Characterization and Quantitation of Antiestrogen Binding Sites in Estrogen Receptor-Positive and -Negative Human Breast Cancer Cell Lines1

(CANCER RESEARCH 43, 3094-3100, July 1983]

Characterization and Quantitation of Antiestrogen Binding Sites in Estrogen Receptor-positive and -negative Human Breast Cancer Cell Lines1

Margaret Ann Miller and Benita S. Katzenellenbogen2

Department ol Physiology and Biophysics, University of Illinois, and University of Illinois College of Medicine, Urbana, Illinois 61801

ABSTRACT gens. It is possible, however, that these antiestrogen binding sites might influence the distribution of antiestrogens and, hence, Antiestrogens are useful in the treatment of endocrine-respon their accessibility to estrogen receptor in estrogen receptor- sive breast cancers in humans. In an attempt to understand the positive cells, or they might mediate actions of antiestrogens mechanisms underlying their estrogen antagonism and antitumor that are unrelated to estrogen antagonism. character, we have examined the interaction of antiestrogens with three human breast cancer cell lines that differ markedly in INTRODUCTION their estrogen receptor content and in their sensitivity to growth suppression by antiestrogens. MCF-7 cells have high levels of Antiestrogens are intriguing compounds that are able to an estrogen receptor, and their growth is inhibited markedly by tagonize many of the effects of estrogens. Although these non- antiestrogens; T47D cells contain low levels of estrogen recep steroidal triphenylethylene compounds were developed initially tor, and their growth is suppressed weakly by antiestrogens; by pharmaceutical companies as fertility-regulating agents, they and MDA-MB-231 cells contain no detectable estrogen recep are of particular interest and importance today because of their tors, and their growth is unaffected by antiestrogens. In addition clinical efficacy in controlling the growth and spread of hormone- to binding to the estrogen receptor, antiestrogens are found to dependent mammary and uterine tumors; with them, it appears be associated with binding sites that are distinct from the estro to be possible to achieve noninvasively the same hormonal gen receptor. These estrogen-noncompetible but antiestrogen- effects and tumor regression that normally follow the more competible binding sites are present in the 800 and 12,000 x g devastating endocrine ablative surgeries (ovariectomy, adrenal- supematants of all three breast cancer cells. The antiestrogen ectomy, and hypophysectomy). Over the past 10 years, clinical binding sites are pelleted upon centrifugation at 100,000 or trials using antiestrogens have documented that antiestrogen 180,000 x g and appear to be associated with microsomal treatment is an effective endocrine therapy for breast cancer membranes, while the majority of the estrogen receptor remains with fewer side effects than are associated with pharmacological soluble at all centrifugation speeds. Although these cells differ hormone therapy (5,13,17,18, 27, 41). markedly in their estrogen recepto* content and sensitivity to While the mechanism by which antiestrogens evoke their growth inhibition by the antiestrogen, tamoxifen, all three cell antitumor effects is incompletely understood, most experimental lines contain similar quantities of estrogen-noncompetible anties data are consistent with the hypothesis that antiestrogens exert trogen binding sites (MCF-7 cells, 390 Â±50 (S.E.); T47D cells, their effects through the estrogen receptor system of target cells 360 Â±50; and MDA-MB-231 cells, 260 Â±50 fmol/mg protein) (8-10, 18, 22, 24, 27). Nonsteroidal antiestrogens bind directly that have a similar affinity (rv, = 2 to 4 nw) for tamoxifen. The to estrogen receptors in cytosol preparations from breast cancer affinity of a series of antiestrogens and related compounds cells, and these antiestrogen receptor complexes become local for these antiestrogen sites follows the order c/s-tamoxifen > ized in the nucleus (9, 10, 18, 19). Furthermore, the affinity of a-|p-[2-(1 -pyrrolidino)ethoxy]phenyli -4-methoxy-a' -nitrostil- different antiestrogens for the estrogen receptor correlates well bene (CI628) > frans-tamoxifen = frans-hydroxytamoxifen > with their potency in inhibiting tumor cell growth (8, 10, 34). a-|p-[2-(1-pyrolidino)ethoxy]phenyl(-4-hydroxy-a'-nitrostilbene Hence, the estrogen antagonism of antiestrogens appears to be (CI628M) > 6-hydroxy-2-(p-hydroxyphenyl)benzo(b)thien-3-yl-p- mediated, at least in large part, by antiestrogen interaction with [2(1-pyrrolidinyljethoxy]phenyl ketone (LY117018). This order of the estrogen receptor system. affinities of different antiestrogens for the antiestrogen binding Recently, it has been reported that triphenylethylene com sites does not parallel their affinity for the estrogen receptor nor pounds also bind to additional saturable sites present in the the potency of these compounds as antiestrogens. cytosol of rat and guinea pig uterus, chick oviduct, and human These findings raise questions about the role of these estro breast cancers (12,15, 28, 36-39). These sites are distinct from gen-noncompetible sites in mediating directly the estrogen an the estrogen receptor and are most readily distinguishable by tagonism of antiestrogens in breast cancer cells, and suggest the fact that they bind triphenylethylene antiestrogens but, in that interaction with the estrogen receptor is most likely the contrast to the estrogen receptor, they do not bind steroidal or mechanism underlying the growth-inhibitory effects of antiestro- nonsteroidal estrogens (38). These sites have been termed as "antiestrogen-specific" or "estrogen-noncompetible" binding 1Supported by NIH Grant CA18119 (USPHS) from the National Cancer Institute sites. Since these sites are present in estrogen target tissues [B. S. K.]. A portion of this work was presented at the 64th Annual Endocrine Society Meeting, June 1982 (35). which are responsive to antiestrogen, they could be important in 2To whom requests for reprints should be addressed, at Department of Physi mediating and/or modulating the actions of antiestrogens. ology and Biophysics, 524 BumÂ«Hall, University of Illinois, 407 South Goodwin To aid in defining the role of these antiestrogen binding sites Avenue, Urbana, III. 61801. Received October 20,1982; accepted March 22,1983. in influencing the action of antiestrogens in breast cancer cells,

