Investigation of Hormone-Receptor Interactions by Means of Fluorescence Labeling1

Home , Equilenin, Estrone acetate, Estrone benzoate

[CANCER RESEARCH 38, 4212-4224, November 1978] 0008-5472/78/0038-OOOOW2.00 Investigation of Hormone-Receptor Interactions by Means of Fluorescence Labeling1

Walter B. Dandliker, R. James Brawn,2 Mao-Lin Hsu, Peter N. Brawn, Jacques Levin, Cal Y. Meyers, and Vera M. Kolb

Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037 [W. B. D., R. J. B., M. L. H.Â¡;Department of Pathology, University of California, San Diego, La Jolla, California 92037 [P. N. B.Â¡;Burroughs Corporation, Fort Lauderdale, Florida 33302 [J. L.]; and Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, Illinois 62901 [C. Y. M., V. M. K.Â¡

Abstract of sequelae specific for each hormone results. These in clude smooth muscle contraction, change in metabolic rate Fluorescent-labeled hormones can be used to study and rates of protein synthesis, mobilization of specific hormone-receptor interactions by means of fluorescence metabolites, and the secretion of enzymes, other hor polarization, visualization by fluorescence microscopy, or mones, or specific effector molecules. An elucidation of the separation methods, e.g., dextran-coated charcoal. Sub- primary interaction at the molecular level between hormone cellular fragments, single cells, and tissue preparations and receptor underlies our ability to understand normal are amenable to study; in this work rat uterine cytosol hormonal control mechanisms, to understand their patho was used unless otherwise noted. logical derangements, and to devise methods for assessing Estrone labeled with fluorescein at position 17 gives and modulating hormonal action. 50% inhibition in the radiometrie dextran-coated charcoal Much of what we now know about hormone-receptor assay at 8.3 x 10 M as compared to 3.4 and 3.5 x 10 " M interaction has evolved from studies with radiolabeled hor for diethylstilbestrol and estradiol, respectively. mones. These have proved useful for studies at the level of Scatchard plots from fluorescence polarization are hy the whole animal, organs, tissues, single cells, and subcel perbolic and consistent with two classes of binding sites lular fractions. A new tool, the fluorescent-labeled hor having association constants 5.6 x 10'" and 6.4 x 10" M '. mone, complements the radiolabeled hormone in many Binding by high-affinity sites, which were present at about types of experiments and enables certain measurements to 3 times the concentration of "specific" sites (radiometrie be made with greater speed and accuracy than possible dextran-coated charcoal assay), was abrogated by estra with radiolabels. More importantly, fluorescent hormones diol or diethylstilbestrol. open up experimental approaches that afford types of Kinetic measurements showed that binding sites that information not hitherto accessible (19). can be blocked by excess estradiol or diethylstilbestrol In this paper we report on (a) the types of experiments on are those that are both slowly associating and slowly hormone-receptor interactions that are facilitated by fluo dissociating. rescence labeling and how these experiments can be inter Staining of tissues by estrone labeled with fluorescein preted so as to yield extensive physicochemical insight into at position 17 as seen in the fluorescence microscope the nature of the interaction, (o) our progress in under showed specificity. In normal rat uterus only epithelial standing molecular structural features that influence the cells were stained. In one human infiltrating ductal carci strength and specificity of binding to estrogen receptors in noma only the malignant ductoid elements stained, while uterine cytosol, (c) the synthesis of a fluorescent estrogen in another there was essentially no staining. (17-FE)3 that binds considerably more strongly to cytosol receptors than did derivatives that we synthesized earlier, Introduction and (d) what has been learned of the estrogen-receptor interaction when 17-FE is used in conjunction with homog- Hormone action provides an important mechanism for communication between different organs and cell types; it enates, cells, and tissue sections. constitutes an important aspect of central nervous system control over the entire organism. The primary step in the Materials and Methods action of hormones at the effector cell is a highly specific, reversible binding of the hormone molecule to a receptor Estradiol, estrone, and several substituted derivatives were obtained from Steraloids, Wilton, N. H. 11^-Hydrox- molecule; the presence or absence of receptor for a partic yestradiol and 11/3-hydroxyestrone were gifts from G. D. ular hormone determines the capability of a cell to respond to that hormone, and the amount of receptor may determine Bearle and Co., Chicago, III. Frozen rabbit uterine tissue quantitatively the magnitude of any graded response. was purchased from Pel Freeze, Rogers, Ark., and held at -80Â° until use. After primary binding has occurred, a diverse spectrum Buffers. Buffers used are: 0.01 M Tris-HCI:0.0015 M tet- 1 Presented at the John E. Fogarty International Center Conference on Hormones and Cancer, March 29 to 31, 1978. Bethesda, Md. Supported by Research Contract N01-CB-43905 from the National Cancer Institute, Re 3The abbreviations used are: 17-FE, estrone labeled with fluorescein in search Grant GB-31611 from the National Science Foundation, and PHS position 17; DTE, dithioerythritol; DTT, dithiothreitol; BSA, bovine serum Research Grant No. 23980 from the National Institute of General Medical albumin; DCC, dextran-coated charcoal; RA, relative binding activity; THF, tetrahydrofuran; TLC, thin-layer chromatography; 6-FE, estrone labeled with Sciences. 2 Present address: University of Oregon Medical Center, Portland, Oreg. fluorescein in position 6; DES, diethylstilbestrol.

