Binding Characteristics of a Major Protein in Rat Ventral Prostate Cytosol That Interacts with Estramustine, a Nitrogen Mustard Derivative of 17ÃŸ-Estradiol

Home , Androstane, Estradiol (medication), Estramustine, Estramustine phosphate, Metabolite

[CANCER RESEARCH 39, 5155-5164, December 1979] 0008-5472/79/0039-OOOOS02.00 Binding Characteristics of a Major Protein in Rat Ventral Prostate Cytosol That Interacts with Estramustine, a Nitrogen Mustard Derivative of 17ÃŸ-Estradiol

BjÃ¶rnForsgren, Jan-Ã€ke Gustafsson, Ake Pousette, and Bertil Hogberg

AB Leo Research Laboratories. Pack. S-251 00 Helsingborg, Sweden [B.F.. B.H.], and Departments of Chemistry and Medical Nutrition. Â¡J-Ã„G.. A P.Â¡and Pharmacology [BH.], Karolinska Institute!. S-104 01 Stockholm 60. Sweden

ABSTRACT causes atrophy of the testes and accessory sex organs; re duces the uptake of zinc by the prostate; depresses the 5a- The tissue distribution of [3H]estramustine, the dephospho- reductase, arginase, and acid phosphatase activities in the rylated metabolite of estramustine phosphate (Estracyt), in the male rat was compared to that of [3H]estradiol 30 min and 2 hr prostate; and affects lipid and carbohydrate metabolism (10, 11, 20, 29, 31 -33, 42). Although the observed effects in many following i.p. administration. In contrast to estradiol, estramus respects are similar to estrogenic effects, several experimental tine was found to be efficiently concentrated in the ventral and clinical results indicate that estramustine phosphate affects prostate gland by a soluble protein. The binding characteristics normal and neoplastic prostate tissue in a way that cannot be of this protein were studied in vitro using cytosol preparations attributed solely to its antigonadotropic or weak estrogenic of the gland. With a dextran-coated charcoal technique, the properties (15, 16, 30, 38). protein was found to bind estramustine with a broad pH opti Plym Forshell and Nilsson (35), using labeled compounds (1 mum between pH 7 and pH 8.5, with an apparent Kd of 10 to to 10 mg/kg body weight), found considerably higher levels of 30 nw, and with a binding capacity of about 5 nmol/mg cytosol radioactivity in the rat ventral prostate following i.v. administra protein. The estramustine/protein complex was not retained tion of estramustine phosphate than after administration of by DNA-cellulose. None of the natural steroids tested inhibited the binding of 10 nw [3H]estramustine by more than 35% estradiol phosphate or estradiol. The main metabolite of estra mustine phosphate in the ventral prostate was identified as its (progesterone), even when added in 4500-fold excess. The dephosphorylated congener estramustine. Estramustine was presence of a nitrogen mustard moiety at position 3 of the also the main metabolite in peripheral human blood after i.v. or steroid was necessary for high-affinity binding to the protein. P.O. (single dose) administration of estramustine phosphate The protein was calculated to constitute about 20% of the total (34). cytosol protein content. Using estramustine of high specific radioactivity, Forsgren and Hogberg (8) and Hpisaeter (17) have shown the presence INTRODUCTION of a macromolecule in the cytosol from the rat ventral prostate that binds estramustine. This macromolecule has a molecular Estramustine phosphate [Estracyt; 1,3,5(10)-estratriene- weight of 40,000 to 50,000, a Stokes' radius of 2.9 nm, a 3,17/?-diol-3-A/-bis(2-chloroethyl)carbamate 17/?-dihydrogen frictional ratio of 1.2, a sedimentation coefficient of 3.5 to 4S, phosphate (NSC 89199)] was introduced in 1966 as a thera and an isoelectric point of 5 (9). peutic agent in the treatment of prostatic carcinoma. It has Since this macromolecule appeared to occur in substantial been used mainly in patients with advanced carcinoma of the quantities in the rat ventral prostate, it was conceivable that it prostate who do not respond to conventional endocrine ther might play an important role in the tissue localization of estra apy. Clinical experience shows that objective and/or subjective mustine and in the mechanism of action of estramustine phos remissions are obtained in 20 to 50% of these cases, including phate in prostatic tissue. We have therefore investigated the regression of original tumor and soft tissue and skeletal mÃ©tas binding kinetics and ligand specificity of this macromolecule tases, as well as relief of pain (for references, see Ref. 18). as well as estramustine uptake in different organs. The mechanism of action of estramustine phosphate is com plex. Studies in humans and animals have shown that the drug decreases testosterone1 and gonadotropin levels in serum; MATERIALS AND METHODS

1The abbreviations and trivial names used are: testosterone, 17/i-hydroxy-4- Chemicals androsten-3-one; estradiol, 17/i-estradiol; dihydrotestosterone, 17/i-hydroxy-5a- androstan-3-one; dihydroepiandrosterone, 3/i-hydroxy-5-androsten-1 7-one; an- The radioactive compounds used were [3H]estramustine drostanediol, Su-androstane-3/J. 17/i-diol; ethynylestradiol. 17o-ethynyl- ([2,4,6,7-3H]estradiol, 3-A/-bis(2-chloroethyl)carbamate; 80 to 1.3.5(10)-estratriene-3,17/j-diol; BSA. bovine serum albumin; TLC, thin-layer 107 Ci/mmol), [3H]estradiol ([2,4,6,7-3H]estradiol; 91.3 Ci/ chromatography; TEK(01)DG, 0.01 M Tris-HCI/0.001 M EDTA/0.25 mM dithiothreitol/10% (v/v) glycerol/0.01 M KCI; TEK(15)DG, 0.01 M Tris-HCI/0.001 M mmol), and [3H]dihydrotestosterone ([1,2,4,5,6,7-3H]dihydro- EDTA/0.25 mM dithiothreitol/10% (v/v) glycerol/0.15 M KCI; TEK(40)DG, 0.01 testosterone; 80 Ci/mmol). [3H]Estramustine was synthesized M Tris-HCI/0.001 M EDTA/0.25 mM dithiothreitol/10% (v/v) glycerol/0.40 M at AB Leo, and [3H]estradiol and [3H]dihydrotestosterone were KCI; DCC, dextran-coated charcoal; androstenediol, 5-androstene-3/i,1 7/i-diol; pregnenolone, 3/J-hydroxy-5-pregnen-20-one; androstenedione. 4-androstene- obtained from New England Nuclear Chemicals GmbH, Dreiei- 3,17-dione; 19-nortestosterone, 17/3-hydroxy-19-nor-4-androsten-3-one; dexamethasone, 9Â«-fluoro-1iÃŸ.17o,21 -trihydroxy-lou-methyl-l ,4-pregnadiene- chenhain, West Germany. The compounds were routinely pu 3,20-dione; diethylstilbestrol.3,4-bis(p-hydroxyphenyl)-3-hexene. rified before use to at least 99% purity by chromatography on Received March 8. 1979; accepted September 10, 1979. Sephadex LH-20 using the solvent systems toluene/methanol

