using the same receptor preparation. This sug gests either that not all sites are shared or that the differ REFERENCES ences in the thermostabilities observed for these steroid receptor complexes are reflected in the measurements of 1. Atger, M., Baulieu, E. E., and Milgrom, E. An Investigation of Progester binding capacities. This has not yet been resolved. one Receptors in Guinea Pig Vagina, Uterine Cervix, Mammary Glands, Similar to the serum proteins, a,-acid glycoprotein and Pituitary and Hypothalamus. Endocrinology, 94: 161-167, 1974. 2. Boylan, E. S. , and Wittliff, J. L. Specific Estrogen Binding in Vivo in the progesterone-binding globulin , the intracellular [3H]pro R3230AC Mammary Adenocarcinoma of the Rat. Cancer Res. , 33: 2903- gesterone-binding proteins from these mammary tumors 2908, 1973. 3. Brooks, W. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. Characteristics of Progesterone-binding Components in Neoplastic Mammary Tissues of the Rat

James E. Goral and James L. Wittliff

Cancer Res 1976;36:1886-1893.

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