4212 CANCER RESEARCH VOL. 38

rasodium EDTA:0.001 M NaN,:0.001 M DTE or DTT, pH 7.6; initial value and was evaluated graphically. This simple 0.01 M Tris-HCI:0.0015 M tetrasodium EDTA:0.001 M measure of binding activity rather than some derived pa NaN3:0.001 M DTE:0.4 M KCI:0.05% BSA, pH 7.6; 0.01 M rameter such as the association constant has been used Tris-HCI:0.0015 M tetrosodium EDTA:0.001 M NaN3:0.001 M because of the complications in interpreting results of DTE, pH 7.6, to which 0.01% rabbit -y-globulin purified by separation methods (3). ammonium sulfate precipitation or by DEAE-cellulose chro- Fluorometric DCC Assay. This assay is similar to the matography was added. radiometrie assay except that fluorescent-labeled hor Culture Medium. Hanks' balanced salt solution (10 ml; mones instead of radiolabeled ones are utilized. In a typical 10x ; Flow Laboratories, Rockville, Md.) without phenol red procedure a mixture of 1 ml of cytosol, 100 /il of 17-FE (6 or sodium bicarbonate was mixed with 2 ml of 50x amino x 10~8M), and 100 //.I of 0.01 M Tris-HCI:0.0015 M tetraso acids (Flow Laboratories), 1 mi of 200 rriM glutamine (Flow dium EDTA:0.001 M NaN3:0.4 M KCI:0.001 M DTT:0.01% rabbit -y-globulin buffer, pH 7.6, or of estradiol (6 x 10~6M) Laboratories), 0.1 ml of 10% BSA, 0.1 g of dextrose, and distilled water to about 90 ml. The pH was adjusted to 7.4 was incubated overnight (12 to 16 hr) at 4Â°.Ablank (1 ml of with 1 M NaHCO3, and water was added to 100 ml. The cytosol and 200 Â¿Â¿Iofbuffer) and a control for total fluores solution was sterilized by filtration and stored at -20Â°.Cell cence (1 ml of cytosol and 100 /Â¿Iofbuffer) were incubated viability by trypan blue dye exclusion was 90% or more after similarly. Isolab DCC columns were prerinsed with 0.5 ml of 24 hr storage at 2Â°inthis medium. buffer, and each sample was passed through a separate Preparation of Cytosol. The method used follows that of column and followed by a 0.5-ml buffer rinse. In the sample for total fluorescence only, 100 /Â¿'of 17-FE (6 x 10~8 M) Puca ef al. (27) with minor modification. Uterine tissue (100 g) stored frozen at -80Â°was held at -20Â° overnight. The were added to the column effluent. The volume of each frozen mass was wrapped in Mylar film and covered with effluent was then made up to 5 ml, and the fluorescence nylon tricot. The entire mass was broken with a hammer intensities were measured. and homogenized in 220 ml of 0.01 M Tris-HCI:0.0015 M Measurements of Binding Equilibrium. Scatchard plots tetrasodium EDTA:0.001 M NaN3:0.001 M DTE buffer, pH of hormone-receptor binding data obtained by radiolabel- 7.6, in a Waring Blendor surrounded by ice with three 30- ing methods are found usually to be linear, although many sec intervals of grinding separated by 30-sec intervals of data in the literature are over a very restricted range of cooling. The temperature never rose above 2Â°.Thehomog- bound to free and hence cannot be regarded as exacting enate was centrifuged (100 min at 100,000 x g), and the tests of binding site uniformity. The plots are normally clear supernatant fluid (200 ml) was drawn off with a syringe obtained after DCC separation or sucrose density gradient and needle avoiding aspiration of the fatty material at the centrifugation and represent a best compromise for elimi meniscus. Sufficient 4 M KCI was then added to bring the nating most of the low-affinity binding while retaining most KCI concentration to 0.4 M. The solution was flushed with of the highest-affinity binding. nitrogen and stored at 2Â°.Protein content was determined Scatchard plots from fluorescence polarization titrations by the method of Lowry ef al. (20) as modified by Geiger show all the binding sites (high and low affinity). The result and Bessman (12). Cytosol prepared in this way is stable for ing plot is highly curved (concave upward) and cannot be perhaps 1 month or more as measured by the radiometrie interpreted in the usual simple ways. The problem of how DCC method; stability of cytosol has been noted by others best to treat curved Scatchard plots has been considered by occasionally (21). many workers in the past (9, 15, 29, 30), and a variety of Radiometrie DCC Assay. Inhibitors to be tested were approaches have been developed. Basically, all result in dissolved in ethanol at 2.4 x 10~3M. For this concentration fitting theoretical curves for a limited number of different 10 /Â¿Iwereevaporated under N2and redissolved in 2 ml of kinds of binding sites present at different concentrations to 0.01 M Tris-HCI:0.0015 M tetrasodium EDTA:0.001 M the experimental data. Recently (7), it has been shown NaN3:0.4 M KCI:0.001 M DTT:0.05% BSA, DTT + BSA buffer, theoretically that if only 2 classes of sites are present the pH 7.6, which was used throughout the assay. Dilutions resulting Scatchard plot is a hyperbola. This fact enables were made in 3.16-fold steps to give final inhibitor concen one to evaluate readily on a sound theoretical basis both trations in the incubation with cytosol ranging from 10~sto the association constants and the site concentrations from 10 8 M or, for estradici itself, from 10~7to 10 9 M. Portions the experimental curve. Knowing these parameters then (100 /il) of the several concentrations of the inhibitor were enables computation of the entire theoretical Scatchard mixed with 100 Â¿Â¿Iof[3H]estradiol (New England Nuclear, plot for comparison with experimental results. The basis for Boston, Mass.; 3.4 x 10~8M), followed by 1 ml of cytosol. this computation is given in the "Appendix" (Equations The mixture was incubated for 18 to 20 hr at 4Â°and then 3a, 4a, and 5a). passed through a DCC column (QS-1; Isolab, Inc., Akron, The data for the utilization of these equations are ob Ohio), rinsed through with 300 /Â¿Iofbuffer, and collected in tained from the following type of experiment. Cytosol is a scintillation vial. Biofluor (New England Nuclear), 16 ml, diluted 10- to 100-fold in 0.01 M Tris-HCI:0.0015 M tetraso was added, and the mixture was counted. Total uptake with dium EDTA:0.001 M NaN3:0.4 M KCI:0.001 M DTT:0.01% no inhibitor was about one-half of the radiolabel added. rabbit y-globulin buffer, pH 7.6, to a final volume of 3 ml. The percentage of total uptake was calculated for each Up to 15 /Â¿.Iofappropriate dilutions of estradiol or other point with inhibitor, and results were plotted on a log inhibitor together with up to 15 /il of fluorescent-labeled molarity scale. RA as defined by Korenman (16) is the ratio estrogen are added; after mixing and equilibration for 30 of the molar concentration of estradiol to other substances min or more at room temperature, the fluorescence polar required to reduce [3H]estradiol binding to one-half of its izations and intensities are measured. Stock solutions

NOVEMBER 1978 4213

(â€”103 M) of labeled or unlabeled hormones are made in but 0.2 ml of the supernatant fluid was withdrawn. The ethanol; subsÃ©quent dilutions are made in buffer to keep sedimented cells were suspended in the remaining fluid by the final ethanol concentration very low. vortex mixing; 25 /Â¿\were transferred to a slide and covered Determination of Dissociation Rate Constants by Direct with a coverglass for viewing or photography in the fluores Observation of the Back Reaction (Dilution Jump). Infor cence microscope. Two different instruments were used; mation on the dissociation rate constant, k,,, can be ob one (Zeiss) is fitted with an excitation filter transmitting tained by monitoring the rates of dissociation of preformed from 270 to 480 nm and a barrier filter peaked at 500 nm, complexes following rapid dilution (dilution jump). Depend and the second (Leitz) utilizes interference filters peaked at ing upon the rate constants involved, the jump may have to 490 and 520 nm. Photomicrographs were usually made at be very fast (a few msec); for slow reactions a slower jump x160. Exposure times varied from 1 to 8 min with Ekta made by manual mixing may be adequate. Attention must chrome 200. also be given to the dilution factor which is limited to Preparation of 17-FE. A solution of 3.00 g of estrone in perhaps 103 by practical considerations. The greater the 111 ml of warm THF, 5 ml of hydrazine hydrate, and 0.5 ml dilution factor the less important the recombination reac of 12 M HCI was evaporated to near dryness on a hot plate. tion will be. This enables the dissociation reaction to go The residue was dissolved in 50 ml of THF, and the solution nearer to completion before equilibrium effects become was diluted with 250 ml of water. Crystals that formed were significant. Investigation of a large fraction of the dissocia isolated by filtration, washed with 800 ml of water, and tion reaction is important in revealing any heterogeneity in dried. This product was dissolved in 80 ml of hot THF, 3 ml binding affinities. of hydrazine hydrate were added, and the solution was In the dilution jump technique, the dissociation of com refluxed for 3 min. Addition of 250 ml of water again plexes differing in stability is partially resolved in time. If induced crystal formation yielding 3.10 g after being the dissociation is first order, as would be expected for washed with water and dried. A portion of this product, unimolecular reactions, then one-half of the complex pres 1.56 g, was washed successively with 22 ml of 1,2-dime- ent at time zero will have disappeared by the half-time thoxyethane, 25 ml of chloroform, and 15 ml of 95% regardless of its initial concentration. Thus, with time the ethanol. The residue, 1.35 g, was recrystallized from 55 ml reaction mixture becomes relatively enriched in the more of hot THF, providing 0.58 g of estrone hydrazone which stable complexes that can be observed after the weaker decomposed without melting when heated above 250Â°.IR complexes have largely disappeared. After initial formation (Nujol): 1661 cm ', medium intensity, C=N stretch (1, 2) of complexes, a DCC separation may be advantageous. (the characteristic estrone, C=O stretch at 1709 cm 1, was This step removes free fluorescent hormone as well as any absent) and 3400 to 3000 cm 1, broad, intense, NH and OH bound to rapidly dissociating sites. In this way sensitivity is stretch (1, 2). Nuclear magnetic resonance (dimethyl sulf- increased and initial readings, extrapolated to zero time, oxide-d(Â¡): fi 0.80, sharp singlet, angular CH:1 (the related allow quantification of the total fluorescent hormone bound singlet at 8 0.85 of estrone was missing). to high-affinity sites. The total concentration of high-affinity sites as well as the parameter pb, the polarization of com Estrone hydrazone (C,BH.MN2O) Calculated: C 76.02, H 8.51, N 9.85 pletely bound fluorescent hormone, are needed to interpret Found: C 75.12, H 8.59, N 8.90 the dissociation curve itself. An experimental way to simu late the effect of very large dilution factors is to determine To a stirred solution of 0.28 g (1 mmol) of this hydrazone the behavior at several different dilutions and then to make in 19 ml of warm THF, a solution of 0.39 g (1 mmol) of an empirical extrapolation to infinite dilution. Equations for fluorescein isothiocyanate, Isomer I (Sigma Chemical Co., the interpretation of dilution jump data are given in the St. Louis, Mo.), in 20 ml of warm THF was added dropwise. "Appendix"; cr. especially Equations 7a, 10a, 13a, 14a, and This solution stood at 25Â°for 2 hr; 0.67 ml of concentrated 35a to 39a. ammonium hydroxide was then added, and the mixture was Cell Staining by Fluorescent Hormones. Tissues, fresh allowed to stand for an additional 4 hr. The solvent was or formalin fixed, were sectioned with a cryostat micro removed in a vacuum leaving 0.73 g of dry residue. This tome. Contiguous tissues were embedded in paraffin, sec product was dissolved in 3 ml of dimethylformamide and tioned, and stained with hematoxylin and eosin for standard fractionated by TLC on ethanol-washed No. 5766 plates (E. histopathological analysis. Suspensions containing single Merck, Elmsford, N. Y.) in a developing mixture of chloro cells and cell clusters were prepared from fresh or frozen form :95% ethanol (7:1, v/v). tissue by the techniques of Lasfargues and Ozzello (17) or Three distinct fluorescent bands (RK 0.07, 0.25, and 0.55) Vaage (34). Tissue sections were stained on slides by resulted, and the material in the center band gave a large incubation in 40 ml of culture medium containing appropri increase in fluorescence polarization when added to a ate concentrations of fluorescent hormones with or without dilution of uterine cytosol. This band (RK 0.25) combined unlabeled hormone. In some instances the section was from several plates was eluted with 95% ethanol; 0.5 ml of preincubated with unlabeled hormone before labeled hor 5 M acetic acid and 10 ml of water were added, and the mone was added. After staining, the slides were drained solvents were removed in a vacuum leaving 0.094 g (0.14 and wiped, and the sections were protected with a cover- mmol, 14% of theory) of 17-FE, a yellow crystalline solid. glass. For cell suspensions 200 Â¿ilwere added to 9.8 ml of For measurements of binding to uterine cytosol, a small hormone solution, and the mixture was incubated at room amount of this material was further purified by TLC in temperature for 30 min with occasional swirling. The sus chloroform:ethanol (7:1) and then in benzene:ethyl acetate pensions were centrifuged (400 x g, 10 min, 22Â°),and all (7:3; RF ~0). This additional purification was a precaution