3094 CANCER RESEARCH VOL. 43

described above. After this time, cells from 2 flasks were harvested and we have studied the distribution, quantities, and binding char counted with a Coulter Counter as described by Butler ef al. (7) (Day 0). acteristics of these sites in 3 human breast cancer cell lines that The medium was then changed to Eagle's minimal essential medium differ in their estrogen receptor content and in their sensitivity to supplemented as described above except containing 2% CDCS and growth suppression by antiestrogens: MCF-7 cells contain high tamoxifen (IO"6 M) or ethanol vehicle (0.1%). At several time points levels of estrogen receptor, and their growth is inhibited markedly during the 12-day growth period, triplicate flasks of cells were counted. by antiestrogens; T47D cells contain low levels of estrogen The procedure was similar for the T47D and MDA-MB-231 cells. T47D receptor, and their growth is inhibited weakly by antiestrogens; cells (seeded at 3 x 105 cells/flask) were grown in regular culture medium and MDA-MB-231 cells contain no detectable estrogen recep throughout the growth period. MDA-MB-231 cells (seeded at 0.5 x 10 / flask) were grown in Leibovitz's Medium L-15 supplemented as descnbed tors and their growth is unaffected by antiestrogens. Our results show that, despite marked differences in the responsiveness above but containing 2% CDCS. Binding Studies. The cells from nearly confluent T-150 flasks were of these cells to growth inhibition by the antiestrogen, tamoxi- harvested by incubating cells at 37Â°for about 10 min in Hanks' balanced fen (i-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1-[Z]- salt solution (calcium and magnesium free; Grand Island Biological Co.) ene), all 3 cell lines possess similar quantities of estrogen- with 1 mw EDTA. The cells were washed twice with a buffer of either 5 noncompetible, antiestrogen binding sites with similar affinities mM sodium phosphate-10 mM thioglycerol-10% glycerol, pH 7.4, or TEA for tamoxifen. In addition, the subcellular distribution of these and homogenized in a Dounce homogenizer (40 to 50 strokes with a B- antiestrogen binding sites and the relative binding affinities of pestle). various antiestrogens for these sites are similar in the 3 cell lines. In preliminary studies, the subcellular localization of the antiestrogen These findings raise questions about the role of these sites in binding sites was determined by centrifuging MCF-7 cell homogenates mediating directly the estrogen antagonistic effects of antiestro at either 800 x g for 10 min; 12,000 x g for 30 min; or 100,000 or 180 000 x g for 60 min. In subsequent fractionation experiments, cell gens in breast cancer cells. homogenates were sequentially centrifuged at 800,12,000, and 180,000 x g The 12,000 and 180,000 x g pellets were resuspended in buffer MATERIALS AND METHODS and thoroughly homogenized with a Kontes glass-glass homogenizer. Chemicals. The pure trans isomer of [3H]tamoxifen (12.2 Ci/mmol) For the Scatchard analysis and competition studies, the 12,000 x g used to assay the antiestrogen binding sites was synthesized by Dr. D. supernatant was used. The concentration of antiestrogen binding sites was determined by Robertson and Dr. J. Katzenellenbogen (32), University of Illinois, Urbana, III. Estrogen receptor content was assayed with [3H]estradiol (54 Ci/ incubating 250 n\ of cell supernatant or resuspended pellet with 5 n\ of 150 nM [3H]tamoxifen in dimethylformamide and with 5 n\ of ethanol or mmol) from New England Nuclear (Boston, Mass.). The nonradioactive IO'4 M estradiol in 5 Ml of ethanol. After a 30-min preincubation at 0Â°to antiestrogens, frans-tamoxifen and frans-4-hydroxytamoxifen [1,4-(2- dimethylaminomethoxy)phenyl-1 -(4-hydroxyphenyl) - 2 - phenylbut-1 -(Z)- fill estrogen receptor sites, samples were diluted with 220 n\ of TEA buffer prior to the addition of 20 v\ of competitor in dimethylformamide ene] and the estrogen, c/s-tamoxifen (ICI 47,699), were from ICI Amer as described previously (36). Samples were incubated for 16 hr at 0-4Â°, icas Ine (Wilmington, Del); CI6283 was from Parke-Davis & Co. (Ann Arbor, Mich.); U23, 469, c/s-i3-[p-(1,2,3,4-tetrahydro-6-methoxy-2- and then 88 Â¿of dextran-coated charcoal (5% Norit A and 0.5% dextran phenyl-1-napthyl)phenoxyl]-1,2-propanediol| was from Upjohn Co. (Ka- in TEA buffer) were added. The charcoal was pelleted by centrifuging at lamazoo, Mich.); and LY117018 was obtained from Eli Lilly & Co. 12,800 x g for 10 min, and an aÂ«quotof the supernatant was removed (Indianapolis, Ind.). The demethylated derivative of CI628, designated for determination of bound radioactivity. Estrogen receptor content was assayed in similar incubations contain CI628M, was synthesized by Dr. D. Robertson and Dr. J. Katzenellen ing 10~8 M radiolabeled estradiol in the presence and absence of 10 M bogen (23). All media, sera, and antibiotics used to culture the cells were unlabeled estradiol for 16 hr at 0-4Â° and treating with dextran-charcoal obtained from Grand Island Biological Co. (Grand Island, N. Y.). Insulin, hydrocortisone, estradiol, progesterone, testosterone, dihydrotestoster- as described above. Enzyme Assays and Protein and DNA Determinations. The micro- one, and diethylstilbestrol were purchased from Sigma Chemical Co. (St. somal marker enzyme, glucose-6-phosphatase, was assayed by the Louis, Mo.). Cells and Culture Conditions. MCF-7 cells were obtained from Dr. method of Hubscher and West (20). Acid phosphatase which served as a lysosomal marker enzyme was assayed as described by Szego et al. Charles McGrath of the Michigan Cancer Foundation (Detroit, Mich.) and were grown in plastic T-150 flasks in Eagle's minimal essential medium (40). The P, liberated in these assays was analyzed using a kit for the colorimetrie determination of inorganic phosphorus (Sigma). Glucose-6- supplemented with 10 rriM 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic phosphate dehydrogenase, a soluble cytoplasmic marker, was assayed acid buffer, gentamicin (50 ^g/ml), pencillin (100 units/ml), streptomycin by the method of Clock and McLean (14). Protein was determined by (100 iig/ml), insulin (1.5 ng/ml), hydrocortisone (3.75 ng/ml), and 5% calf the method of Peterson (31). DNA was assayed by the diphenylamine serum that had been treated with dextran-coated charcoal for 45 min at 55Â°to remove endogenous hormones (CDCS) (10). reaction (6). The T47D and MDA-MB-231 cells were obtained from EG & G Mason Research Institute (Worcester, Mass.). Medium for the T47D cells was RESULTS the same as that for the MCF-7 cells except that the serum was 5% charcoal-treated fetal calf serum. The MDA-MB-231 cells were grown in Subcellular Localization of Antiestrogen Binding Sites. Al though other investigators have reported that estrogen-noncom- Leibovitz's Medium L-15 supplemented with all of the additives used in the MCF-7 cell medium plus glutathione (16 mg/liter) and 5% calf serum petible binding of the antiestrogen, tamoxifen, occurs in cytosol that was not charcoal treated. preparations from MCF-7 cells (28, 39), we had always found Cell Growth Experiments. To determine the effect of tamoxifen on antiestrogen and estrogen binding in cytosol to be nearly mu- breast cancer cell growth, MCF-7 cells were seeded into T-25 flasks (1.5 tuaHy competitive (9); we, therefore, found little evidence for x 105 cells/flask) and grown for 2 days in medium supplemented as antiestrogen-specific binding sites in our high-speed MCF-7 cy tosol preparations. However, when we undertook subcellular 3The abbreviations used are: CI628, a-|p-[2-(1-pyrrolidino)ethoxy]phenyl|-4- methoxy-a'nitrostilbene; LY117018, 6-hydroxy-2-(p-hydroxyphenyl)benzo(/J)thien- fractionation studies, we found that substantial amounts of es- 3-ylW-<1-pyrrolidinyt)ethoxy]phenyl ketone; CI628M, a-|p-{2-(1-pyrroiKlinoÂ£ trogen-noncompetible, antiestrogen binding sites do exist in low- ethoxylphenyl|-4-hydroxy-a'-nitrostilbene; CDCS, charcoal-dextran-stnpped calf serum; TEA, 10 mw Tris-1.5 IDM EDTA-0.2% sodium azkte (pH 7.4). speed supernatants. 3095 JULY 1983