DECEMBER 1979 5155

(85/1 5, v/v) for estramustine and estmadiol and toluene/meth 55 sec for cooling. The homogenate was centrifuged for 1 hr anol (90/1 0, v/v) for dihydrotestostemone. at 105,000 x g (Beckman L2-65B on L5-65 ultracentnifuge In addition to estramustine phosphate (LS 299) and estra with an SW 56 or SW 27 rotor; Beckman Instruments, Inc., mustine (LS 275), the following unlabeled nitrogen mustard Palo Alto, Calif.). The cytosol (supennatant) was freed from the steroid derivatives synthesized at AB Leo were used: estrone, floating lipid layer and, when not used immediately, stored in 3-N-bis(2-chloroethyl)carbamate (LS 271); estradiol, 17$-ace 0.5-mI portions at â€”30Â°.Allpreparation steps were carried out tate, 3-N-bis(2-chloroethyl)cambamate (LS 289); estradiol, 17$- at 0â€”4Â°.Theprotein concentration of the cytosol preparations, N-bis(2-chloroethyl)carbamate (LS 298); estradiol, 3-N-bis(2- determined according to the method of Lowry et a!. (24) using chloroethyl)cambamate, 17$-propionate (LS 358); androster BSA as a standard, was in the range of 10 to 30 mg/mI. For one, 3a-N-bis(2-chlomoethyl)cambamate(LS 451 ); dehydroepi the in vitro studies, the cytosol was diluted with a suitable androstemone,3$-N-bis(2-chloroethyl)carbamate (LS 452); es volume of TEK(O1)DG buffer. tradiol, 3,1 7$-bis[N-bis(2-chlomoethyl)carbamate](LS 453); es tmadiol,3-N-bis(2-chlomoethyl)cambamate,17$-tnimethyl acetate Scintillation Counting (LS 470); androstenediol, 3$-N-bis(2-chloroethyl)carbamate (LS 524); dihydrotestostemone, 17$-N- bis(2 - chlomoethyl) Scintillation counting was performed using a Packard Tn cambamate(LS 543); ethynylestradiol, 3-N-bis(2-chloroethyl)- CambModel 2650 liquid scintillation spectrometer on a Philips cambamate(LS 675); estniol, 3-N-bis(2-chloroethyl)cambamate PW 4510/00 liquid scintillation analyzer (N.y. Philips' Gloei (LS 161 1); estniol, 16a-acetate, 3-N-bis(2-chloroethyl)carba lampen-fabnieken, Eindhoven, The Netherlands). Both instru mate (LS 2035); and 17a-estmadiol, 3-N-bis(2-chloroethyl)- ments were equipped with an external standard for calculation cambamate(LS 2179). of dpm. Soluene-treated samples, however, were analyzed The estrogen sulfates used were synthesized at AB Leo, using the internal standard procedure, involving recounting of while estrogen glucumonidesand other unlabeled steroids were the samples following addition of tnitiated toluene. obtained from Sigma Chemical Co., St. Louis, Mo., as were Tris, dithiothmeitol, dithioerythnitol, charcoal (Nomit A), BSA, Calculations various proteases, RNase A, DNase I, and calf thymus DNA All calculations and curve constructions except for those in (type I, sodium salt). Cellulose (Cellex 410) was purchased Chart 7 were made by means of a Hewlett-Packard 9820A from BioRad Laboratories, Richmond, Calif., and purified ac calculator equipped with a Model 9862A calculator plotter cording to the method of Alberts and Hemnick(1) before use. (Hewlett-Packard Calculator Products Division, Loveland, Gelatin was bought from Difco Laboratories, Inc., Detroit, Mich. Cob.). EDTA and precoated aluminum sheets (aluminum oxide 60 F254,neutral, type E) for TLC were purchased from E. Merck In Vivo Studies AG, Dammstadt, West Germany. Chemicals were of analytical grade quality from E. Merck AG and BDH Chemicals Ltd., Distribution Studies. Two experiments were performed, one Poole, England. Sephadex LH-20, Sephadex G-25 Medium, with [3H]estmamustineand the other with [3H]estradiol. In each Blue Dextran 2000, and Dextran T 70 were purchased from experiment, 2 groups of animals (5 in each) were used, one Phammacia,Uppsala, Sweden. Ultrogel AcA-54 was obtained killed 30 mm and the other 2 hr after i.p. administration of the from LKB-Produkter AB, Bromma, Sweden. Combusto-Cones, tracer dissolved in acetone, 100 @zI/animalcontaining 35 @sCi Soluene, InstaGel, and tritiated toluene were bought from Pack (0.44 nmol; 0.8 @tg/kgbody weight) of [3Hjestramustine on 43 and Instrument Co., Inc., Downers Grove, Ill. @.tCi(0.47nmol; 0.5 @tg/kgbody weight) of [3H]estnadiol. The animals were bled to death under ether anesthesia. Various Animals tissues were dissected out and stored at _300 until an alyzed. Duplicate samples (100 to 500 jsg) of blood plasma Male Wistar rats, 8 to 10 weeks old, with an average body and dissected tissues, with the exception of the epiphysis, weight of 250 g, were used in all experiments. The animals pituitary gland, and thyroid gland, were dried in Combusto were orchiectomized 24 hr before the experiments. Cones for combustion by means of a Packard Tni-Carb Model Buffers 306 sample oxidizer. The recovery was checked by use of corresponding tissue samples from untreated animals contain The various buffers used were: TEK(01)DG buffer, TEK ing known amounts of [3Hjestmamustineon [3H]estnadiol. The (1 5)DG buffer, and TEK(40)DG buffer; and DCC buffer [0.05 entire epiphysis, pituitary gland, and thyroid gland were dis M Tnis-HCI, 0.001 M EDTA, 0.1 m@ dithioerythmitol, 10% (v/v) solved at 50Â°in 0.5 to 1 ml of Soluene and counted for glycerol, 0.1 % (w/v) gelatin, and 0.05% (w/v) Dextran T 70]. radioactivity after addition of 10 ml of lnstaGel/0.5 M HCI (9/ All buffers were adjusted to pH 7.2 to 7.4 at 25Â°. 1, v/v). For all experimental purposes, glass-distilled, deionized wa AnalysisofRadioactivityFoundInProstateCytosolfollow ten was used. ing Labeling in Vivo. Fourratsweregiveni.p. injectionsof 20 @.tCi(0.25nmol) of [3H]estnamustinein 100 @zlofacetone. Two Preparation of Cytosol hr later, the ventral prostate glands were removed, pooled, and Ventral prostate cytosol was prepared by homogenization of homogenized. The cytosol was prepared, and protein-bound the tissue in 3 to 10 volumes (w/v) of ice-cold TEK(O1)DG radioactivity was recovered by gel chromatography on a 1.4- buffer by means of an Ultra-TummaxtypeTP 18/2 homogenizer x 27-cm column of Ultrogel AcA-54 (separation range, 6,000 (Janke and Kunkel, K.G., Staufen im Breisqau, West Germany) to 70,000). Fractions of 0.5 ml were collected and measured at a setting of 34 to 36 for 6 periods of 5 sec with intervals of for absorption at 280 nm and for radioactivity. Radioactive

