The Effects of Androgens and Antiandrogens on Hormone Responsive Human Breast Cancer in Long-Term Tissue Culture1

Home , Antiandrogen, Nafoxidine

[CANCER RESEARCH 36, 4610-4618, December 1976] The Effects of Androgens and Antiandrogens on Hormone responsive Human Breast Cancer in Long-Term Tissue Culture1

Marc Lippman, Gail Bolan, and Karen Huff MedicineBranch,NationalCancerInstitute,Bethesda,Maryland20014

SUMMARY Information characterizing the interaction between an drogens and breast cancer would be desirable for several We have examined five human breast cancer call lines in reasons. First, androgens can affect the growth of breast conhinuous tissue culture for andmogan responsiveness. cancer in animals. Pharmacological administration of an One of these cell lines shows a 2- ho 4-fold stimulation of drogens to rats bearing dimathylbenzanthracene-induced thymidina incorporation into DNA, apparent as early as 10 mammary carcinomas is associated wihh objective humor hr following androgen addition to cells incubated in serum regression (h9, 22). Shionogi h15 cells, from a mouse mam free medium. This stimulation is accompanied by an ac many cancer in conhinuous hissue culture, have bean shown celemation in cell replication. Antiandrogens [cyproterona to be shimulatedby physiological concentrations of andro acetate (6-chloro-17a-acelata-1,2a-methylena-4,6-pregna gen (21), thus suggesting that some breast cancer might be diene-3,20-dione) and R2956 (17f3-hydroxy-2,2,1 7a-tnima androgen responsive in addition to being estrogen respon thoxyastra-4,9,1 1-Inane-i -one)] inhibit both protein and siva. DNA synthesis below control levels and block androgen Evidence also indicates that tumor growth in humans may mediahed stimulation. Prolonged incubahion (greahenhhan be significantly altered by androgens. About 20% of pahianhs 72 hn) in antiandrogen is lethal. The MCF-7 cell line conhains with metastahic breast cancer will show objective tumor high-affinity receptors for androganic steroids damonstra regressions when treated wihh androgens (1). A somewhat bla by sucrose dansihy gradients and competitive protein higher proportion of patients will respond to adnenalechomy binding analysis. By cross-competition studies, androgan (4). The efficacy of hhis latter therapy has bean attributed to receptors are distinguishable from estrogen receptors also the removal of androgen precursors of adrenal origin, pni found in this cell line. Concentrations of steroid that sahu manly dehydnoepiandrostamone and t@4-androstenedione. mateandrogen receptor sites in vitro are about 1000times Clean-cut evidence that human breast cancer is androgen lower hhan concentrations hhat maximally shimulate the dependent has not, however, been previously available. cells. Changes in quantity and affinity of androgen binding Finally, andmogen receptor proteins have recently been to intact cells at 37Â°ascompared with usual binding tech demonstrated in some human breast cancer samples (3, 9, niques using cyhosol preparation at 0Â°do not explain this 12) and have been shown to be distinct from both estrogen difference between dissociation of binding and affect. How receptor and sex steroid-binding globulin. It is clear that ever, this difference can be explained by conversion of [3H]- most if not all steroid hormone actions are mediated by an 5a-dihydmohastosherona to 5a-andnoshanediol and mona po initial interaction of steroid hormone and receptor (h3). Ianmatabolihas at 37Â°.Anexamination of incubation media, Since estrogen receptor determination in human breast cytoplasmic extracts, and crude nuclear pellets reveals cancer appears to be of value in guiding therapeutic deci probable conversion of [3H]lashoslerone to [3H]-5a-dihydro sions in the management of metashaticbreast cancer by testosterone. Our data provide compelling evidence that predicting which patients will respond ho hormonal man ipu some human breast cancer, at least in vitro, may be andro lahions (14), it is reasonable to hope that further delineation gen dependent. of hhe mechanism of inheraction of androgens wihh breast cancer might also havetherapeutic implications. Study of these processes, however, has been hampered iNTRODUCTION by the lack of a suitable modal system in which the action of one hormone could be studied independently of the actions We have recently examined several call lines of human of ohhemImophic hormones. In addition, in vivo model sys breast cancer maintained in long-term tissue culture. The tems for androgen action are significantly perturbed by hormone responsiveness of some of these cell lines to interactions of androgen with sex steroid-binding globulin estrogen (6, 7) and glucocorticoid (8) encouraged us to as well as by effects of androgens on concentrations of examine the effects of androgens and antiandrogens. many ohhenhormones including gonadotrophins, prolachin, and estrogen (18). I This is Paper 3 in a series on hormone-responsive human breast cancer cell lines in long-term tissue culture. We now report the characterization of an androgen-re Received November 4, 1976; accepted September 10, 1976. sponsive human breast cancer call line grown in continuous