When tamoxifen binding was investigated in homogenates of MCF-7 cells centrifuged at different speeds for various periods O of time, "antiestrogen-specific" binding sites were present in the H 100 800 and 12,000 x g for 30 min low-speed supernatants of cell homogenates, but were pelleted upon centrifugation at 100,000 or 180,000 x g for 60 min (Chart 1). Chart 1 shows the results 75 of assays in which a trace amount of tritiated tamoxifen (1.5 nw) was incubated in the presence of increasing concentrations of unlabeled estradici or unlabeled tamoxifen (after a 30-min pre LL CL 50 treatment with 10~6 M estradici to occupy estrogen receptors O Z5 sites fully). The difference between the estradici curve and the o o 25 tamoxifen plus 10~6 M estradici curve is considered to represent (r o the estrogen-noncompetible, but antiestrogen-competible ("an Lu CO Q- tiestrogen-specific") binding sites. The subcellular fractionation

pattern for "antiestrogen-specific" sites differs from that of the BOOXg 12,000 X g l2,OOOXg ISO.OOOXg ISO.OOOXg SUPERNATANT SUPERNATANT PELLET SUPERNATANT PELLET estrogen receptor (Chart 1, right of each panel) which remained soluble at all centrifugation speeds. However, the 100,000 and B 180,000 x g supernatants contain approximately 50 to 70% of tÂ£< or the estrogen receptors in the 800 and 12,000 x g supernatant >u- 300 fractions and, therefore, some estrogen receptors may be pel I- H OZ ut ^ leted at the higher centrifugation speeds (as noted by others for < < y< S estrogen and androgen receptors) (25, 30). 200 It is important to note that, since the different subcellular fractions contain a different proportion of "antiestrogen-specific" Â£Â£ w;2 binding sites and estrogen receptor sites, the distribution of the tritiated tamoxifen between these sites varies in the different supernatant preparations. Hence, the apparent increase in effec 38 H oo tiveness of estradiol in competing for tritiated tamoxifen binding (estradiol curves) in the 100,000 and 180,000 x g supernatant eOOXg IZ.OOOXg IZ.OOOXg ISO.OOOXg ISO.OOOXg preparations is most probably attributable to the relative increase SUPERNATANT SUPERNATANT PELLET SUPERNATANT PELLET in estrogen receptor content versus antiestrogen binding sites Chart 2. Subcellular distribution of antiestrogen sites, estrogen receptors, and and nonspecific binding, and not to an increase in the affinity of several enzyme markers upon sequential centrifugation of the 10-mm 800 x g tamoxifen for the receptor. MCF-7 supernatant. Levels of antiestrogen sites, estrogen receptors, and the In an attempt to define the subcellular localization of antiestro- activity of 3 enzyme markers were determined in the 10-min 800 x g supernatant fraction. The 800 x g supernatant was then centrifuged at 12,000 x g for 30 min at 0Â°,and the level and activity of the antiestrogen sites, estrogen receptors, and enzyme markers were determined in the 12,000 x g supernatant and in the 8OOXgXIOmins/t IOO,OOOXgX60min resuspended 12,000 x g pellet; 12,000 x g supernatant was then centrifuged at 180,000 x g for 1 hr, and these parameters were again monitored in the supernatant Q en and resuspended pellet. In A. the total activity of each parameter in the 800 x g supernatant was set at 100% to allow a comparison with the subsequent distri X "0 bution between supernatant and pellet fractions. In B, the relative specific activity (level or activity per mg protein) of each parameter in the 800 x g supernatant was E set at 100% so that changes in specific activity during fractionation can be a a. O â€” observed. The absolute specific activities of the 5 parameters in the 800 x g 0 -10 -9 -6 -7 -6 O -IO -9 -8 -7 -6 _l supernatant fraction were: antiestrogen sites, 290 fmol per mg protein; estrogen LJ O receptors, 336 fmol per mg protein; glucose-6-phosphatase, 13.0 pg P, released U- l2,OOOXgX30mins/t l80,OOOXgX60mins/t o x per mg protein per hr; acid phosphatase, 476 nmol a-naphthol per mg protein per O hr; and glucose-6-phosphate dehydrogenase, 12.4 nmol NADPH per mg protein per min. Values are means of 2 separate fractionation studies. Bars, S.E.