4214 CANCER RESEARCH VOL. 38

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1978 American Association for Cancer Research. Fluorescent-labeled Hormones to avoid the possibility of minor contamination by estrone, estradiol, DES, and 17a-ethynylestradiol. The pattern of estradici, or estrone hydrazone. specificity shown indicates that the binding observed is due Estrone hydrazone hydrate can also be coupled to fluo- to cellular receptors and not to serum binding proteins (14). rescein isothiocyanate. This gives rise to various chroma- The RA (16) for 17-FE from Chart 2 is found to be 0.03 to tographically separable fluorescent products one of which 0.05. is identical with 17-FE with regard to Chromatographie Table 2 provides a summary of the effects of changing behavior and receptor binding activity. For elemental anal one substituent at a time on the estradiol or estrone mole ysis this product was purified by TLC, 2 times in chloro- cule. The RA's are taken from Table 1 which is intended to form:ethanol (7:1) and once in benzene:ethyl acetate (7:3). be a complete listing of all structures with RA > 0.01. These data include our own and many values from the literature. Other structures with RA < 0.01 should nevertheless be Calculated: C 69.52, H 5.24, N 6.24, S 4.75 Found: C 64.36, H 5.73, N 5.36, S 4.74, ash 2.64 mentioned. The doisynolic and dehydrodoisynolic acids such as fenocylin (23) and the 16,16-dichlorodoisynolic It was reported (5) that estrone hydrazone, prepared by acids (22) are known to be active as estrogens in vivo. heating estrone with excess hydrazine hydrate in ethanol, However, we have found that neither the 3-hydroxy nor the melts at 244-246Â°.We prepared presumably the same ma 3-methoxy forms of these compounds show more than a terial but have characterized it as estrone hydrazone hy modicum of receptor-binding activity in vitro. We have drate. Preparation of the hydrazone has also been reported found the following RA's in this series: 3-hydroxyfenocylin, by others (28), but no characterization was published. In -0.02; 16,16-dichlorodoisynolic acid, -0.004; 3-methoxy- our preparation of the hydrate, a solution of 0.135 g (0.5 16,16-dichlorodoisynolic acid, <0.001 ; 16,16-dibromodo- mmol) of estrone and 0.100 g (2.0 mmol) of hydrazine isynolic acid, 0.01 and 3-methoxy-16,16-dibromodoisynolic hydrate in 4 ml of 95% ethanol, 4 ml of THF, and 1 drop of acid, <0.001. The low activity of the 3-methyl ethers is 12 N HCI afforded, after 10 hr at 25Â°,amass of white needle perhaps not surprising in view of the well-known low in clusters; dilution of the filtrate with water provided an vitro activity of estradiol-3-methyl ether and the full in vivo additional batch of needles. The total crystalline product, activity due to metabolic demethylation. However, the low thoroughly washed with water and dried under high vacuum in vitro activity of the free phenols is surprising and is being (25Â°,12hr), melted at 245-247Â°(with decomposition, orange investigated. The dibromodoisynolic acids have been only oil, gas evolution) and weighed 0.150 g, a quantitative yield recently prepared,4 and no in vivo results are as yet avail calculated for the hydrazone hydrate. TLC (benzene:ethyl able. Several other simple derivatives of estradiol or estrone acetate, 7:3) gave a single spot the RKof which was quite had remarkably low RA's: estradiol-6-iminooxyacetic acid, different from that of estrone. IR and nuclear magnetic <0.003; 11/y-hydroxyestrone, 0.001; estrone-17-iminooxy- resonance were essentially identical with those given for acetic acid, 0.001. estrone hydrazone. Fluorescent-labeled hormones are readily adapted to per forming assays analogous to those with radiolabels. The Estrone hydrazone (C1BH,., N._,O) results of a fluorometric DCC assay can be expressed in Calculated:C 76.02, H 8.51 ,N 9.85 terms of fluorescence intensities (measured as photon counts/min from the sample). In a determination of cytosol Estrone hydrazone hydrate (C,K H2I1N,O2) in which the total fluorescence present was 6.37 x 105, the total uptake (present in the DCC column effluent) was 3.10 Calculated :C 71 .49, H 8.67, N 9.26 x 10s while that present after inhibition with a 100-fold excess of estradiol was 1.57 x 105. The magnitude of the Found:C 71.88,H 8.74,N 8.78 cytosol blank alone was 2.11 x 105. These results indicate Results the presence of 210 fmol of specific sites per mg of cytosol protein as compared to 171 fmol per mg by radiolabeling. We have prepared several fluorescent-labeled estrogens The receptor binding of 17-FE is further illustrated by and have investigated, by means of radiolabeling and fluo Chart 3. Here the results of a fluorescence polarization rescence methods, their binding to rabbit uterine cytosol titration are shown as a Scatchard plot extending over a receptors. Structures of 3 of these compounds are shown wide range of concentrations. The plot is highly curved but in Chart 1. The first of these to be prepared (6-FE) binds is accurately fitted by a hyperbola (Equation 3a). This fit is with high specificity to antiestradiol antibody (8) but only consistent with the presence of only 2 classes of binding weakly to estradiol receptors. Insertion of a 5-carbon spacer sites, 1 and 2, with K, = 5.6 x 10'Â°ivr1 at a concentration of improves the binding to receptors but still results in a RA 5.6 x 10-" M and K2 = 6.4 x 107 NT1at a concentration of well under 0.01. 1.5 x 10~8M. Chart 3 also demonstrates that only the high- Attachment of the fluorescein label to position 17 of affinity sites can be demonstrably inhibited by preincuba- estrone yields a more highly specific compound, 17-FE. The tion with estradiol or DES. results presented in this paper deal with the properties and The results of dilution jump measurements on complexes binding specificity of 17-FE examined in a number of ways. preformed between 17-FE and cytosol receptors are shown Our methodology illustrates the types of data made acces sible by fluorescent-labeled hormones. In Chart 2 the ability of 17-FE to inhibit the binding of â€¢¿C.Y. Meyers and V. M. Kolb. a,a-Dibromination-Cleavage of Camphor, Estrenes, and Estradiols into Gem-dibromo Carboxylic Acids with the [3H]estradiol to cytosol receptors is compared to that of CBrCls-KOH-t-BuOH Reagent, manuscript in preparation.