4610 CANCER RESEARCH VOL. 36

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1976 American Association for Cancer Research. Androgans, Breast Cancer, and Tissue Cu!tura tissue culture that obviates the above difficulties. And rogan Variation in cell counts for any group of dishes was about effects are demonstrable in totally defined medium that 10%. contains no additional hormonal or serum factors. We are Thymidine and Leucine Incorporation Studies. Calls optimistic that such a model system may significantly aid in growing in log phase were harvested in hmypsin-EDTAand hha study of mechanisms of androgen responsiveness. plated in 6-place Linbno dishes (BalIco Glass, Inc. , Vine land, N. J.) in MEM supplemented with 5% fetal calf serum. The next day the medium was changed to MEM without serum. The following day the serum-free medium was again MATERIALSAND METHODS replenished, and hormones ware added as noted in the various chart legends. Two hr before harvesting, radioac hive nucleoside and/on amino acid were added to each well, Cells and CuftureTechniques.Methodsfor propagation and the cells wane harvested as previously described (6, 8). of cells are as previously described (6, 7).2All cell lines have Whole-Cell Binding Experiments. Calls growing in MEM been maintained for ah least 1 year without changes in plus 5% fatal calf serum were changed to MEM without responsiveness. All lines have been tested for Mycop!asma serum on the day prior to the start of the experiment. Calls (Flow Laboratories, Rockville, Md.) and wane shown to be were harvested by suspension in tmypsin-EDTA and were free of contamination. All cell lines used in the present washed twice in PBS. The cells ware than dispersed in MEM shudy synthesize enzymahically active a-lactalbumin. (M. E. supplemented with 25 m@i N-tmis(hydmoxymethyl)methyl Lippman and B. Vondarhaan, unpublished observations; a- glycina (pH 7.2) at a densihy of about h06/ml. Calls ware lactalbumin activity was detected using an enzymahic assay then added to 12- x 75-mm glass tubes containing [3H]- in which call homogenates are incubated with [â€˜4C]UDP 5a-DHT at various concentrations with on without a 1000- galactose and formation of [â€œC]lactosaismeasured.) fold excess of unlabeled 5a-DHT. Tubas were shaken in a SucroseDensityGradientAnalysisandBindingStudies. waler bath at either 0Â°for 3 hr or 37Â°for 1 hr. At the and Analysis of androgan receptors on sucrose gradients and by of the incubation, the medium was diluted with 3 ml of iced competitive protein binding assay was as previously de PBS, and the cells ware collected by cantnifugation for 30 scnibed (6, 8). Briefly, 100,000 x g supemnatants of cell sac in a Clay Adams sarofuge. The calls were washed twice homogenates were prepared and incubated with 5 x 10@ M in ice-cold PBS, suspended in 0.5 ml of distilled water, and [3H]-5a-DHT3 with or without competitor. After treatment sonically dispersed for 4 sac at the lowest salting in a Bran wihh dextran-coated charcoal, these extracts were layered son sonicaton; aliquohs wane taken for protein datenmina onto 5 to 20% sucrose gradients in 10% glycerol. The purity lion by the technique of Lowry at a!. (11) or counted in of [3H]DHT(107Ci/mmole; AmershamSearleCorp., Evans Aquasol (New England Nuclear, Boston, Mass.) in a Pack ton , Ill.) was checked by thin-layer chromatography (10) and and liquid scintillation counter (efficiency for Inilium, was shown to be more than 98% radiochemically pure. For â€”40%).Results ware analyzed by the method of Scatchand studies designed to examine the specificity of binding, au (20). quots of cyhoplasmic extract ware pipetted into tubas con Conversion of 5a-DHT to 5a-Androstane-3a,17/JdiOl. T taming various concentrations of unlabeled steroid as 1000- 75 flasks of confluent MCF-7 cells ware incubated at 37Â°in fold concentrates in ethanol. Radiolabeled steroid, eihhen 10 ml of serum-free MEM containing 10@ M [3H]-5a-DHT (44 1 0@ M [3H]-5a-DHT as above on 1 7f3-[3H]estradiol (1 00 Ci/ Ci/mmole) for 0, 1, 4, and 8 hr. The medium at each lime mmola; Amarsham Seanla), was added immediahaly thareaf point was collected and extracted with 2 volumes of methyl her. Labeled steroid was used at 10@ M, which is only ene chloride. The organic phase was separated by centnifu slightly above the dissociation constant for the receptor and gation and evaporated to dryness under nitrogen. The resi therefore not saturating, in order to keep nonspecific bind dues were redissolvad in 1 ml of methylene chloride con ing below 10% of the total binding. taming 200 @geach of androshanediol, andnostenadione, Effect of Androgens on Cell Growth. Cells growing loga testosterone, and 5a-DHT. Fifty @lwereapplied to duplicate nithmically were harvested with sterile 0.02% EDTA and thin-layer Silica Gel G plates (20 x 20 cm) (Analtech, Inc., plated in 60-mm sterile plastic PaIn dishes in MEM with Newark, Del.), and the chromatognams were developed by serum. After 24 hr, the medium was changed to medium ascending chromahography in chloroform :methanol (98:2, wihhouh serum. 5a-DHT or R2956 (h7f3-hydnoxy-2,2,17a-tni v/v). The steroid spots on each plate were visualized by methylestna-4,9,11-hniana-3-Ona) at a concentration of 10@ streaking the ascending paths of each sample wihh M in ethanol was added ho one-third of the dishes, respec H2SO4:methanol (1:1, v/v) and heating at 90Â°for 2 hn; the lively, ho give a final concentration of 10@ M. Dishes in other plate was quantihated by counting 0.5-cm sections of hniplicata were harvested daily, and cells were either the silica in Aquasol (New England Nuclear) in a Packard counted in a hemocytometer or assayed for total protein. liquid scintillahion counter. ConversionofTestosteroneto5a-DHT.The samepnoce dune as above was followed utilizing 10@ M [3H]teshosterone a The MCF-7 cell line was generously provided by Marvin Rich of the (82 Ci/mmole) as hhesteroid to be studied . Extractions were Michigan Cancer Foundation. Dr. Ronald Herberman of National Cancer done after 1 and 4 hr of incubation. However, the plates Institute supplied the MDA-231, G-11, and HT-39 cell lines. S The abbreviations used are: 5a-DHT, 5a-dihydrotestosterone (5cr-an were developed hwicahoensure adequahaseparahionof the drostane-1713-ol-3-one). DHT, dihydrotestosterone; MEM, Eagle's minimal 5a-DHT from the testosherone. Recovery, monitored with essential medium; PBS, Dulbecco's phosphate-buffered saline; 5B-DHT, 5/3- [14C]tastosterona, is given in â€œResults.â€• dihydrotestosterone (5/3-androstane-17/3-01-3-one).