gen binding sites, MCF-7 cell homogenates were ultracentrifuged

O1-1-// sequentially, and the supernatant fractions and resuspended O -IO -9 -8 -7 -6 O -IO -9 -8 -7 -6 pellets were assayed for marker enzyme activities and for an LOG MOLAR CONCENTRATION tiestrogen binding site and estrogen receptor content. The re Chart 1. Subcellular fractionation of antiestrogen binding sites and estrogen receptors from MCF-7 cells. Cells were homogenized, and the nomogenate was sults of these studies are presented in Chart 2. When the 800 x centrifugea at the different speeds for the times indicated. After centrifugation. the g supernatant fraction is centrifuged for 30 min at 12,000 x g, supernatants (s/i) were incubated with [3H]tamoxifen (1.5 nnÂ«)atone (O), [3H] tamoxifen with increasing (10~8 to 10~6 M) concentrations of estradiol (Â£2,O). or over 80% of the antiestrogen binding sites and estrogen recep [3H]tamoxifen plus 10"* M estradiol for 30 min at 0Â°to fill estrogen receptor sites tors remain soluble as does the soluble cytoplasmic marker, (â€¢)prior to addition of tamoxifen (7am; 10~10 to 10"6 M; â€¢).Incubations were glucose-6-phosphate dehydrogenase. Approximately half of the maintained for 18 hr at 0Â°prior to charcoal-dextran treatment. Additional aliquots of the supernatants from each centrifugal fraction were incubated with 13H(estradici microsomal marker, glucose-6-phosphatase, and the lysosomal (10 DM) in the absence and presence of 10 '' M unlabeled estradiol to monitor marker, acid phosphatase, were pelleted during this centrifuga estrogen receptor levels p, â€¢).Values are expressed as pmol [3H]-tamoxifen bound per mg cell DMA (left ordinate) and as pmol [3H]estradiol bound per mg cell tion. When the 12,000 x g supernatant is subsequently centri DMA (right ordinate). Please note that the scale for antiestrogen binding sites is fuged at 180,000 x g for 1 hr, virtually all of the antiestrogen expanded for clarity in the 100,000 x g and 180,000 x g fractions. binding sites and a portion of the microsomal marker activity are