5156 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1979 American Association for Cancer Research. Estramustine in Rat Ventra! Prostate peaks were collected, and the radioactive ligands were char binding at different times was calculated. actenized by TLC after extraction of the aqueous phase with The remaining portion of each incubation mixture was used diethyl ether. After mixing with unlabeled estradiol, estrone, to examine the degradation rate of the estramustine-binding estramustine, and the nitrogen mustard derivative of estrone protein at the same temperatures as used for the association (LS 271 ), the samples were analyzed on aluminum oxide TLC and dissociation studies. At various times, duplicate samples sheets developed in chloroform/methanol (99/1 , v/v). Unla (200 @I)wereanalyzed for protein-bound radioactivity. Specif baled standards were run simultaneously on both sides of the ically bound [3H]estramustine was calculated as described samples and visualized by spraying with concentrated sulfuric above. acid/absolute ethanol (70/30, v/v) and drying at 110Â°for 5 EnzymaticDigestion.Theeffectof variousenzymesonthe mm. The sample lanes (2 cm wide) were divided into 1-cm [3H]estramustine/pmotein complex was examined. Samples of pieces, which were extracted for 20 mm with 1 ml of ethyl cytosol [0.5 ml, 10 ,.sgprotein, and 4 mg BSA per ml TEK(01 )DG acetate in counting vials and counted for radioactivity after buffer] were labeled at 0Â°for 18 hr with 0.5 nM [3H]estramus addition of 10 ml of InstaGel. tine. Following addition of 1 mg enzyme in 0.5 ml TEK(01)DG In Vitro Studies buffer to each sample, incubation was performed at 37Â°for 30 mm. After cooling on ice, a portion of each incubation mixture In all in vitro incubation studies except in case of the DNA was counted for total radioactivity, and the remaining part was binding experiments, the cytosol protein concentration used examined for protein-bound radioactivity by filtration through was 1 to 20 @sg/mI.Theincubationswere performed in the a Sephadex G-25 Medium column (0.8 x 12.5 cm) in presenceof 2 mgBSApenml or 0.1% (w/v) gelatinin orderto TEK(01)DG buffer using Blue Dextran 2000 as a marker. avoid adsorption of ligand or binding of protein to the glass Effect of Various Factors on the Binding of [3H]Estramus wall of the incubation tube. For each test sample, a background tine. The bindingof [3H]estmamustinebyprostatecytosol was sample without cytosol was nunand connectedfor. examined between pH 4 and pH 9. Cytosol samples prepared Separation of Free and Bound Ligand. In the in vitro expem in TEK(01)DG buffer were diluted with 2 volumes of 0. 1 M iments,the DCCadsorptiontechniquewas usuallyappliedto acetate buffer (pH 3.6 to 5.6), 0.1 M phosphate buffer (pH 6.0 remove unbound and weakly bound ligand from the incubated to 7.6), or 0.1 MTnis-HCIbuffer (pH 7.4 to 8.8), giving samples cytosol sample. In this procedure, the incubated sample (200 with varying pH containing 10 @tgcytosolprotein and 2 mg BSA or 300 psI)was cooled (0Â°)and treated with 0.5 ml of ice-cold per ml. Incubation was performed at 15Â°for 18 hr with 200-s1 DCC suspension consisting of 1.5% charcoal (washed with samples in the presence of 10 nM [3Hjestramustine. Protein waterto removefines,dried at 120Â°for24 hn,and stored in a bound ligand was determined by the DCC technique. desiccator) in DCC buffer. Following addition of the DCC sus Samples (200 @tl)containing10 to 20 @sgcytosolprotein and pension to the sample, the mixture was vortexed for 1 sec and 2 mg BSA per ml were also incubated with 1 to 5 nM [3H]- stored for 20 mm at 0Â°.After centnifugation at 3000 x g for 20 estramustine in the presence of varying amounts of N-bmomo mm at 0â€”4Â°,a200- or [email protected] of the supemnatant, succinimide, p-hydroxymercunibenzoate, dithiothneitol, abso containing the DCC-nesistant estramustine/protein complex, lute ethanol, and KCI. wascountedfor radioactivity. LigandSpecificityandBindingCharacteristicsofthe Es Rate of Association.Themateofassociationbetweenthe tramustine-binding Protein. The specificity of the estramus estramustine-binding protein and [3Hjestramustine was studied tine-binding protein was studied by incubation of duplicate at 5 temperatures(0Â°,15Â°,22Â°,30Â°,and37Â°)bymeasuring samples [200 j@l,1.6 zg cytosol protein pen ml TEK(01 )DG the amount of DCC-nesistantligand/protein complex formed at buffer containing 0.1 % gelatin] with 10 nM [3H]estmamustinein different times following incubation of duplicate samples of the presence of various unlabeled steroids and steroid deniva cytosol[200 ,@I,10 @.tgprotein,and 2 mg BSA per ml TEK(01)DG tives in concentrations ranging from 1 nM to 45 @.tM.After18 hr buffer] with 10 nM[3H]estramustine. No corrections were made at 15Â°,the bound radioactivity was determined by DCC treat for rates of degradation or dissociation. ment. The result was expressed as percentage of the amount Ratesof DIssociationandDegradation.Cytosolsamples of radioactivity bound in the absence of unlabeled competitor [10 ,sg protein and 2 mg BSA per ml TEK(01)DG buffer] were and plotted following logit transformation against the logarithm incubated with 5 nM [3H]estramustine for 18 hr at 15Â°in the of the ratio between unlabeled competitor and labeled marker absence or presence of a 250-fold excess of unlabeled estra according to the method of Rodband et a!. (36). The resulting mustine. Each incubation mixture was divided into 2 equal displacement curve was considered parallel to the standard portions. One portion of each kind was used to study the curve of estramustine when p > 0.05 when tested on the dissociation rate of the estramustine/protein complex. In order assumption of nonparallelism. â€˜â€˜Parallelism'â€˜wasconsidered to to removeunbound ligand, these portions were treated with represent competitive inhibition of [3Hjestramustine binding. DCC suspension (1 % charcoal) for 5 mm at 0Â°and centrifuged Comparison between the different concentrations required for at 12,000 x g for 5 mmat 2Â°.Thesupernatantswerechecked 50% inhibition of [3H]estramustine binding was made only in for radioactivity (zero time sample), and unlabeled estramustine case of curves parallel to the standard curve. Nonpanallelism (in absolute ethanol) was added to a final concentration of (p< 0.05)wasinterpretedasthepresenceofdifferentbinding approximately I 0 @sMinorder to inhibit reassociation of disso sites on the protein for the competing compound and estra ciated [3H]estramustine. The decrease of bound radioactivity mustine and/or that the displacing effect was caused by other was followedat the sametemperaturesas usedin the associ factors than pure competition for the estramustine-binding site. ation study. By subtracting the nonspecific binding of [3H]- Maximal specific binding and apparent dissociation constant estramustine (i.e. , binding in the presence of a 250-fold excess were calculated according to the method of Scatchard (37) of ligand)from the total binding of[3H]estnamustine, the specific correcting for nonspecific binding according to the method of