4611 DECEMBER1976

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1976 American Association for Cancer Research. M. Lippman at a!. RESULTS lines that lack androgan receptor activity and are unstimu lahed by lower concentrations of 5a-DHT are also inhibited The affects of h0@ M 5a-DHT or the anhiandrogan R2956 by 5f3-DHT at 10@ M. This result is similar to that seen with (2) on growth of the MCF-7 call line are shown in Chart 1. high molar concentrations of estrogen and human breast 5a-DHT stimulates cell division above control levels. Inhibi cancer cell lines as previously observed (7). lion by 10@ M R2956 is apparent after 2 days in culture. A The overall stimulation of precursor incorporation into stimulatory effect of androgen and an inhibitory effect of protein or nucleic acid may exceed the net increment in call antiandrogen on total protein par dish are also seen. The division shown in Chart 1 because of the growth-limiting, exact mechanism of the R2956 affect may not be via an serum-free conditions used which probably enhance cahab interference with an androgen-stimulated pathway as dis olism of cellular macromolecules. cussed below, since androgans incompletely reverse the If the cells are responsive to 5a-DHT, one would predict effects of anliandrogen treatment. In the experiment that they would contain specific cytoplasmic androgen me shown, the calls were in serum-free medium. We observe captors. Five to 20% sucrose density gradients in 10% glyc that the calls grow more slowly under serum-free conditions anol of cytoplasmic extracts incubated with radiolabalad 5a- even when the medium is supplemented with hormones. DHT are shown in Chart 3 for the andmogen-rasponsiva This is not surprising considering the many potentially MCF-7 cell line. [3H]-5a-DHT (5 x 10@ M) is bound ho a growth-promoting components in serum. Nonetheless, it is molecule that sediments at about 8 5. This peak of binding clear that androgen stimulates cell division above control. is totally competed by the addition of a 100-fold excess of The effects of various concentrations of 5a-DHT on unlabeled 5a-DHT ona 1000-fold excess of the antiandrogen [3H]thymid me and [14C]Ieucine incorporation inb macro cypnoherone acetate (6-chloro-1 7a-acehate-1 ,2a-methylene molecules in the MCF-7 human breast cancer call line are 4,6-pmegnadiene-3,20-diona). A 1000-fold excess of unla shown in Chart 2. Incorporation of amino acid on nucleoside baled tamoxifen (ICI 46474; trans isomer of 1-[4-(2-dimahhy begins to increase oven control levels at a 5a-DHT concan laminoathyoxy)phenyl]-1 ,2-phanyl-1-but-1-ene citrate), an tration of 5 x 10-s M. 5a-DHT at 10@ M stimulates thymidina anhiestrogan, competes with less than 10% of the [3H]-5a- incorporation 300% and leucine incorporation 200% when DHT bound to receptor, suggesting that the [3H]-5a-DHT is incorporation malesare measured 48 hr after the addition of binding to an androgan receptor site. We ware curious as ho hormone. Themeis a rapid decrease in stimulation seen as whether tamoxifen might compete with DHT for androgan 5a-DHT concentrations rise above 10_6M. In the experiment receptor sites, since an appreciable response to antiestro shown, 10@ M 5a-DHT reduces incorporation to control gen therapy has been reported in women with melashalic levels when measured at 48 hr. Frequently, values signifi breast cancer even if tumor samples lack estrogen receptor cantly below control are obtained at this steroid concentra (14). lion. This variation may be due ho metabolic effects dis Binding of [3H]-5a-DHT to cytoplasmic extracts is shown cussed below. The steneoisomen 5f3-DHT is nonstimulahomy in Chart 4. Using a daxtran-coated charcoal assay, a high at any concentration tested (Table 1). If the cells are left in affinity receptor site is demonstrable. The straight line ob 5a-DHT at concentrations >10@ M, the calls round up from tamed by Scatchard analysis (20) of the binding data (Chart the surface of the dish, detach, and die after about 72 hr. 4, inset) suggests that [3H]-5a-DHT is binding to a class of This effect may be nonspecific since 10@ M 5f3-DHT also receptors of uniform affinity (Kd = 8.7 x 10'Â° M, r = 0.982). inhibits these calls. 5f3-DHT, 10@ , is without effect. Cell There are 45 fmoles of [3H]DHT bound par mg of cytoplas