NOVEMBER 1978 4215

6-FE WITH SPACER 6-FE I7-FE

Chart 1. Structures of fluorescent estro gens. Lett, 6-FE. 9

cent staining observed was determined on unfixed sections IOO of immature rabbit uterus. Staining by 17-FE can be clearly seen after exposure of sections to concentrations as low as s 80 2 x 1CT9 M. At higher concentrations the intensity of I S staining increases markedly. The uptake is rapid and incu â€¢¿560 bation can be continued over a period of 3 hr with no further apparent change after about 15 min. The staining 40 observed at 5 x 10 Â»M17-FE was not noticeably inhibited as observed visually by the presence in the incubation mixture of 1.2 x 10~6 M estradiol or DES. 20 Since the potential utilization of formalin-fixed tissue could considerably increase the breadth of application of fluorescent hormone staining, the possible effects of for -log [inhibitor] malin were investigated. Thin sections were prepared from Chart 2. Relative binding activity of 17-FE as compared to estradiol, 17o- immature rat uterus either unfixed or fixed in neutral ethynylestradiol, and DES. The curves show the degree of inhibition of binding of [3H]estradiol to uterine cytosol as measured by the DCC assay. buffered 4% formalin (1% formaldehyde). The sections were Open versus filled symbols, determinations done on different days. O, â€¢¿. then stained for 30 min in 3.5 x 10 e M 17-FE and examined. estradiol; D. â€¢¿17-FE;A, DES; V, 17a-ethynylestradiol. Total cpm in system, 101,200; total uptake with no inhibitor, 43,100. Values of RA (estradiol = 1): In both cases the staining was highly localized to the 17a-ethynylestradiol, 1.9; DES, 1.2; 17-FE, 0.03 to 0.05. endometrial areas with a relative absence of staining in the myometrium. A series of such experiments at different in Chart 4. The slowly dissociating and hence tight com formalin concentrations revealed virtually no change in plexes evidenced by the smaller slope in the upper curve staining pattern up to 10% formalin. For further investiga are absent in the lower curve in which the receptor prepa tion of a different aspect of the possible effects of formalin, ration was preincubated with DES. An analysis of the curves uterine cytosol was treated with HCHO at room tempera utilizing Equations 36a through 39a is consistent with 2 ture, and after a time the receptors were precipitated with classes of sites of which the slowest to dissociate are also ammonium sulfate to remove the formaldehyde. The pre those inhibitable by DES. cipitated receptors were redissolved, and their specific The kinetics of the forward reaction between 17-FE and estrogen-binding activity was measured by [3H]estradiol up cytosol is shown in Chart 5. These data show that only the take against that of controls. In a typical experiment cytosol complexes slowly forming between 17-FE and cytosol are was brought to 4% formalin (1% HCHO) for 1 hr at room inhibitable by estradiol. temperature. After ammonium sulfate precipitation the re A different aspect of the binding specificity of 17-FE is ceptors were taken up in buffer containing DTE. Portions shown in Chart 6. Here the reaction between 17-FE and were incubated with 2.5 x 10~9 M [3H]estradiol either with antiestradiol-17-BSA is shown to be strongly inhibited by or without 2.5 x 10 7 M cold estradiol. After separation on estradiol in the pg/ml range. a DCC column, the differences in uptake with or without Staining by fluorescent hormone derivatives has been estradiol were found. This specific uptake after HCHO studied on various tissues under different methods of prep treatment varied between 20 and 40% of that for the un aration of sections and different staining conditions. The treated control. effect of concentration as related to the amount of fluores- The most characteristic feature of fluorescent hormone

4216 CANCER RESEARCH VOL. 38

Table 1 Table 2 Relative binding activities of estrogens and inhibitors (uterine Effects of substitution on receptor-binding activity in the estradiol cytosol) and estrone molecules Ref.17a-EthynylestradiolCompound RA" Species text.OHm^%^^^Aare from Table 1 or from the 1.91 14 Estrone acetate 1.91 14 17a-Ethynylestradiol 1.9 Rabbit This work Hexestrol 1.8" Calf 18 DES 1.5-2.9 14 DES 1.2 Rabbit This work (+)-17/3-Estradiol 1 (Standard) Estradiol 3-acetate 0.97 14 17-Epiestriol 0.90 14 17Â«-Butylestradiol 0.71 Rabbit This work 3-(4-Hydroxyphenyl)-4-(3- 0.70 13 azido-4- ring B ringringEstradiolC ring D hydroxyphenyljhexane )3-Acetate (1 16a-Estradiol 0.66 14 Estrone 0.47'' Calf 18 6-Keto (0.22) 110-Hydroxy 17a-Ethynyl 16-Epiestriol 0.44 14 (0.97)" (0.02) (1.9) Estrone acetate 0.37 Rabbit This work 3-Methyl 6-Dehydro 17a-Butyl Estrone propionate 0.32 Rabbit This work ether (0.03) (0.10) (0.71) Estrone 0.32 Rabbit This work 6-CMO''-urea 17-Acetate Estradiol 17-acetate 0.29 14 (-0.005) (0.29) 16tt-Hydroxyestrone 0.25 14 6-CMO 16Â«-Hydroxy Erythro-a-ethyl-a'-methyl- 0.24 Rabbit 16 (<0.003) (0.04-0.36) 4,4'-dihydroxybibenzyl 16-Keto (0.14, Equilin 0.24 14 0.05) 6-Ketoestradiol 0.22 Rabbit This work 17-Aceto Coumestrol 0.21 '' Calf 18 (0.13)Estrone Hexestrol 0.20-0.74 14 Dimethylstilbestrol 0.17 Rabbit 16 (-0.4)3-Acetate Estriol 0.16'' Calf 18 16-Ketoestradiol 0.14 14 11/3-Hydroxy 16u-Hydroxy 17-Acetoestradiol 0.13 Rabbit This work (1.9,0.37) -0.001) (0.25) Dimethylstilbestrol 0.11 14 3-Propionate 17-Fluores- Estradiol 3,1 7-diacetate 0.11 14 (0.32) ceinyl thio- 6-Dehydroestradiol 0.10 14 3-Hemisuc- semicaba- Estrone 0.10-0.66 14 cinate zone meso-Butestrol 0.09 14 (0.09) (0.03) Estrone hemisuccinate 0.09 Rabbit This work 3-Benzoate 17-Hydrazone 17a-Estradiol 0.08-0.49 14 (0.02)(0.02)" Equilenin 0.08 14 Numbers in parentheses, RA. 17Â«-Ethynylestradiol 3- 0.08 14 oxime.estrogen'' CMO, carboxymethyl methyl ether Mestranol 0.08 14 ClomipheneB 0.07fc Rabbit 31 receptor content were kindly supplied by Dr. 16-Ketoestradiol 0.05 Rabbit This work Francis Markland, Jr., University of Southern California, Estriol 0.04-0.36 14 Los Angeles, Calif.), one poorly differentiated and one well 17-FE 0.03 Rabbit This work differentiated, were compared as to their staining by 4 x (-)-17 0-Estradiol 0.03 14 10~eM 17-FE. The poorly differentiated tumor showed very Estradiol 3-methyl ether 0.03 14 I.C. 1.46, 474 0.027'' Rabbit 31 little staining in the malignant cell mass and even less Clomiphene (c/s isomer) 0.023 14 elsewhere except or a few intensely stained inflammatory 11/3-Hydroxyestradiol 0.02 Rabbit This work cells. In the well-differentiated tumor (shown in Fig. 1), the Estrone hydrazone 0.02 Rabbit This work Estrone benzoate 0.02 Rabbit This work staining was highly localized to the malignant ductoid 3-Hydroxyfenocylin 0.02 Rabbit This work elements. For comparison with conventional histological U 11,14" 100 0.01 analysis, a hematoxylin-eosin-stained, fixed section of con As defined by Korenman (16). tiguous tissue is also shown in Fig. 1. Cytosols from these publication.staining'' Estimated graphically from 2 breast carcinomas had been independently assayed for estrogen receptor content with standard assays. The poorly thus far observed is the marked localization of the differentiated tumor contained, per mg, 12.29 fmol of 4S staining to discrete areas. This localization evidences a type receptor and zero 8S receptor, while the well-differentiated of chemical specificity that was found in all tissues studied contained, per mg, 7.8 fmol of 4S and 1.5 fmol of 8S and with all fluorescent hormone preparations. In rat uterus receptor. either unfixed or formalin fixed, the staining by 17-FE is The staining of unfixed sections of immature rat uterus localized in epithelial cells. Formalin-fixed samples of 2 hu by several other fluorescent-labeled hormones was also man breast cancers, infiltrating ductal carcinomas (these investigated. With all of these hormones including 6-FE tissues together with the results of standard assays for theirRA's(Chart 1), 6-FE (thiosemicarbazone), estrone labeled with