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1979 American Association for Cancer Research. B. Forsgren et a!. Chamness and McGuime(4). Binding to DNA-cellulose. The interaction of DNA with the 30 â€”491 Â±194 A estramustine/cytosol protein complex was studied using DNA

cellulose synthesized according to the method of Alberts and w

Hemmick(1) by means of calf thymus DNA. Samples [1 ml, 8 to tI) I-. 10 mg cytosol protein per ml TEK(01 )DG buffer] were incu @ 20 bated for 18 hr at 0Â°with 1 to 10 nM [3H]estnamustine or w Lfl [3H]dihydrotestostenone in the absence or presence of a 100- 0 0 fold excess of unlabeled estramustine or dihydnotestostenone. U.0 Portions (5 ml) of DNA-cellulose suspension containing ap

proximately 1200 @gofDNA per g of cellulose (dry weight) w @ 10 were used to prepare columns with a bed volume of about 1.5 ml. After the columns were washed overnight with TEK(15)DG a. buffer, the incubation mixtures were applied on top of the columns. When the samples had been adsorbed on to the DNA-cellulose, the flow was stopped. The columns were incu bated for 2 hr at 0Â°and washed with 10 ml TEK(15)DG buffer Pa Ep Lu Li AG SV CG BC VP Ki Pi PG SG Sp TG BP SM at a mateof 4 to 5 mI/hr. The columns were eluted with 7.5 ml TEK(40)DG buffer containing 0.2 mg BSA pen ml. Samples were also run on columns of pure cellulose to evaluate the 30 background binding of radioactivity.

LU RESULTS

In Vivo Studies 0 20 LU Distribution Studies. The results from the studies on the 8 U. tissue distribution of [3H]estmamustineand [3H]estnadiol are 0 shown in Chart 1. A clean difference is seen in the distnibu tion patterns of the 2 compounds and/or their metabolites. LU 10 Thirty mm following administration of the tracer, the pancreas, a. :D epiphysis, lung, adrenal gland, seminal vesicle, and brain con tex showed a higher uptake of estramustine than of estradiol. Some of these organs, such as the lung, adrenal gland, and seminal vesicle, also retained estramustine more efficiently than they retained estradiol at 2 hr. Four tissues showed increased concentrations of estramustine at 2 hr compared to Chart 1. Uptake of [3H]estramustineand [3H)estradiolin various tissues of the male rat. The results are expressed as percentage of dose Â±S.E. (bars) (n = 5) 30 mm: ventral prostate; preputial gland; submaxillary gland; per g tissue, wet weight, 30 mm (white columns) and 2 hr (hatched columns) and thyroid gland. Although the uptake of estradiol by the after administration of 0.44 nmol [3H)estramustine(A) on0.47 nmol [3Hjestnadiol (B) to onchiectomized rats. The tissues examined were (left to right) pancreas ventral prostate at 30 mm was as high as the uptake of (Pa),epiphysis(Ep),lung(Lu),liven(Li),adrenalgland(AG),seminalvesicle(Stâ€•), estnamustine, the concentration of estradiol in the same tissue coagulating gland (cG), brain cortex (BC), ventral prostate (VP), kidney (Ki), at 2 hr was insignificant. These results indicate that the rat pituitary gland (Pi), preputial gland (PG), submaxillary gland (SG), spleen (Sp), ventral prostate is capable of accumulating estramustine but thyroid gland (TG), blood plasma (Br'), and skeletal muscle (SM). not estradiol. created a sharp maximum in bound radioactivity following Protein-boundMetabolitesof Estramustinein ProstateCy incubation for 10 mm. At 30Â°,decreased binding was observed tosol. Chromatography on Ultrogel AcA-54 of prostate cytosol after 2 hr. The association rate decreased with decreasing recovered from mats2 hr following administration of [3Hjestra temperature. Equilibrium was reached at 22Â°after 2 hr, at 15Â° mustine showed a major radioactive peak eluted at a position after 8 to 12 hr, and at 0Â°after 20 hr. At 0Â°,however, only corresponding to a molecular weight of 40,000 to 50,000 25% of the maximal binding was achieved. For practical nea (Chart 2). The fractions from 19.5 ml to 32.5 ml were pooled sons, cytosol labeling was carried out at 15Â°for 18 hr in most and analyzed by TLC. The main radioactivity had a mobility experiments. corresponding to that of estramustine. A smaller amount of Ratesof Dissociation and Degradation.The rate of disso radioactivity travelled as the estramustine-congenen LS 271 ciation of the estramustine/protein complex increased rapidly (Chart 3). with temperature, with half-lives of about 21 0, 80, 9, and 4 mm In Vitro Studies at 15Â°,22Â°,30Â°,and37Â°,respectively (Chart 5). The half-lives and dissociation rate constants are summarized in Table 1. At Rate of Association. The associationrate of [3Hjestnamus 0Â°,no dissociation was found even after 24 hr. Since the tine with the prostate cytosol protein was very high at 37Â° dissociation rates were high compared to the degradation rates (Chart 4) and almost as high at 30Â°.At 37Â°,however, the (see below), no corrections were made for the latter. The estramustine/protein complex was rapidly degraded, which estramustine-binding protein in rat ventral prostate cytosol

5158 CANCER RESEARCH VOL. 39

1500- -33

1000- -02 '

Q 500 -01 - o

10 20 30 1.0 12 16 ELUTION VOLUME(ml] INCUBATION TIME (hr) Chart 2. Chromatography of [3H]estramustine-labeled prostate cytosol on Ul- Chart 4. Rate of association between [3H]estramustine and the binding protein trogel AcA-54. Cytosol was prepared from ventral prostate glands 2 hr after in rat ventral prostate cytosol at various temperatures. The concentrations of administration of 0.25 nmol [3H]estramustine to each of 4 rats. The first UV- ligand and protein were 10 nM and 10 jig/ml, respectively. At each time, protein- absorbing peak at an elution volume of 16.5 ml corresponds to the exclusion bound 3H-ligand was determined by use of the DCC technique. Corrections for volume of the column. dissociation and degradation were not made.