3096 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1983 American Association for Cancer Research. Antiestrogen Binding Sites in Breast Cancer Cells pelleted. In contrast, most of the estrogen receptors and the Table 1 soluble enzyme marker, glucose-6-phosphate dehydrogenase, Antiestrogen binding sites in human breast cancer cells remain in the 180,000 x g supernatant. The specific activity concentration" (sites/unit protein) of the antiestrogen sites is enriched in the CellMCF-7 (fmol/mgprotein)390 Â±SO6 Â±0.9" 180,000 x g pellet (Chart 2B). Since the antiestrogen binding T47D 360 Â±50 3.6 Â±0.9 sites are pelleted by centrifugation at 180,000 x g for 60 min, MDA-MB-231Site 260 Â±50Ko(nM)3.7 3.4 Â±0.7 they may be associated with the microsomal membranes. There a Concentration of estrogen-noncompetible, antiestrogen binding sites meas is, however, little of the total activity (Chart 2A) of the microsomal ured in the 12,000 x g for 30 min supernatant fraction. marker enzyme, glucose-6-phosphatase, in the 180,000 x g for 6 Mean Â±S.E. from 3 separate experiments with each cell line. 60 min pellet. This enzyme and the lysosomal marker enzyme, MDA-MB-231 acid phosphatase, are found predominantly in the 12,000 x g for 30 min pellet. The separation of the microsomal marker 200 |0 enzyme and antiestrogen binding sites, which also appear to be paniculate, suggests that these 2 components may be located in different areas of the microsomal membranes which are known to be heterogeneous (1). Antiestrogen Inhibition of Growth of Breast Cancer Cells _ _ _ _ and Antiestrogen Binding Sites in Different Breast Cancer O-IO -9 -8 -r -6 O -IO -9 -8 -7 -6 O -IO -9 -8 -7 -6 Cell Lines. To determine if antiestrogen bindingsites are present LOG MOLAR CONCENTRATION in other breast cancer cell lines, we examined tamoxifen binding Kd=2.0nM S-Ka=3.lnM 03 \ 310 fmol/mg protein \260 fmol/mg protein 0.3 in T47D and MDA-MB-231 cells as well as in MCF-7 cells. These 3 cell lines differ markedly in their estrogen receptor content and 0.2 in their responsiveness to antiestrogen (Chart 3). Tamoxifen inhibition of growth was greatest in MCF-7 cells which we find 8 o., O.I CD to contain approximately 7 pmol of estrogen receptor/mg DMA. 0 0.5 1.0 2.0 In T47D cells, estrogen receptor levels were 10 to 15% of the BOUND (nonomolar) values determined for MCF-7 cells (data not shown), and tamox Chart 4. Evaluation of the presence and levels of "antiestrogen-specific" bind ifen decreased growth, but far less than in MCF-7 cells. No ing sites in 3 different human breast cancer cell lines. Cells were homogenized, estrogen receptor was detected in MDA-MB-231 cells, and ta and the 30-min 12,000 x g supernatant was incubated with 1.5 nw [3H]tamoxifen alone (O), pHJtamoxifen with increasing (10~Â°to 10~* M) concentrations of estradiol moxifen did not alter growth in these cells. Hence, tamoxifen (Â£2,O), or [3H]tamoxifen plus 10"* M estradiol for 30 min at 0Â°to fill estrogen inhibited growth only in those cell lines that contained estrogen receptor sites (â€¢)prior to addition of tamoxifen (Tarn; IO"10 to 10"* M, â€¢). receptor, i.e., MCF-7 and T47D cells, and the magnitude of Incubations were maintained for 18 hr at 0Â°prior to charcoal-dextran treatment. Values for [3H]tamoxifen-bound radioactivity were normalized for protein concen growth suppression appears to be related to the estrogen recep tration of the cell supernatants (all about 1.2 to 1.6 mg protein per ml) and are tor content of the cells. expressed as fmol [3H]tamoxifen bound per mg protein. In lower panels, data on In contrast to the differences observed in estrogen receptor antiestrogen binding are analyzed by Scatchard plot analysis from which the KÂ« and number of sites were calculated. levels and in responsiveness to tamoxifen among these 3 cell lines, the concentration of estrogen-noncompetible antiestrogen binding sites and their affinity for tamoxifen were found to be estradiol to fully occupy estrogen receptors). Values have been similar in the 3 cell lines (Table 1 and Chart 4). Chart 4 shows normalized per mg protein. These data are also replotted in the the results of a typical competitive binding experiment in which form of a Scatchard plot (Chart 4, lower panels) where KOvalues a trace amount of tritiated tamoxifen (1.5 nw) was incubated in of 2 to 4 nM are obtained for the binding of tamoxifen. As shown the presence of increasing concentrations of unlabeled estradiol in Table 1, which summarizes data from 3 competitive binding or unlabeled tamoxifen (after a 30-min pretreatment with 10"6 M experiments with each cell line, the antiestrogen-specific binding sites are present at approximately the same concentration in the 3 cell lines, and the affinity of tamoxifen for these sites is likewise ; MDA-MB-231 similar in all of the cell lines. Ligand Binding Specificity of Antiestrogen Binding Sites. Competitive binding assays were used to determine the affinities of various antiestrogens and related compounds for the "anties trogen-specific" binding sites. As seen in Chart 5 and Table 2, these sites are specific for compounds which are triphenylethylene derivatives. None of the steroids (estradiol, hydrocortisone, progesterone, dihydrotestosterone, or testosterone) nor the nonsteroidal estrogen, diethylstilbestrol, competed with [3H]tamoxifen for binding to these sites. The order of competitive effective ness is c/s-tamoxifen > CI628 > frans-tamoxifen = frans-hy- DAYS droxytamoxifen > CI628M > LY117018. Chart 5 also shows Chart 3. Effect of tamoxifen (7am; 10"* M) on the growth of 3 different human that the competitive binding curves are parallel for all of the breast cancer cell lines, MCF-7 cells, T47D cells, and MDA-MB-231 cells. Cells triphenylethylene compounds tested. The competition curve for were grown in the continuous presence of tamoxifen, and media with fresh tamoxifen were renewed every other day. On the days indicated, triplicate flasks LY117018 has a somewhat shallower slope, suggesting that of cells were counted. Values are means of the triplicate determinations. Bars, S.E. interaction of LY117018 with these antiestrogen sites may be