NOVEMBER 1978 4217

275 4

220

I65

55 3 5 10 20 30 40 50 85 95 160 170 380 390 I0~3 x Seconds 10" x Fb Chart 4. Fluorescence polarization dilution jump curve showing the kinet Chart 3. Scatchard plot of fluorescence polarization titration data show ics of dissociation of complexes preformed between 17-FE and cytosol in ing binding of 17-FE (O) to uterine cytosol and inhibition of this binding in the presence (II) or absence (O) of DES. Cytosol protein (8.3 mg/ml) was the presence of 1.6 x 10 ' M DES (D); similar inhibition is produced by reacted with 3 x 10~7M 17-FE in a solution containing no DES; in a separate estradici. Ordinate, ratio of bound:free 17-FE; abscissa, molarity of bound experiment cytosol protein (8.3 mg/ml) previously incubated with 1.2 x 10 * 17-FE. The cytosol was diluted 75-fold so as to give a final concentration of M DES was also reacted with 17-FE. Under these conditions most of the 17- 0.11 mg protein per ml. In the upper curve, high-affinity sites are evidenced FE was bound to cytosol. Both solutions were incubated overnight at 4Â°and by the steep upward curvature at low values of F,,. In the lower curve, this then passed through a DCC column. A sample of the effluent was quickly high-affinity binding is shown to be abrogated in the presence of OES. The collected and diluted 1000-fold, and the polarization was monitored as a upper curve is a theoretical curve (hyperbola; Equation 3a) for only 2 types function of time. After dilution the final concentration of cytosol was 0.008 of binding sites, 1 and 2, K, = 5.6 x 10'Â°M ' at concentration 5.6 x 10 " M. mg protein per ml and 17-FE was 8 x 10 " M. The upper curve after dilution and K, = 6.4 x 10' M~' at concentration 1.5 x 10" M. The concentration of contained no DES, while the lower curve contained 10" M DES. The buffer high-affinity sites is 508 fmol per mg protein; receptor content by radiomet used was 0.01 M Tris HCI:0.0015 M tetrasodium EDTA:0.001 M NaN.,:0.001M rie DCC assay was 170 fmol of specific sites per mg protein. DTE:0.4 M KCI 0.01% rabbit y-globulm Receptor content by radiometrie DCC assay was 170 fmol of specific sites per mg cytosol protein. The dilution jump curves are consistent with the presence of 2 classes of sites present at different concentrations and dissociating at different rates: in the uninhibited fluorescein in position 19, and progesterone labeled in sample, Class 1 is present at 3.38 x 10 " M with k,, = 8.20 x 10 Vsec and position 3 the staining patterns were similar, and in all Class 2 is present at 1.77 x 10'" M with k,, = 2.89 x 10 Vsec: in the DES- inhibited sample, Class 1 is present at 2.75 x 10 " M sites with k., = 2.27 x cases the staining was highly localized to the endometrial 10'Vsec and Class 2 is present at 2.25 x 10 " M sites with kd = 3.17 x 10 V epithelial cells. sec. The very slowly dissociating sites present in the uninhibited sample are absent in the DES-inhibited sample. The analysis to obtain these parameters utilized Equations 14a to 16a and 35a to 39a. Data over only the first 1000 Discussion sec were utilized in order to avoid effects due to the approach to equilibrium at longer times. This paper delineates types of information regarding hormone-receptor interactions afforded by fluorescent-la (6-FE and 6-FE + 5-carbon spacer) are very much lower beled hormones. The interactions may be quantified at than that for 17-FE. The RA for 17-FE (-0.03) is sufficiently thermodynamic equilibrium, and rates of forward and back high to permit realistic, representative experiments to be ward reactions can be independently assessed. Measure carried out; much higher RA's are desirable. Some struc ments are made without the necessity of any separation tures have RA's exceeding 1, e.g., 17a-ethynylestradiol, step such as adsorption or sedimentation; if desired, how DES, hexestrol, and perhaps others. These seem to offer ever, such procedures can be used with fluorescent-labeled attractive possibilities for synthesizing fluorescent deriva hormones. Since only a simple optical measurement is tives that can mimic the binding properties of estradiol needed to obtain the bound:free ratio, measurements are closely. rapid and allow a time resolution not possible where a In establishing the activities of fluorescent hormone de separation step is involved. rivatives, a crucial point must be kept in mind. Assessment A crucial question in the application of fluorescent-la of activity by inhibition of [3H]estradiol binding alone is not beled hormones to receptor studies or assays concerns the sufficient clearly to establish activity of the fluorescent influence of the fluorescent label on the interaction being conjugate, especially if the RA is low (less than 2%). In measured. This influence may be expected, a priori, to take this situation a small amount of an active contaminant or a several forms. First, there may be decreased binding affinity small amount of degradation resulting in liberation of free, of the hormone; alternatively, binding may take place to unlabeled hormone could lead to an inflated estimate of the sites to which the native hormone is indifferent. All of these RA. In addition to [3H]estradiol-binding inhibition, direct possible effects can be experimentally evaluated and elimi measurements of binding to receptors utilizing fluores nated or minimized by appropriate variations in structure, cence methods are essential. Only in this way can assur namely, in the hormone or hormone competitor, in the ance be given that the entire dye:hormone complex acts as point of attachment of the fluorescent label, in the chemical a molecular unit and is indeed behaving like a fluorescent- nature of the bonds linking the dye to the hormone, and in labeled hormone. In addition, obvious controls with simple the nature of the fluorescent dye itself. dye derivatives are necessary to ensure that no binding of A beginning has been made in accumulating the new, these alone occurs. No detectable binding of fluorescein or basic information necessary to optimize these factors. The of A/-acetylfluoresceinamine to receptor preparations oc RA's of the first fluorescent derivatives that we synthesized curs even at concentrations 3 orders of magnitude higher

4218 CANCER RESEARCH VOL. 38

-SÂ«â€”K-B-r-o-0-0 er OI8

â€¢¿5OI6

6 9 OI4 IO"2 x Seconds Charts. Binding kinetics of 17-FE reacting with uterine cytosol in the presence (D) or absence (O) of DES as measured by fluorescence polariza tion. DES (1.6 x 10 ' M) was preincubated for 90 min at room temperature with diluted uterine cytosol (0.11 mg protein per ml) in 0.01 MTris-HCI:0.0015 OI2 M tetrasodium EDTA:0.001 M NaN.,:0.001 M DTE:0.4 M KCI:0.01% rabbit y- 0.2 03 0.4 globulin. A control solution containing no DES was made under similar conditions. 17-FE (4 x 10 " M)was added at zero time, and the fluorescence Estradici (ng/ml) polarization of each solution was monitored. In both cases the fluorescence Chart 6. Fluorescence polarization immunoassay for estradici with the polarization rises rapidly to about 0.21. From this point the solution contain use of 17-FE. For each point goat antibody to estradiol-17-BSA equivalent to ing DES undergoes no further change, while in the absence of DES the 0.22 ml of whole serum (Miles-Yeda, Rehovot, Israel) was diluted into polarization continues to rise further over a period of 25 min to about 0.25. This additional change of 0.04 polarization unit is due to the "specific" buffered NaCI solution (0.15 M NaCI. 0.01 M K..HPO,, 0.005 M KH.PO,. and 0.006 M NaN,) and incubated for 5 min with various final concentrations of binding of 17-FE to cytosol estrogen receptors. Receptor content by radio- estradici as indicated. 17-FE to a final concentration of 10 'Â°Mwas added, metric DCC assay was 170 fmol of specific sites per mg cytosol protein. and after 10 min the fluorescence polarization, p, was read. The polarization of 17-FE is 0.043 in buffer alone and 0.138 in normal goat serum. Most of the latter polarization is probably due to the binding of the estradici and than those used in our experiments. fluorescein moieties by serum albumin. Thus, similar experiments at a Another feature probably desirable in fluorescent hor constant level of 3.3 pig of ammonium sulfate-precipitated IgG per ml gave values of p and of bound:free, respectively, for added 17-FE as follows: mone derivatives is that no change in fluorescence intensity normal goat, 0.051 and 0.038; normal rabbit, 0.055 and 0.060; antiestradiol- would occur when combination with receptor takes place. 6-BSA. 0.064 and 0.111; antiestradiol-17-BSA, 0.213 and 5.03. Values of Constancy of intensity affords partial assurance that the bound:free were calculated from values of p utilizing Equation 41a in conjunction with the values of p, = 0.043, pâ€ž=0.245, and Q,/Qb = 0.96. dye moiety is remaining at its distance and not becoming involved in the reaction. If the dye is involved, almost certainly the binding characteristics of the hormone will be found (Chart 3) is about 3-fold that obtained by the DCC found to have been perturbed. method. This difference may be attributable to several The receptor binding activity of 17-FE has been estab factors such as dissociation of complexes during the DCC lished by both equilibrium and kinetic experiments. From separation, loss of receptor on DCC, or binding of 17-FE to these measurements (Charts 3 to 5), an important concept high-affinity nonreceptor sites; the relative importance of emerges. The high-affinity, slowly binding, and slowly dis these factors is not yet known. In Chart 3 it is evident from sociating sites in cytosol preparations are inhibited by values of bound :free that even in the presence of a large estradici or DES and are, by inference, the specific sites. excess of inhibitor (estradici or DES) a large fraction of the Whether or not the lower-affinity sites have any physiologi 17-FE present is still bound. The same is probably true for cal function is uncertain. estradici itself; the reason that this binding is not seen in Analysis of the Scatchard plot (Chart 3) shows that only 2 conventional measurements (e.g., by DCC separation) is classes of sites need be postulated completely to explain that these complexes are relatively rapidly dissociating, the data. If only 2 classes of binding sites are present, we hence disappear during the DCC separation, and can be have found (Equation 3a) that the theoretical Scatchard plot seen only at thermodynamic equilibrium. The validity of this is a hyperbola. This finding immediately provides a firm conclusion is supported by the results of a fluorometric theoretical basis for resolution of such plots by means of DCC separation with 17-FE. Before DCC separation the B:F the 2 asymptotes of the hyperbola, which according to ratio was 5.9 with 1.2 x 10~9 M 17-FE present. After Equations 4a and 5a give the association constants and separation the B:F ratio was 19.6 with 0.6 x 10~" M 17-FE binding site concentrations independently. The value found present. The initial value of 5.9 shows that only 14.6% of (Chart 3) for the association constant of the high-affinity the 17-FE was originally free, and yet the DCC was able to sites, K, = 5.6 x 1010M"1 present at a concentration of 508 remove one-half of the total fluorescence. The conclusion fmol/mg cytosol protein, is reasonable in the context of is that perhaps 35% of the original 17-FE present before what is known of the K for estradici itself, 1.3 to 1.5 x 10' DCC is bound up in rapidly dissociating complexes. The M~1for immature calf uterus at 4Â°(26),and 7.1 x 10sto 2 x well-known affinity of proteins such as serum albumin for 1010 at 0Â°(16). The binding of dihydrotestosterone by steroids makes it likely that estradici itself is also similarly cytosol from rat anterior hypophysis (33) was also found to bound. yield data consistent with 2 classes of sites as interpreted Kinetic measurements by the dilution jump technique by the method of Rosenthal (29). The site concentration (Chart 4) provide a powerful method to segregate high-