100- E, LS 275 LS 271 400-

300-

Â£ 200-

100-

5 10 15 1 2 3 4 MOBILITY (cm) INCUBATIONTIME (hrl Chart 3. TLC analysis of radioactivity extracted from the main radioactive Chart 5. Rate of dissociation of [3H]estramustine/protein complex in rat ven peak eluted from the Ultrogel AcA-54 column (cf. Chart 2). The mobilities of tral prostate cytosol. The preformed complex was incubated in the presence of standards are indicated E2, estradiol; E,. estrone; LS 275, estramustine; LS271, 10 /ÃŒMunlabeled estramustine at various temperatures. At each time, protein- 17-dehydroestramustine. bound 3H-ligand was determined by use of the DCC technique. Correction for degradation of protein was not made. showed no sign of degradation after 24 hr at 0Â°and degraded with half-lives of about 80, 30, 6, and 1.5 hr at 15Â°,22Â°,30Â°, estramustine decreased by 50% when labeling of the cytosol and 37Â°,respectively (Chart 6; Table 1). was performed in the presence of 1 to 2 ITIMA/-bromosuccinim- Effects of pH, Enzymes, and Chemicals. Binding of estra ideorp-hydroxymercuribenzoate. Dithiothreitol also influenced mustine to rat ventral prostate cytosol occurred with a broad the binding of [3H]estramustine giving a 35% decrease at 5 rnw pH optimum between pH 7 and 8.5 with maximum binding at and a 55 to 60% decrease at 10 rriM. The binding was uninflu pH 7.8 (Chart 7). enced by 10% absolute ethanol or 0.6 M KCI. DNase I and RNase A had no significant effects on the Ligand Specificity of the Estramustine-binding Protein. binding of estramustine, whereas subtilopeptidase A com The efficiency of various steroids and steroid derivatives in pletely destroyed the estramustine/protein complex. Strepto- displacement of 10 nrvi[3H]estramustine from the estramustine- myceus griseus and pancreatic crude protease decreased the binding protein in ventral prostate cytosol (1.6 fig of protein binding by 85 and 45%, respectively. These results indicate per ml) is shown in Chart 8 and in Tables 2 and 3. that the estramustine-binding agent is a protein. Binding of [3H] Most of the tested estramustine congeners carrying the

DECEMBER 1979 5159

B. Forsgren et a!.

Table 1 7060U50403020U10 Rates of dissociation and degradation of the estramustine/protein complex in rat ventral prostate cytoso!

@ tl /2 and k values were6.Rate calculated from results shown in Charts 5 and 0 U

U. degradationTemperature of dissociation Rate of 0 (min')150 t,2 (mm) k_ (min') t,,2 (mm) k_ >- I- 210 0.003 5010 0.0001 > 22 80 0.009 1750 0.0004 U U 4 30 9 0.08 360 0.0019 0 U 37 4 0.16 85 0.0085 0 4 cc U U 0z 0 0 cc n 5 6 7 8 9 pH

Chart 7. Effect of pH on the binding of[3Hjestramustmneinrat ventral prostate cytosol. Cytosol samples (10 @gcytosolprotein per ml) were incubated with 10 mM [3H]estramustine at 15Â°for 18 hr at various pH values. Protein-bound 3H- ligand was determined by the DCC technique.

95 0 cc 90 z 0 U 80 @ 70 @6O

@ 40 @ 30 â€˜20

05 1 2 5 10 20 50 12 16 CONCCOMPETITOR/CONC[3H)LS 275 INCUBATION TIME (hr) 95 Chart 6. Rate of degradation of [3H]estramustine/protein complex in rat yen B tral prostate cytosol. After 18 hr at 15Â°,the samples containing 10 np@i[3H]- 90 estramustine (with or without a 250-fold excess of unlabeled estramustine) and 10 @gofcytosol protein per ml were further incubated at various temperatures. U 80 At each time, protein-bound 3H-Iigand was determined by use of the DCC @ 70 technique. @ 60 @5C nitrogen mustard group at position 3 gave rise to displacement (N40 @ curves â€œparallelâ€•(p> 0.05) to the estramustine standard 30 curve with concentrations for 50% inhibition ranging from 15 @r20

to 25 nM (estramustine) to 6 to 8 @.tM(estramustinephosphate) C @ (Chart 8). LS 271 (the estrone nitrogen mustard) and LS 2179 10 cc0 (the 17a-estmadiol,3-N-derivative) were as efficient competitors 5 05 1 2 5 10 100 1000 10000 as was estramustine. Substitution on the 0-ring of the 17$- CONCCOMPETITOR/CONCE3H1LS275 estradiol moiety diminished (LS 289, LS 358, LS 675) on Chant8. Competition of various steroid mustard derivatives for binding of virtually (LS 299, LS 161 1, LS 2035) or totally (LS 298, LS [3H)estramustinein rat ventral prostate cytosol. The resulting inhibitory curves 453, LS 470) abolished the affinity of the competitor for the are linearized by plotting logit V versus log X, where Y is the ratio between the estramustmne-bindingprotein. The saturated compound LS 451 amount of [3H]estramustinebound in the presence and the absence of unlabeled competitor, and X is the ratio between concentration (conc.) of unlabeled corn (the nitrogen mustard derivative of androstemone)was a weak petitonand concentration of [3Hjestnamustineincubated, respectively. Each point competitor, whereas the dehydnoepiandrostemoneand andro represents the mean of duplicate samples. For compound code number, see â€œMaterialsandMethods.â€•A,LS 275 (â€¢),LS271 (0), LS 289 (t), L5 675 (L, stenediol derivatives (LS 452 and LS 524, respectively) were and LS 358 (x). The curve of LS 2179 coincided with that of LS 275 and is not efficient competitors. Tables 2 and 3 show the relative binding drawn. B, LS 275 (â€¢),LS452 ED),LS 524 (A), LS 2035 (Es),L5 161 1 (C), L5 affinity calculated at 50% inhibition for the compounds giving 451 (x), and L5 299(0). displacement curves â€˜â€˜parallel'â€˜(p > 0.05) to that of estramus tine. mustine binding by more than 10% when added in 4500-fold When present in 4500-fold excess, progesterone, estrone, excess. pregnenobone, and androstenedione inhibited the binding of Binding Characteristics of the Estramustine-binding Pro [3H]estnamustine by 20 to 35% (Table 4). None of the other tein. Analysisofthe bindingcharacteristicsoftheestramustine steroids or steroid conjugates examined inhibited [3H]estna binding protein according to the method of Scatchard (37)

5160 CANCERRESEARCHVOL. 39

Table 2 Relative binding affinity of various estrogen nitrogen mustard derivatives for the estramustine-binding protein in rat ventral prostate Cytosol samples (200 @l,1.6 @gprotein pen ml) were incubated at 15Â°for 18 hr with 10 nM [3H]- estramustine in the presence of varying amounts (1 n@to 45 @M)ofunlabeled estrogen nitrogen mustard derivatives. Bound radioactivity was analyzed by use of the DCC technique described in â€œMaterialsand Methods.â€•For compounds giving curves not parallel (p < 0.05) with that of estramustine (L5 275), relative binding affinity (ABA) was not calculated.