JULY 1983 3097

cells to growth suppression by tamoxifen are markedly different. In MCF-7 cells, the concentrations of estrogen receptor and "antiestrogen-specific" binding sites are approximately equal while, in T47D cells, the concentration of receptor is only one- eighth that of the antiestrogen sites. These 2 binding compo nents are, however, readily distinguishable because: (a) the ligand binding specificities of the estrogen receptor and anties trogen sites are very different; (b) the binding capacity of the estrogen receptor is destroyed by heating for 60 min at 37Â°, while the antiestrogen sites are stable to this treatment (data not presented); and (c) the antiestrogen binding sites are pelleted by centrifugation at 180,000 x g for 60 min, whereas much of the estrogen receptor remains soluble. The antiestrogen binding sites in human breast cancer cell -IO -9 -8 -7 lines appear to be similar to the antiestrogen binding sites LOG MOLAR CONCENTRATION described in rat tissues. We have recently reported that high- affinity (Kd 1 to 3 nw) estrogen-noncompetible antiestrogen bind Chart 5. Relative affinity of different triphenytethylenederivatives, and the ste roid hormones estradici (E2),dihydrotestosterone (DHT), hydrocortisone (Hydro- ing sites are present in the 12,000 x g for 30 min supernatants con.), progesterone (Prog.),testosterone (T), and the nonsteroidalestrogen, dieth of estrogen receptor-positive (uterus, ovary, liver, and brain) as ylstilbestrol (DES),for anliestrogen binding sites. Assays used the 30-min 12,000 x g supernatant fraction of MCF-7 cells which was incubated with 1.5 HM|-'H|- well as estrogen receptor-negative (esophagus, lung, and spleen) tamoxifen plus 10"'Â°to10~Â°Mestradiolto assess[3H]tamoxifenbindingto estrogen rat tissues (36). In the immature rat uterus, the antiestrogen receptor sites; or with 1.5 MM[3H]tamoxifenplus 10~'Â°to10~*M diethylstilbestrol binding site content is about one-tenth that of the estrogen dihydrotestosterone,hydrocortisone,progesterone,or testosterone, which all gave identicalcurves and hence have been combined; or with 1.5 nM[3H]tamoxifenplus receptor. As in MCF-7 cells, these 2 binding components can be io hMestradici for 30 min at 0Â°to fill estrogen receptor sites prior to addition of readily distinguished by differences in ligand specificity, fraction- IO"10to 10"* M concentrations of the competitors to be tested. Incubations with [l-IJtamoxifen alone gave values identical to those containing 10~toM competitor ation pattern, and protease and heat sensitivity (36). and hence are not plotted. Incubations were maintained at 0Â°for 18 hr prior to The antiestrogen binding sites we find in the 3 breast cancer charcoai-dextrantreatment. Values are [3H]tamoxifencom bound per assay. cell lines are, in most respects (affinity, quantity, and ligand specificity), very similar to the estrogen-noncompetible antiestro qualitatively different from that of tamoxifen and compounds gen sites described by other investigators (11, 12, 15, 28, 37- structurally related to tamoxifen. 39). However, our results differ in one respect from these other The relative binding affinities of the various triphenylethylene reports. Whereas others (11, 37-39) report that antiestrogen and related compounds for these antiestrogen sites do not binding sites are present in the cytosolic fraction, we find that parallel the potency of these compounds as antiestrogens or estrogen-noncompetible binding sites are always pelleted by their affinity for the estrogen receptor (Table 2). For example, centrifugation at 180,000 x g for 60 min. Moreover, our fraction- the affinity of c/s-tamoxifen for this binding site is about 2-fold ation experiments indicate that these sites may be membrane greater than that of frans-tamoxifen, but c/s-tamoxifen has no associated. If this is the case, differences in buffer tonicity and/ antiestrogenic effects and is only a weak estrogen in MCF-7 or homogenization techniques may result in liberation of these cells4 (8) and in rat uterus (16, 21, 33). In addition, LY117018 is sites to the supernatant and, hence, account for the differences a very potent antiestrogen (2, 3, 34) but binds weakly to these in apparent localization of these sites. sites, and CI628M and frans-hydroxytamoxifen, which are more As reported by Sutherland ef a/. (38), the antiestrogen binding potent antiestrogens than tamoxifen and CI628 (8, 10), have sites are specific for compounds with a triphenytethylene struc- affinities for these sites only equal to or less than that of tamox ifen. Table2 Affinities of antiestrogens and other compounds for estrogen receptor and A similar order of competitive effectiveness of different anties antiestrogen binding sites of MCF-7 breast cancer cells trogens was found in the 12,000 x g supernatant of T47D or MDA-MB-231 cells (data not presented) and, in all 3 cell lines, binding sites9 the antiestrogen, LY117018, competed very poorly with [3H]- receptor (%; frans-ta- (%; estradiol = moxifen = tamoxifen for binding to these antiestrogen binding sites, and Compoundfrans-Tamoxifen 100%)2 100%)100 the competition curve with LY117018 was shallower than that Â± 0.56 of tamoxifen and other triphenytethylene compounds. c/s-Tamoxifen 0.2 Â± 0.1 267 Â±20 CI628 3 Â± 2 156 Â± 7 DISCUSSION frans-Hydroxytamoxifen 167 Â±20 110Â± 8 CI628M 22 Â± 4 60Â± 4 LY117018 115 Â±26 19 Â± 8C The results of our studies demonstrate that antiestrogen bind Estradiol or diethylstilbestrol 100 <0.001 ing sites distinct from the estrogen receptor are present in the Progesteroneor hydrocortisone <0.01 <0.001 MCF-7, T47D, and MDA-MB-231 breast cancer cell lines. The Testosterone or dihydrotestosteroneEstrogen <0.01Antiestrogen <0.001 ''All values determined by competitive binding assays using |'H|estradioi or concentration of the antiestrogen binding sites and their affinity [3H]tamoxifenfor measurementof estrogen receptor or antiestrogen sites, respec for tamoxifen are similar in the 3 breast cancer cell lines, although tively. the concentration of estrogen receptor and the sensitivity of the Mean Â±S.E.of 3 determinations. c The competition curve with LY117018 has a shallowerslope than that of irans- tamoxlfen. This value has been determined from the concentration giving 50% 4B. S. Katzeneflenbogenand M. J. Norman, unpublished results. competition.