NOVEMBER 1978 4219

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1978 American Association for Cancer Research. W. B. Dandliker et al. affinity from low-affinity binding. After dilution jump all is true for 6-FE (8). The type of measurement shown in complexes begin to dissociate but with characteristic rate Chart 6 has an obvious practical value as a fluorescence constants and half-times. As a result, if the rate constants polarization immunoassay for estradiol. are quite different most of the rapidly dissociating com Perhaps the most promising and far-reaching application plexes will have disappeared after a time and the effects of of fluorescent-labeled hormones is in staining cells and slowly dissociating complexes can be observed independ tissues observed by fluorescence microscopy. In Fig. 1 the ently. The value of dilution jump measurements has been staining of human breast tumor tissue by conventional realized previously as, for example, by Gardner and Wittliff hematoxylin and eosin staining is compared to that pro (11), who obtained curves by the DCC method. The disso duced by 17-FE. The most important feature observed with ciation constants determined by the DCC method are in the 17-FE staining is the marked specificity involved; i.e., only same range as those determined from Chart 4. In the curves a few of the cells (the tumor cells) are stained. We have shown (Chart 4), the preformed complexes were passed observed analogous types of specificity with other speci through a DCC column before the dilution to remove mens of both tumor and normal tissue. For example, in complexes that dissociate in less than â€”¿1min.The initial normal immature rat uterine tissue, staining is most intense part of the dilution jump curve is indicative of the changes at epithelial cells. Because many factors may be involved in that proceed on an even faster scale during the DCC the staining observed, we cannot at present attribute the separation. If an analysis of the entire dissociation is re staining exclusively to any one, such as the localized quired, the DCC separation can be omitted. Dilution jump presence of estradiol receptors. Further work with a series analysis permits resolution of experimental curves so as to of fluorescent derivatives differing in their properties and yield concentrations and rate constants for each class of applied to cells and subcellular fractions of different types site present (Equations 35a to 39a). The difference seen in (4, 24, 25) will help to elucidate the significant parameters. inhibited versus uninhibited curves (Chart 4) makes it evi Such a series of derivatives may make it possible to deline dent that preincubation with DES eliminates slowly disso ate rapidly a hormonal sensitivity profile for a tissue under ciating complexes preferentially. investigation. This type of information may prove to be of Measurements on the forward rate of combination (Chart considerable value to the investigator and clinician alike. 5) show that the slowly dissociating sites are specifically inhibitable. Weak nonspecific combinations occur rapidly; APPENDIX at the specific sites the initial combination is probably followed by slow conformational rearrangements finally In this appendix derivations of the relationships necessary to interpret leading to a very tight combination. These phenomena are measurements of the binding equilibria and kinetics of hormone-receptor interactions are given. These relationships permit a quantitative interpreta strikingly similar to those occurring in antibody-hapten tion of the curved Scatchard plots and of dilution jump curves obtained on reactions with weakly binding versus strongly binding anti systems containing 2 classes of binding sites differing in affinity and/or concentration. The equations given here complement and extend those body (6). In fact, the entire concept of specificity as it published previously for simpler systems (7, 19). The general type of reaction applies to the immunochemical vis-Ã -visthe hormone area assumed in the treatment below is that in which a ligand .' binds to a can be discussed in very similar terms. The interaction of receptor A, to form reversibly a complex 3-9t: the small molecule with receptor or antibody owes its specificity to the favorable interaction between atomic groupings on the small molecule with complementary ones Measurements to characterize this type of reaction may be made on the equilibrium, on the forward rate (complex formation), or on the backward on the receptor. The kinds of bonds responsible for these rate (dissociation). interactions can be enumerated easily since the nature of Symbols Subscript indicating "bound" the groupings present is known. There can be electrostatic b charge interactions, hydrogen bonding, dipole-dipole inter e In kinetic equations, subscript indicating value at equilibrium F Molar concentration of ' actions, and hydrophobic interactions. These interactions Subscript indicating "free" f occur between numerous atomic groupings on the steroid The maximum value of F,,\taken to be equal to the total molar and those on the receptor (amino acid residues). The concentration of receptor sites steroid, being relatively rigid, can be imagined first to Similar to F6.maxexcept for 2 discrete classes (1 and 2) of binding sites interact at one site and then to settle slowly into place Second-order rate constant for the association reaction achieving a minimum of free energy as more and more k:, First-order rate constant for the dissociation reaction Kf.,l Association constants for 2 discrete classes (1 and 2) of groups come into play. The fact that some structures, like K, Â¿I receptor sites DES, hexestrol (32), and 17a-ethynylestradiol, seem to bind M The total molar concentration of f in both free and bound even more tightly than estradici implies that estradiol has forms o Subscript indicating value at zero time not evolved to make the tightest possible fit with receptor p The polarization of the excess fluorescence, i.e., p = (Av - and hence that the 3-dimensional electron density distribu Aft)/(Av + A/7), where Av and Ah are the intensities in arbitrary units of the components in the excess fluorescence tion of estradiol does not complement that of the receptor (above that of the blank) polarized in the vertical and quite as well as do other realizable structures. horizontal directions, respectively The ability of 17-FE to react with antibody against estra- 0 Molar fluorescence of a mixture of free and bound forms of 3- as they exist in a solution under observation, i.e., 0 = (Av diol-17-BSAand for this reaction to be inhibited by estradiol + AM/M is shown in Chart 6. There is a marked specificity deter ( Time mined by the point of attachment of the fluorescent dye; i.e., 17-FE reacts strongly with antiestradiol-17-BSA and Analysis of Scatchard Plots When 2 Types ot Binding Sites Are Present only weakly with antiestradiol-6-BSA, whereas the converse If 2 types of noninteracting receptor sites are present, separate mass law

4220. CANCER RESEARCH VOL. 38

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1978 American Association for Cancer Research. Fluorescent-labeled Hormones expressions can be written for each class of site: tion curve can be expressed as the sum of 2 exponentials (cf. Equation 10a): F,., + FM = F,.nâ€ž.1e-*'-" + Fj.^e-*Â«' (13a) da)