RBA â€”Excessof estramustine required for 50% inhibition â€”Excessofcompetitorrequiredfor50%inhibition

Under the experimental conditions used, the concentration of estramustine required for 50% inhibition was 20 nM; i.e., 2-fold excess and saturation of the estramustine binding sites were accomplished at approxi mately 100 rn@estrarnustine (cf Chart 9). R1 (@@2@R3

C)CH2CH2\N C@0@'@ C)CH2CH2'

bind affinityL52710H1.16LS275OH A1A2A3Relative ing

0HH1.00LS289OCCH3HH0.77LS

299? OP(OH)2 0HH0.002LS

358OCCH2CH3 0HHNC8LS

470OCC(CH3)3 0HHNCLS

453OCN(CH2CH2CI)[email protected]

00.01LS2035OHHOCCH30.02LS2179HOHH1.00a

NC, not calculated. yielded a mean apparent Kdof about 17 nM[16.6 Â±2.6 (S.E.); this estramustine-binding agent was partially characterized in n = 7]. The mean value of the maximal binding capacity (Bmax) a previous paper as a macromolecule with a molecular weight was about 5 nmol/mg cytosol protein (4.6 Â±0.2; n = 7). A of 40,000 to 50,000 (9). representativeanalysisis shownin Chart9. The binding capacity of the estramustine-binding molecule Bindingof the Estramustine/Protein Complexto DNA-Cel is resistant to treatment with RNase on DNase but is reduced lulose. Practically no estramustine/protein complex was me or abolished following incubation with various proteases. These tamed by DNA-cellulose (Chart 10). In agreement with reported results indicate that the estramustine-bmndingmacromolecule results (27), however, the dihydnotestosterone/androgen me is a protein. Scatchard analysis of the binding data obtained ceptor complex was significantly retained on the DNA-cellulose from incubations of [3H]estmamustinewith prostatic cytosol column. suggests that the estramustine/protein complex has an appar ent Kd of 10 to 30 nM and that about 5 nmol of estnamustine DISCUSSION are bound pen mg of total cytosol protein. Assuming that the estramustine-bmndingprotein has one estramustine-binding site The presentinvestigationshowsthat the ventralprostateof per molecule and that the molecular weight of the protein is the rat accumulates estramustine more efficiently than estra 45,000, it can be calculated that the estramustine-binding diol. Furthermore, the retention of estramustine was more protein accounts for about 20% of the total protein content of significantin the prostatethan in most other tissues studied, the rat ventral prostatic cytosol. although the preputial gland, submaxillary gland, and thyroid The estramustine-binding protein in mat ventral prostate gland also showed retention of the drug. The most probable seems to be quite specific for estramustine and closely similar explanation for this capacity of the ventral prostate to accu compounds. None of the tested steroids lacking the nitrogen mulate estramustine is the occurrence in the gland of an agent mustard moiety inhibited the binding of estramustine by more that has a high capacity to bind the drug. As mentioned above, than 35% (progesterone in 4500-fold excess).

DECEMBER1979 5161

3Relative Table Table 4 binding affinity of some non-estrogen nitrogen mustard derivatives and Effect of various steroids and steroid conjugates on the binding of @HJ prostateForL5 298 for the estramustine-binding protein in rat ventral cytosolCytosol estramustine by rat ventral prostate further details,2.0 see legend to Table 18hr samples (200 @tl,1.6 ,cgprotein per ml) were incubated at 15Â°for 45MM)with 10 flM [3H)estramustinein the presence of varying amounts (1 flM to Relative withDCC.of unlabeled steroids and steroid conjugates. Following a treatment II binding aspercentagethe radioactivity bound in the presence of competitor was expressed R = â€”Câ€”N-â€”(CH2CH2CI)2 Leo no. affinity showsthe of radioactivity bound in the absence of competitor. The table i.e.,acytosol binding of [3H)estramustinein the presence of 45 @cMcompetitor, [3H]-estramustine.4500-fold excess of competitor in proportion to the concentration of Estrarnustine gave 50% inhibition in 2-foldexcess.Bound [3H]estramustlne(1 L5275 1.00 setCompetitor 0 nM) (% of control R0@Q@@ 100)No (45 RM) to 100Progesteronecompetitor 65Estrone 70Pregnenolone 75Androstenedione 80Dehydnoepiandrostenone 901 >90Dihydnotestostenone7$-Estradiol LS452 0.23 >90Testosterone >901 R0@ >905a-Androstane-3$,9-Nortestosterone >90Corticosterone 17$-diol >90Cortisol >90Dexamethasone >90Diethylstilbestrol >90Ethynylestradiol >90Estniol >90Estnone LS524 0.16 >901 sulfate >9017f1-Estnadiol, 3-sulfate R0@ >90Estrone7$-Estradiol, 3,1 7-disulfate >901 glucuronide 7$-Estradiol, 17-glucuronide >90

PD L:I:@I@:@@ LS 451 0.007

C (\1S (I)

0z :a HOX@I@@'@ LS298 NC8 0 cci

8 NC, not calculated.

The results presented in this study and those reported earlier Chart 9. Saturation and Scatchard analysis of bindin9 13H]estnamustlnetorat (8, 9, 17) clearly distinguish the estramustine-binding protein ventral prostate cytosol. Each point represents the mean of duplicate samples. from the androgen receptor that occurs in rat ventral prostate The Scatchand plot (inset) shows the specific binding after connectionaccording to the method of Chamnessand McGuire (4). MaxImal specific binding was 8 np.i cytosol. This receptor has a molecular weight of 270,000 to giving 5 nmol pen mg protein (1.6 @gcytosolprotein per ml incubate) with an 280,000 (25) and a sedimentation coefficient of 7 to 85 (25). apparent K8 of 13 nM (r â€”â€”0.943). Furthermore, the androgen receptor has ligand specificity quite different from that of the estramustmne-binding protein (22). estramustine corresponding to that of matventral prostate cy Also, the estramustine/protein complex does not bind to DNA tosol could be shown in rat plasma (9), indicating that the cellulose, whereas such binding occurs in the case of all steroid estramustine-binding protein does not originate from blood. receptors (26). Another characteristic of steroid receptors is Following i.v. injection of [3H]estnamustine, Appelgmenet a!. their low tissue concentration (in the range of fmol/mg cytosol (2), using an autoradiognaphic technique, found a time-de protein) which contrasts to the high tissue concentration of the pendent transport of radioactivity from the epithelial cells into estramustine-binding protein. the luminal secretory content of rat ventral prostate. This result The apparent Kd of the estramustine-binding protein (10 to indicates that the estramustine-binding protein is a secretory 30 nM), is of the same order of magnitude as the Kd's of the protein formed in the prostate cell and transported into the steroid-binding plasma proteins (41). However, no binding of lumen of the prostate Iobulus. The function of this major protein