3098 CANCER RESEARCH VOL. 43

ture. The affinity of different antiestrogens for these sites does 6. Burton, K. A. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetrie estimation of deoxyribonucteic acid. Biochem. J. not, however, correlate with their potency as antiestrogens. For 62:315-323,1956. example, c/s-tamoxifen binds to these sites with twice the affinity 7. Butler, W. B., Kelsey, W. H., and Goran, N. Effects of serum and insulin on of frans-tamoxifen but displays no estrogen-antagonistic prop the sensitivity of the human breast cancer cell line MCF-7 to estrogen and antiestrogens. Cancer Res., 41: 82-88, 1981. erties (16, 21, 33). Other compounds (i.e., LY117018 and 8. Coezy, E., Borgna, J-L., and Rochefort, H. Tamoxifen and metabolites in MCF- CI628M) show high antiestrogen potency and high estrogen 7 cells: correlation between binding to estrogen receptor and inhibition of cell growth. Cancer Res., 42: 317-323, 1982. receptor affinity (4, 9, 23, 34) but low or reduced affinity for the 9. Eckert, R. L., and Katzenellenbogen, B. S. Physical properties of estrogen antiestrogen sites. Because these sites also have affinity for receptor complexes in MCF-7 human breast cancer cells: differences with compounds that are estrogens (e.g., c/s-tamoxifen), it is not fully antiestrogen and estrogen. J. Biol. Chem., 257: 8840-8846, 1982. appropriate to consider these as "antiestrogen-specific" binding 10. Eckert, R. L., and Katzenellenbogen, B. S. Effects of estrogens and antiestro gens on estrogen receptor dynamics and the induction of progesterone recep sites. tor in MCF-7 human breast cancer cells. Cancer Res., 42: 139-144,1982. Although the antiestrogen binding sites are present in all 3 cell 11. Faye, J. C., Jozan, S . and Bayard, F. Mechanism of action of antiestrogens of the triphenylethylene type in MCF-7 cells. In: proceedings of the 64th Annual lines, only those cells which contained estrogen receptors Endocrine Society Meeting, Abstract 376, p. 173. Baltimore: Williams & Wilkins, showed growth inhibition following tamoxifen treatment. This 1982. result, coupled with the finding that the affinity of antiestrogens 12. Faye, J. C., Lassere, B., Bayard, F. Antiestrogen specific high affinity saturable binding sites in rat uterine cytosol. Biochem. Biophys. Res. Commun., 93: for these sites does not parallel antiestrogenic potency, suggests 1225-1231,1980. that antiestrogen binding to these sites probably does not me 13. Fisher, B., Redmond, C., Brown, A., and other NSABP Investigators. Treatment of primary breast cancer with chemotherapy and tamoxifen. N. Engl. J. Med.. diate the effects of antiestrogens on cell growth. 305:1-6,1981. While our data shed doubt on the role of these antiestrogen 14. Clock. G. E., and McLean, P. Further studies on the properties and assay of sites in the growth suppression evoked by antiestrogens, it is glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver. Biochem. J., 55: 400-408,1953. conceivable that a defect in a biochemical process that occurs 15. Gulino, A., and Pasqualini, J. R. Heterogeneity of binding sites for tamoxifen after tamoxifen binding to the antiestrogen sites could account and tamoxifen derivatives in estrogen target and nontarget fetal organs of for the tamoxifen resistance of cells, such as the MDA-MB-231 guinea pig. Cancer Res., 42: 1913-1921,1982. 16. Harper, M. J. K., and Walpole, A. L. A new derivative of triphenylethylene and cells, that contain antiestrogen binding sites but do not display mode of action in rats. J. Reprod. FÃ©rtil.,J3: 101-119,1967. growth inhibition by tamoxifen. Surely this is possible, since there 17 Heuson, J. C., Engelsman, E., Bkxik-Vander Wijst, J., Maass, H., Drochmans, A., Michel, J., Nowakowski, H., and Gorins, A. Comparative trial of nafoxidine is clear evidence for the presence of estrogen receptors in some and ethinyloestradiol in advanced breast cancer: a European Organization for human breast cancers that are unresponsive to estrogens and Research on Treatment of Cancer study. Br. Med. J., 2: 711-713,1975. antiestrogens (26,29). In addition, Faye ef al. (11) have presented 18. Horwitz, K. B., and McGuire, W. L. Antiestrogens: mechanism of action and effects in breast cancer. In: W. L. McGuire (ed.). Breast Cancer: Advances in evidence for a variant of MCF-7 cells that appears to have lost Research and Treatment, Vol. 2, pp. 155-204. New York: Plenum Publishing the antiestrogen binding site and sensitivity to growth inhibition Corp., 1978. 19. Horwitz, K. B., and McGuire, W. L. Estrogen control of progesterone receptor by tamoxifen while retaining estrogen receptor activity. in human breast cancer. J. Biol. Chem., 253: 2223-2228,1978. Nevertheless, the presence of antiestrogen binding sites in 20. HÃ¼bscher,G., and West, G. R. Specific assays of some phosphatases in antiestrogen-responsive and -nonresponsive breast cancer cells subcellular fractions of small intestinal mucosa. Nature (Lond ) 205:799-800. 1965. as shown here, as well as in many rat tissues (36, 38), suggests 21. Jordan, V. C., Haldemann, B., and Allen, K. E. Geometric isomersof substituted that these sites are probably not mediating directly the estrogen triphenytethylenes and antiestrogen action. Endocrinology, 108: 1353-1361, antagonism of antiestrogens. These sites may represent binding 1981. 22. Katzenellenbogen, B. S., Bhakoo, H. S., Ferguson, E. R., Lan. N. C., Talee, sites for other natural ligands for which the triphenylethylene T., Tsai, T. L., and Katzenellenbogen, J. A. Estrogen and antiestrogen action compounds bear a structural resemblance. While the natural in reproductive tissues and tumors. Recent Prog. Horm. Res., 35: 259-300, 1979. ligands for these sites and the physiological importance of these 23. Katzenellenbogen, B. S., Pavlik. E. J., Robertson, D. W., and Katzenellenbo- sites is not known presently, it seems likely that these sites gen. J. A. Interaction of a high affinity antiestrogen (,,-|4-pyrrolidmoethoxy| might alter the distribution and pharmacokinetics of antiestro phenyl-4-hydroxy-Â«-nitrostilbene, CI628M) with uterine estrogen receptors. J. Biol. Chem., 256: 2980-2915, 1981. gens in vivo and might mediate effects of antiestrogens unrelated 24. Ã¼ppman,M., BolÃ¡n,G., and Huff, K. The effects of estrogens and antiestrogens to estrogen antagonism. Studies in our laboratory are continuing on hormone-responsive human breast cancer in long-term tissue culture. in an attempt to determine the physiological role of these sites Cancer Res.. 36: 4595-4601, 1976. 25. Maclndoe, J. H., Woods, G. R., and Lee, F. J. The specific binding of androgens and their role in the action of antiestrogens. and the subsequent distribution of androgen-receptor complexes within MCF- 7 human breast cancer cells. Steroids, 38: 439-452, 1981. 26. McGuire, W. L. Steroid receptor sites in cancer therapy. Adv. Intern. Med., 24 ACKNOWLEDGMENTS 127-140,1979. 27. McGuire, W. L., Chamness, G. C., Horwitz. K. B., and Zava, D. T. Hormones We thank Frederick Monsma. Jr., Mary Jane Norman, and Dr. Katsuichi Sudo and their receptors in breast cancer. In: B. O'Malley and L. Birnbaumer (eds.), for their contributions to these studies. Receptors and Hormone Action, Vol. 2, pp. 401 -441. New York: Academic Press, Inc., 1978. REFERENCES 28. Murphy, L. C.. and Sutherland. R. L. Modifications in the aminoether side chain of clomiphene influence affinity for a specific antiestrogen binding site in MCF- 1. Arnar-Costesec, A., and Beaufay, H. A structural basis of enzymatic hetero 7 cell cytosol. Biochem. Biophys. Res. Commun., 700: 1353-1369,1981. geneity within liver endoplasmic reticulum. J. Theor. Biol., 89: 217-230,1981. 29. Nawata, H., Bronzert, D., and Ã¼ppman.M. E. Isolation and characterization of 2. Black, L J., and Goode, R. L. Uterine bioassay of tamoxifen, tnoxifene and a a tamoxifen-resistant cell line derived from MCF-7 human breast cancer cells. new estrogen antagonist (LY117018) in rats and mice. Life Sci., 26: 1453- J. Biol. Chem., 256. 5016-5021,1981. 1458. 1980. 30. Parikh, I., Anderson, W. L., and Neame, P. Identification of high affinity estrogen 3. Black, L. J., and Goode, R. L. Evidence for biological action of the antiestrogens binding sites in calf uterine microsomal membranes. J. Biol. Chem., 255: LY117018 and tamoxifen by different mechanisms. Endocrinology, 709: 987- 10266-10270,1980. 989.1981. 31. Peterson, G. L. A simplification of the protein method of Lowry e( al. which is 4. Black, L. J., Jones, C. 0., and Goode, R. L. Differential interaction of antiestro more generally applicable. Anal. Biochem.. 83 346-356,1977. gens with cytosol estrogen receptors. Mol. Cell. Endocr., 22:95-103,1981. 32. Robertson, D. W., and Katzenellenbogen, J. A. Synthesis of the E and Z 5. Bloom, H. J. G., and Boesen, E. Antioestrogens in treatment of breast cancer: isomers of the antiestrogen tamoxifen and its metabolite, hydroxytamoxifen, value of nafoxidine in 52 advanced cases. Br. Med. J., 2; 7-10, 1974. in tritium-labeled form. J. Org. Chem., 47: 2387-2393.1982.