The following analysis provides a method for determining values of the and parameters Fs.mâ€ž,â€žFbM^Â¿,ka,,, and fc,,,2from the composite curve. For simplicity in notation express Equation 13a in the form (2a) y = Ae" + Be"' (14a)

The experimental data consist of pairs of values of (FM + F6i2)= and (F,,,, in which a and ÃŸare both negative. Solutions to this equation are found + Fti2)/F, â„¢¿8-Adding Equations 1a and 2a and substituting <Â¿>and0 gives: from measurements taken at different times, namely, 0, 7, 2T, and 47 where 47" is the longest time over which measurements are feasible. Let - f,,,,20,max., ,.â„¢,.., (3a) -(<<â€ž.,F6.mâ€ž,,+Kf.2FÃ,max.2)Â»=0 (15a)

This is the equation of a hyperbola with asymptotes and

Â»= K, ..,FÂ».mâ€ž.,- K,.,* (4a) (16a) A set of 3 linear relations between A and B at times i = 0, 7",and 27 can and then be written: Ã¶= Kt.2Fft.max.2- K, ,2* (5a) A + B = yâ€ž (17a) Hence evaluation of the slopes and intercepts of the 2 asymptotes gives K,..,, KF& Fs.max.i,and FÂ».max.!.separately. Au + Bv = yÂ¡ (18a)

Au' + Bv1 = y2 (19a) Analysis of Dilution Jump Measurements in which y,>,y,, and y2 correspond to times 0, 7, and 27, respectively. In this technique the reaction starts with complex, y.Ji, present only, and A and 8 are eliminated by setting the determinant of this set of equations the reaction is followed after dilution. The simplest case physically is that in equal to zero: which only a single kind of complex is present and in which both the dissociation and the association reactions are first order with respect to 1 1 yÂ» each concentration. Under these circumstances the rate equation for the u v y,|= 0 (20a) entire system may be written:

= MF,.mâ€ž-Ft)(F,)- (6a) which is the equation at dt (v - u){y2 - y,(v + u} + yauv} = 0. Integration of Equation 6a (10) yields the "integrated rate equation" (7a) (21a) which determines all concentrations for all t after any arbitrary dilution factor Since u * v (otherwise there would be only one exponential), Equation 21a for a system in which only one type of binding site is present. becomes:

y0u v - y,(u -I- v) + y2 = 0 (22a) In (F. - Ft.,)(F6.max) ' F..... - FÂ«,, (7a)

An alternative form is: (23a) â€”¿Ffif)(Fbjnax â€”¿F/) yÂ«YÂ« In ,.â„¢ - F,) - (F,.â„¢, - F,.,)]Ft.â€ž (8a) This is the equation of a hyperbola with asymptotes u = yÂ¡/yÂ«andv = yÂ¡/ya and intersections with the u and v axes at u = y,/y, and / = y2/y(. = *â€ž ft.max Fjif) In summary, the solution pair lu, i/ must satisfy Equation 23a as well as the following inequalities: Equation 7a may also be expressed as: 0< u < 1 (24a) t.^x(F/x + F,) - 0 < v < 1 ,., - F,) (9a) A second set of linear relations (similar to Equations 17a. 18a, and 19a) between A and ÃŸcan be obtained for times i = 0, 27, and 47: If the dilution is so large that the dissociation goes practically to comple tion, then Equations 7a to 9a take on their limiting first-order forms: A + B = yâ€ž (25a)

Au2 + Bv2 = y.. ,n^ (10a) (26a)

Au' ~ Bv* = yt (27a) In terms of polarization, p, Equation 10a becomes:

- p) + 0,(p - p,) In this case the determinant In 0/(p - p,) (11s 1 1 yÂ»\ u2 v2 y2J = 0 (28a) IfO, = OÂ»then: u' v' yj

(12a)

(v2 - u2){y< - y2(u2 + v2) + y^'v*} = 0 (29a) which permits the evaluation of ka from a linear plot. If instead of only one type of binding site, there are 2 types of sites which, Again since v * u Equation 29a becomes: however, do not mutually interact and if the dilution factor is practically infinite so that the association reaction can be neglected, then the dissocia- yâ€žu*v2- y-f(u2 + v2) + y, = 0 (30a)

NOVEMBER 1978 4221

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1978 American Association for Cancer Research. W. B. Dandiiker et al. or as estradici will give rise to virtually completely depolarized fluorescence; some polarization will be retained as molecular size increases. Considering 2 molecules of equal size, the fluorescence of the more asymmetrical rigid (31a) y yÂ» yÂ» structure will be more highly polarized. The essential feature of applying these phenomena to hormone binding Now u and v may be found as simultaneous solutions of Equations 23a consists in labeling the hormone molecule with a fluorescent label and and 31a. Substituting for convenience new coefficients observing the degree of polarization of the fluorescent light or, in certain cases, the intensity of fluorescent light when measured quantities of the m â€¢¿y./XÂ« (32a) labeled hormone and receptor are allowed to interact. The dependence of polarization (and also occasionally the fluorescent intensity) upon the extent 7)2= y.Jya (33a) of reaction between the hormone and binding site forms the basis for the quantification. Reaction between the hormone and binding site results in an TI<= yjyÂ« (34a) increase in size of the kinetic unit and in a retardation of the rotary brownian motion subsequently manifested as an increase in the polarization of After the variable, v. is eliminated, an equation in u alone is obtained: fluorescence. In the presence of added unlabeled hormone, a smaller percentage of the labeled hormone is bound to the binding site; in this (la ~ 112!"* + (2T)|2T)2 - 7)4 - 7)22)U2 + 27),(7)4 - 7)j2|U + 7)j3 - 7),27)4 = 0 (353) circumstance, the polarization observed will be lower. From the observed degree of inhibition, the binding characteristics of the native, unlabeled The variable v satisfies an identical equation (in which u is replaced by v}. hormone can be deduced. From measurements of polarization the final, Hence, the 2 roots of Equation 35a such that 0 < u < 1 give both u and v: derived parameters that can be obtained are (a) binding site concentration, i.e., u = uÂ¡and v = u.,. (b) degree of heterogeneity of the sites, (c) association constants for the Equation 35a may be solved by Newton s method using initial values such binding of the native, unlabeled hormone, and (d) forward and backward that: rate constants for binding, together with the form of the rate law. From the magnitude of these parameters and their dependence on temperature and composition of the medium, a definitive picture of the binding can be constructed. Basically, fluroescence polarization and intensity measurements are use ful because they provide a direct and rapid measure of the bound:free ratio:

F= 0,/p-pA (41a) F, 6 \ptt - p)