5162 CANCERRESEARCHVOL. 39

ventral prostate. z [3H] LS 275 [3HJDHT w The presence of a major estramustine-binding protein in the matventral prostate raises the question whether a similar protein cr0 20 occurs in the human prostate. If so, this would provide an a, E explanation for the efficiency of Estracyt in the treatment of

0 prostatic carcinoma; the estramustine-bmnding protein would E concentrate estramustine from the blood and provide the tissue z with large amounts of the drug. Studies are now in progress to < 10 CD investigate the possible existence of estramustine-bmndingpro @1 tein in normal, hyperplastic, and carcinomatous human pros

C,) tate. z ACKNOWLEDGMENTS :@ 0 @ r We are indebted to Per Bjbrk, Ulf Jbnsson, RoIf Kant, and Birthe Ljungberg abcd abcd for their skilled experimental assistance; to Inger Persson for expert animal operations; to AndensAhrent for excellent preparations of charts: and to Ingrid Chart 10. Binding of [3Hjestnamustine and [3Hjdihydrotestostenone (DHT)/ Ohrstnbmfor invaluable secretarial assistance. protein complexes to DNA-cellulose. [3HJLS275/protein and [3H]dihydrotestos terone/protein complexes were prepared from rat ventral prostate cytosol as descrIbed In â€œMaterialsandMethods.â€•About100 fmol oflabeled ligand/protein REFERENCES complex were applied on each column. The radioactivity that eluted with TEK(40)DGbufferbut not withthe washing TEK(15)DGbufferwas regarded as 1. Alberts, B., and Herrick, G. DNA-cellulose chromatography. Methods En retained on the column (27). Retention was measured after chromatography on zymol., 21: 198â€”217,1971. cellulose (Column a) and DNA-cellulose (Columns b to d) following incubation of 2. Appelgren, L.-E., Forsgnen, B., Gustafsson, J.-A., Pousette, A. . and Hog the tnltlated Ilgand in the absence (Columns a and b) or presence of a 100-fold berg, B. Autonadiographic studies of 3H-estramustine in the rat ventral excess of unlabeled LS 275 (Column c) ondihydrotestosterone (Column d). prostate. Acta Pharmacol. Toxicol., 43: 368â€”374,1978. 3. Baulieu, E.-E., and Jung, I. A prostatic cytosol receptor. Biochem. Biophys. Res. Commun., 38: 599-606, 1970. can only be speculated upon, but its quantitative importance 4, Chamness, G. C., and McGuire, W. L. Scatchard plots: common errors in makes it reasonableto assumethat the protein may be an correction and interpretation. 5teroids, 26: 538â€”542,1975. essential component of the seminal fluid. The capacity of the 5. Fang, S., Anderson, K. M., and Liao, S. Receptor proteins for androgens: on the role of specific proteins in selective retention of 17a-hydroxy-5a- protein to bind estramustine and its close congenems with high androstan-3-one by rat ventral prostate in vivo and in vitro. J. Biol. Chem., affinity and high capacity may be only a fortuitous phenomenon 244: 6584-6595, 1969. 6. Fang, S., and Liao, S. Dihydrotestostenone binding by androphilic proteins but may also indicate that the protein has a ligand-binding site of natventral prostate. Fed. Proc., 28: 846, 1969. that binds a naturally occurring compound. 7. Fang, S., and Liao, S. Androgen receptors: steroid- and tissue-specific Severalreports havebeen publishedon the bindingof ste retentionofa 17a-hydroxy-5a-androstan-3-one-proteincomplexbythecell nuclei of ventral prostate. J. Biol. Chem., 246: 16â€”24,1971. roids to rat ventral prostate cytosol proteins sedimenting at 3 8. Fonsgren, B., and HOgberg, B. Binding of a nitrogen mustard derivative of to 58 and with molecular weights of 45,000 to 70,000 (3, 5â€”7, estradiol to rat ventral prostate gland and cytosol. Res. Steroids, 7: 431 â€” 25, 26, 28, 39, 40). Karsznia et a!. (1 9) in 1969 reported the 433, 1977. 9. Forsgren, B., Hogberg, B., Gustafsson, j.-A, and Pousette, A. Binding of presence in rat ventral prostate of a pregnenolone- and pro estramustine, a nitrogen mustard derivative of estnadiol-17@6,incytosol from gesterone-binding protein sedimenting at 3.55 and with a rat ventral prostate. Acta Pharm. Suec., 15; 23â€”32,1978. molecular weight of 45,000 to 50,000. Simultaneously, Liao et 10. FossÃ¡,S. 0., FossÃ¡,J., and Aakvaag, A. Hormone changes in patients with prostatic carcinoma during treatment with estramustine phosphate. J. Urol., a!. (23) reported on a dihydnotestostenone-binding protein des 118: 1013â€”1018,1977. ignated as â€œa-protein'â€˜inrat ventral prostate cytosol sedi 11. Gustafson, A., Nilsson, S., Persson, B., Tisell, L.-E., Wiklund, 0., and Ohlson, R. Treatmentwith oral estramustine phosphate (Estracyt) in prostatic menting at 3 to 3.55 that was distinct from the androgen carcinoma.Invest.Urol.,15:220â€”224,1977. receptor (â€œfl-protein'â€˜).Recently,Heyns et a!. (12â€”14)have 12. Heyns, W., and De Moor, P. Prostatic binding protein: a steroid-binding described a protein in rat ventral prostate cytosol with â€˜â€˜peculiar protein secreted by rat prostate. Eur. J. Biochem., 78: 221â€”230,1977. 13. Heyns, W., Peetens,B., Mous, J., Rombauts, W., and De Moor, P. Purification steroid-binding properties' â€˜andwith a molecular weight of and characterisation of prostatic binding protein and its subunits. Eur. J. 51,000 and a seÃ§iimentationcoefficient of 3.75. The steroids Biochem., 89: 181-186, 1978. that were bound with highest affinity were pregnenobone and 14. Heyns, W., Van Damme, B., and De Moor. P. Secretion of prostatic binding protein by rat ventral prostate: influence of age and androgen. Endocrinol androstenedione. It was calculated that this protein accounted ogy, 103: 1090â€”1095,1978. for about 15% of the total protein in prostate cytosol. A high 15. Heisaeter, P. A. Incorporation of 3H-thymidine into nat ventral prostate in organ culture: influence of hormone-cytostatic complexes. Invest. Urol., 12: concentrationof the proteinwas found in prostaticfluid, mdi 479-489, 1975. cating that the protein is secreted by the prostate. Lea et a!. 16. Heisaeter, P. A. Incorporation of 3H-thymidine and â€˜4C-aminoacidsinto the (21 ) have also reported on the isolation and characterization of ventral prostate after in vivo treatment with estradiol-3N-bis(2- chloroethyl)carbamate-1 7/3-phosphate (Estracyt) and its estrogen and cy a protein constituting 20 to 30% of the total protein in mat tostatic parts. Invest. Urol., 14: 85â€”92,1976. ventral prostate cytosol and with a molecular weight of 46,000. 17. H@isaeter,P.A. In vitro binding of tritiated hormone-cytostatic complexes in This protein bound dihydrotestostenone with low affinity and the cytosol of various rat tissues and the incorporation of these complexes into nuclei. Scand. J. Urol. Nephnol., 11: 135â€”141, 1977. high capacity. It was localized in the lumen and epithelium of 18. Jonsson, G., Hogberg, B., and Nilsson, T. Treatment of advanced prostatic the prostate lobuli and was also found in high concentration in carcinoma with estramustine phosphate (EstracytÂ®).Scand. J. Unol. Ne seminal plasma, indicating that it is secreted by the prostate. phrol., 11: 231â€”238,1977. 19. Kansznia, R., Wyss, R. H., LeRoy Heinrichs, W., and Herrmann, W. L. The similarities between the estramustmne-bmndingproteinand Binding of pregnenolone and progesterone by prostatic â€˜â€˜receptor'â€˜proteins. the steroid-binding proteins described above are conspicuous. Endocrinology, 84: 1238-1 246, 1969. 20. Kaer, T. B., Nilsson, T., and Madsen, P. 0. Effect of estramustine phosphate Work must now be carried out to establish the relationship on plasma testosterone during treatment of carcinoma of prostate. Urology, between these quantitatively important constituents of the rat 5:802â€”804,1975.