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33. Robertson, D. W., Katzenellenbogen,J. A., Long, D. J., Rorke, E. A., and 1983. Katzenellenbogen,B.S. Tamoxifenantiestrogens.A comparisonof the activity, 37 Sutherland, R. L. and Foo, M. S. Differential binding of antiestrogens by rat pharmacokinetics,and metabolic activation of the cis and frans isomers of uterineand chick oviduct cytosol. Biochem. Biophys. Res. Commun.,92:183- tamoxifen. J. Steroid Biochem., 76:1-13,1982. 191,1979. 34. Scholl,S., Huff, K., and Ã¼ppman,M.E. Effects of LY117018 on MCF-7 human 38 Sutherland,R. L., Foo, M. S., Green,M. D., Wayboume, A. M., and Krozowski, breast cancer cells. In: Proceedings63rd Annual Endocrine Society Meeting, Z. S. High affinity antiestrogen binding site distinct from the estrogen receptor. Abstract 175, p. 126. Baltimore: Williamsand Wilkins, 1981. Nature (Lond.),288: 273-275,1980. 35. Sudo, K., Miller, M. A., Monsma. F. J., Jr., and Katzenellenbogen, B. S. 39 Sutherland, R. L., and Murphy, L. C. The binding of tamoxifen to human AntÃestrogen-specificbindingsites: subcellular localization, distribution and mammary carcinoma cytosol. Eur. J. Cancer, 76: 1141-1148,1980. iigandspecificity in rat tissues and human breast cancer cells. In: Proceedings 40 Szego, C. M., Seeler, B. J., Steadman, R. A., Hill, D. F., Kimura, A. K., and 64th AnnualEndocrineSociety Meeting,Abstract 57, p. 94. Baltimore:Williams Roberts,J. A. The lysosomalmembranecomplex: focal point of primarysteroid and Wilkins, 1982. hormone action. Biochem. J., J23: 523-538,1971. 36. Sudo, K., Monsma,F.J., Jr., and Katzenellenbogen,B. S. Antiestrogenbinding 41 Tormey, D. C., Simon, R. M., Lippman, M. E., Bull, J. M., and Myers, C. E. sites distinct from the estrogen receptor: subcellular localization, Iigandspec Evaluation of tamoxifen dose in advanced breast cancer: a progress report. ificity, and distribution in tissues of the rat. Endocrinology, 112: 425-434, Cancer Treat. Rep., 60:1451-1459,1976.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1983 American Association for Cancer Research. Characterization and Quantitation of Antiestrogen Binding Sites in Estrogen Receptor-positive and -negative Human Breast Cancer Cell Lines

Margaret Ann Miller and Benita S. Katzenellenbogen

Cancer Res 1983;43:3094-3100.

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