Once u and v have been found. Â«and ÃŸare determined from and

a = - log u (42a) (36a) F, 0-0Â«,

In order to utilize these equations, the constants Q/, Q6, p,, and pt must be determined for a particular system under study. No problem is posed in finding Q, and p,, since these come directly from a measurement on the ÃŸ=j\ogv (37a) labeled component alone. The determination of 0,, and p,,. however, implies measurements on a state in which the fluorescent-labeled material is com A and B are then found from pletely bound to its complementary partner. Since complete binding cannot be realized physically, an extrapolation is involved. If equilibrium values ofp plotted against M are extrapolated to M = 0, p approaches a limit, p'. Values (38a) ofp' for different receptor concentrations plotted against (p' - p,) divided Au + Bv = y, (39a) by receptor concentration in any arbitrary convenient units give p,, as the intercept of a straight line (7). Fluorescence Polarization Q,(p' - p,) P = Pt - (43a) The light emitted from fluorescent solutions is partially polarized; it consists of a mixture of linearly polarized and unpolarized light. The origin of this partial polarization and its implications concerning the kinetic unit This procedure makes it unnecessary to know absolute values of , carrying the fluorescent moiety can be seen from the following considera beforehand. A similar relationship facilitates the determination of Q,, tions. Classically, the emission from a single molecule may be regarded as ,, ,, 0,-0' radiation from a single oscillating dipole. This radiation has an oscillating (448) electric field parallel to the direction of oscillation of the dipole and is said to be polarized in the same direction. If a randomly oriented assembly of molecules is excited by fully polarized light, their fluorescence is only "partially" polarized, even if the molecules are prevented from rotary Acknowledgments brownian motion in solution. For simplicity assume that the directions of the The authors are indebted to Dr. Henry Rapoport, University of California, absorption and emission oscillators in a single molecule are the same and Berkeley, and to Dr. Charles Perrin, University of California. San Diego, for that they are rigidly fixed with respect to the geometric axis of the molecule. their helpful suggestions. Furthermore, assume the molecule rigidly fixed in position during the interval between absorption and emission (typically a few nsec). The proba bility of absorption of light is proportional to the square of the magnitude of References the component of the electric vector of the exciting light in the direction of 1. Bellamy, L. J. The Infra-red Spectra of Complex Molecules. London: the oscillator. If the absorption and emission oscillators are parallel, the emitted light will be partially polarized with a degree of polarization, p. This Methuen and Co., Ltd., 1958. quantity is defined in terms of intensities, /. polarized either parallel or 2. Bellamy, L. J. Advances in Infrared Group Frequencies. London: Me perpendicular to the incident electric field thuen and Co., Ltd.. 1968. 3. Blondeau, J. P., and Robel, P. Determination of Protein-Ligand Binding Constants at Equilibrium in Biological Samples. European J. Biochem , P = (40a) 55:375-384,1975. 4. Brecher, P. I., Vigersku, R., Wotiz, H. S., and Wotiz, H. H. An In Vitro System for the Binding of Estradici to Rat Uterine Nuclei. Steroids, 10: For randomly oriented molecules in a rigid medium, the maximum value 635-651, 1967. of p observed with linearly polarized light is one-half. If, instead of being 5. Cavallini, G., Massarini, E., Mazzucchi, F., and Ravenna, F. Some rigidly fixed, the molecules are subject to rotary brownian motion, the Hydrazides-Hydrazones and Isonicotinoylhydrazones, II. Farmaco Sci. molecular rotation taking place between the times of absorption and emis Tec. Pavia, 7: 397-404, 1952. sion may be expected to result in values of p lying between one-half and 6. Dandliker, W. B. Investigation of Immunochemical Reactions by Fluores zero. The extent of this rotation is a function of molecular dimensions and cence Polarization. Immunochem. Proteins, 7: 231-261, 1977. structure, solvent, and temperature. Low-molecular-weight compounds such 7. Dandliker. W. B.. Dandliker, J., Levison, S. A., Kelly, R. J., and Hicks. A.

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N. Fluorescence Methods for Measuring Reaction Equilibria and Kinet 21. Mester, J., Robertson, D. M., Feherty, P., and Kellie, A. E. Determination ics. Methods Enzymol., 48F: 380-415, 1978. of High-Affinity Oestrogen Receptor Sites in Uterine Supernatant Prepa 8. Dandliker, W. B., Hicks, A. N., Levison, S. A., and Brawn, R. J. rations. Biochem. J., 120: 831-836, 1970. Fluorescein-Labeled Estradiol: A Probe for Anti-Estradiol Antibody. Res. 22. Meyers, C. Y., and Kolb, V. M. Facile and Selective Chlorination- Commun. Chem. Pathol. Pharmacol., 18: 147-156, 1977. Cleavage of Some Cyclanones and Cyclanols with the CCr,-KOH-f-BuOH 9. Ekins, R., Newman, G. B., and O'Riordan, J. L. H. Theoretical Aspects Reagent. In situ Conversion of Estrones and Estradiols into Dichlorodo- of "Saturation" and Radioimmunoassay. Radioisotopes in Medicine: In isynolic Acids. J. Org. Chem., 43: 1985-1990, 1978. Vitro Studies. Division of Technical Information, U.S. Atomic Energy 23. Miescher, K. On Doisynolic Acids, a New Class of Estrogens. Chem. Commission. Oak Ridge Symposium, pp. 59-100, November 13, 1967. Rev., 43: 367-384, 1948. 10. Frost, A. F., and Pearson, R. G. Kinetics and Mechanism. Complex 24. Noteboom, W. D., and Gorski, J.Stereospecific Binding of Estrogens in Reactions, p. 187. New York: John Wiley & Sons, Inc., 1953. the Rat Uterus. Arch. Biochem. Biophys., 111: 559-568, 1965. 11. Gardner, D. G., and Wittliff, J. L. Specific Estrogen Receptors in the 25. Pietras, R. J., and Szego, C. M. Specific Binding Sites for Oestrogen at Lactating Mammary Gland of the Rat. Biochemistry, 12: 3090-3096, the Outer Surfaces of Isolated Endometrial Cells. Nature. 265: 69-72, 1973. 1977. 12. Geiger, P. J., and Bessman, S. P. Protein Determination by Lowry's 26. Puca, G. A., and Bresciani, F. Association Constant and Specificity of Method in the Presence of Sulfhydryl Reagents. Anal. Biochem., 49: Oestradiol-Receptor Interaction. Nature, 223: 745-747, 1969. 467-473,1972. 27. Puca, G. A., Ã‘ola, E., Sica, V., and Bresciani, F. Purification of Estrogen 13. Katzenellenbogen, J. A. Photoaffinity Labeling of Estrogen Receptors. Receptors. Methods Enzymol., 36: 331-365, 1975. Federation Proc., 37: 174-178,1978. 28. Robinson, C. H., and Finckenor, L. E. Estratriene Hydrazone Derivatives. 14. King, R. J. B., and Mainwarmg, W. I. P. Steroid-Cell Interactions. U.S. Patent 3,257,423, June 21, 1966, cited in Chem. Abstr.,65. 12263b, Oestrogens, pp. 216-218. Baltimore: University Park Press, 1974. 1966. 15. Klotz, I. M., and Hunston, D. L. Properties of Graphical Representations 29. Rosenthal, H. E. A Graphic Method for the Determination and Presenta of Multiple Classes of Binding Sites. Biochemistry, 10: 3065-3069, tion of Binding Parameters in a Complex System. Anal. Biochem., 20: 1971. 525-532, 1967. 16. Korenman, S. G. Relation between Estrogen Inhibitory Activity and 30. Scatchard, G., Coleman, J. S., and Shen, A. L. Physical Chemistry of Binding to Cytosol of Rabbit and Human Uterus. Endocrinology, 87: Protein Solutions VII, Binding of Some Small Anions to Serum Albumin. 1119-1123, 1970. J. Am. Chem. Soc., 79: 12-20,1957. 17. Lasfargues, E. Y., and Ozzello, L. Cultivation of Human Breast Carcino 31. Skidmore, J. R., Walpole, A. L., and Woodburn. J. Effect of Some mas. J. Nati. Cancer Inst., 21: 1131-1140, 1958. Triphenylethylenes on Oestradiol Binding In Vitro to Macromolecules 18. Lee, Y. J., Notides, A. C., Tsay, Y.-G., and Kende, A. S. Coumestrol, from Uterus and Anterior Pituitary. J. Endocrinol., 52: 289-298, 1972. NBD-Norhexestrol, and Dansyl-Norhexestrol, Fluorescent Probes of Es 32. Solmssen, U. V. Synthetic Estrogens and Relation between Their Struc trogen-Binding Proteins. Biochemistry, 16: 2896-2901,1977. ture and Their Activity. Chem. Rev., 37: 481-598, 1945. 19. Levison, S. A., Dandliker, W. B., Brawn, R. J., and VanderLaan, W. P. 33. Thieulant, M. L., Mercier, L., Samperez, S., and Jouan, P. Dihydrotes- Fluorescence Polarization Measurement of the Hormone-Binding Site tosterone-Protein Binding in the Cytosol of Rat Anterior Hypophysis In Interaction. Endocrinology, 99: 1129-1143, 1976. Vitro: Evidence for a Specific Receptor. J. Steroid Biochem., 6: 1257- 20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein 1260, 1975. Measurement with Folin Phenol Reagent. J. Biol. Chem., 193: 265-275, 34. Vaage, J. A Mechanical Technique for Obtaining High Yields of Viable, 1951. Dispersed Tumor Cells. Transplantation, 6. 137-139, 1968.

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Fig. 1. A, frozen thin section of moderately well- differentiated infiltrating ductal carcinoma of the breast stained with 17-FE. 400 p\ at 3.5 x 10'8 M for 30 min, followed by rapid washing in pH 7.4- buffered 0.15 M NaCI solution. Fluorescence photomicrography was performed in the Zeiss fluorescence microscope. Exposure, 120 sec with Kodak Ektachrome 200. Note the fluorescence localization to the malignant ductoid elements. x 160. B, thin section of contiguous tumor tissue. Note the malignant ductoid elements and the ratio of malignant epithelial to nontransformed mesen- chymal tissue. H & E, x 160.

4224 CANCER RESEARCH VOL. 38

Walter B. Dandliker, R. James Brawn, Mao-Lin Hsu, et al.

Cancer Res 1978;38:4212-4224.

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