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21. Lea, 0. A., Petnusz,P., and French, F. S. Isolation and characterization of Pharmacoclinical study of oral estramustine phosphate (Estracyt) in ad prostatein: a major secretory protein of rat ventral prostate. Fed. Proc., 36: vanced carcinoma of the prostate. Invest. Urol., 12: 65â€”68,1974. 780, 1977. 32. MUntzing,J., Varkarakis, M. J., Yamanaka,H., Murphy, G. P., and Sandberg, 22. Liao, S., Liang, T., Fang, S., CastaÃ±eda,E.,and Shao, T.-C. Steroid structure A. A. Studies of antiprostatic agents in the baboon (38204). Proc. Soc. Exp. and androgen activity: specificities involved in the receptor binding and Biol. Med., 146: 849â€”854,1974. nuclear retention of various androgens. J. Biol. Chem., 248: 6154â€”6162, 33. Nilsson, T., and MUntzing,J. Histochemical and biochemical investigation of 1973. advanced prostatic carcinoma treated with estramustine phosphate, Estra 23. Liao, S., Tymoczko, J. L., Liang, T., Anderson, K. M., and Fang, S. Androgen cytÂ®.Scand.J. Urol. Nephrol., 7: 18â€”22,1973. Receptors: 17fJ-hydroxy-5a-androstan-3-one and the translocation of a 34. Plym Forshell, G., MÃ¼ntzing,J.,Ek, A., Lindstedt, E., and Dencker, H. The cytoplasmic protein to cell nuclei in prostate. Adv. Biosci., 7: 155â€”163, absorption, metabolism, and excretion of Estracyt (NSC 891 99) in patients 1971. with prostatic cancer. Invest. Unol., 14: 128â€”131, 1976. 24. Lowry, 0. H., Rosebnough, N. J., Farr, A. L., and Randall, R. J. Protein 35. Plym Forshell, G., and Nilsson, H. The distribution of radioactivity after measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265â€”275, administration of labelled estramustine phosphate (EstracytÂ®),estradlol 1951. 17$-phosphate, and estradiol to rats (abstract). Acta Pharmacol. Toxicol., 25. Mainwaning, W. I. P. A soluble androgen receptor in the cytoplasm of rat 35(Suppl. 1): 28, 1974. prostate. J. Endocrinol., 45: 531â€”541,1969. 36. Rodbard, D., Rayford, P. L., Cooper, J. A., and Ross, 0. T. Statistical quality 26. Mainwaring, W. I. P., and Irving, R. The use of deoxyribonucleic acid control of nadioimmunoassays.J. Clin. Endocrinol. Metab., 28: 1412â€”1418, cellulose chromatography and isoelectric focusing for the characterization 1968. and partial purification of steroid-receptor complexes. Biochem. J., 134: 37. Scatchard, G. The attractions of proteins for small molecules and ions. Ann. 113â€”127,1973. N. V. Acad. Sd., 51: 660â€”672,1949. 27. Mainwaning, W. I. P., and Mangan, F. R. The specific binding of steroid 38. Smolev, J. K., Heston, W. D. W., Scott, W. W., and Coffey, D. S. Character receptor complexes to DNA: evidence from androgen receptors in rat ization of the Dunning R3327H prostatic adenocarcinoma: an appropriate prostate. Adv. Biosci. 7: 165â€”177,1971. animal model for prostatic cancer. Cancer Treat. Rep., 61: 273â€”287,1977. 28. Mainwaring, W. I. P., and Peterken, B. M. A reconstituted cell-free system 39, Unhjem, 0. Metabolization and binding of oestradiol-1 7/@by rat ventral for the specific transfer of steroid-receptor complexes into nuclear chromatin prostate in vitro. Res. Steroids, 4: 139â€”143,1970. isolated from rat ventral prostate gland. Biochem. J., 125: 285â€”295,1971. 40. Unhjem, 0., Tveten, K. J., and Aakvaag, A. Preliminary characterization of 29. McMillin, J. M., Seal, U. S., and Doe, R. P. Effect of oral estramustine an androgen-macromoleculan complex from the rat ventral prostate. Acta phosphate on pituitary, gonadal, and adrenal function in the green monkey Endocrinol., 62: 153â€”164,1969. (Cercopithecus aethiops sabaeus). Invest. Unol., 15: 151â€”154,1977. 41. Westphal, U. Steroid-protein interactions. Monogn.Endocrinol., 4:213â€”214, 30. MUntzing, J., Kindani, R. V., Saroff, J., Murphy, G. P., and Sandberg, A. A. 367, 1971. Inhibitory effects of Estracyt on R-3327 rat prostatic carcinoma. Urology, 42. Yamanaka, H., Shimazaki, J., lmai, K., Sugiyama, V., and Shida, K. Effect of 10: 439â€”445,1977. Estracyt on the rat prostate. Invest. Urol., 14: 400â€”404,1977. 31. MUntzing, J., Shukla, S. K., Chu, T. M., Mittelman, A., and Murphy, G. P.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1979 American Association for Cancer Research. Binding Characteristics of a Major Protein in Rat Ventral Prostate Cytosol That Interacts with Estramustine, a Nitrogen Mustard Derivative of 17β-Estradiol

Björn Forsgren, Jan-Åke Gustafsson, Åke Pousette, et al.

Cancer Res 1979;39:5155-5164.

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