0 45

I (I) --0 Chart 1. The effects of 10' M 5a-DHT or the anti 0 androgen R2956 on net protein synthesis in MCF-7 U, 35 @@ human breast cancer. Details are given in Materials a. and Methods.'@ 2 U, 0 a.

2 3 4 5 9

TIME (Days(

4612 CANCER RESEARCH VOL. 36

0 0 .C C) C rn a' C 0 C) a 2 0) m 0@ z C) 0 0

a- Chart 2. The effects of 5a-DHT on the incorporation 0 -@> of [3Hjthymidine or [â€œC]leucinein acid-precipitable z 0 material. Values are means of quadruplicate determina 0 z I- tions Â±S.D. 0

-C 0 Co 0 V 2 0 U, CD z 0 0 0 C >. I I- I I

DHT CONCENTRATION(LogM)

Table 1 mic protein in the experiment shown. Our previous analysis Effects of 5a-DHTand 5/3-DHTonfâ€•CJthymidineincorporationin of estradiol receptor in this same call line revealed 83 MCF-7human breast cancer fmoles of [3H]eslradiol binding pan mg of cytoplasmic pro lain with a similar Kd of 6.8 x 10_b (r = 0.986) (7). We (dpmincorporationx 103/pg protein/ invariably observe nearly twice the number of estrogen hr)Control32.8SteroidConcentrationThymidine binding sites in paired cytosol preparations. These differ 3â€¢5a5a-DHT107 Â± ences in number of binding sites do not allow one to differ antiata between estrogen and androgen receptors in these Â±4.2 0.955f3-DHT10@ 10-651.7 63.4 Â± cells. We therefore examined the receptors for differences in Â±2.8 specificity of binding in the cross-competition studies 1.0a 10-i33.5 35.0 Â± shown in Charts 5 and 6. In Chart 5 are shown results of Means of triplicatedeterminationsÂ± S.D. incubating 17f3-[3H]eshradiol with cytoplasmic extracts from MCF-7 human breast cancer in the presence of various concentrations of unlabeled competing steroids. Unlabeled 1 7f3-estradiol and diathylshilbestrol compete with [3H]eslradiol for receptor sites. The failure to observe the exactly predicted competition by unlabeled 17f3-eslradiol 6 may be due to the sequence of addition of unlabeled steroid 5x10'M to cytosol followed by labeled steroid. The antieshmogens 5 nafoxidine, tamoxifan, and Cl 628 (tniphenylathylena demiva lives) all competed at least 80% of the specifically bound 0 1000xTamoxifen 4 [3H]estmadiolfrom receptor when present in 2000-fold molar excess. At 2000-fold molar excess, 5a-DHT, testosterone, a. 0 @4-androslenedione,and the anhiandrogan R2956 all fail to I 3 compete significantly with [3H]ashradiol for binding sites. This strongly suggests that [3H]estradiol is bound to a me 2 captor with a restricted binding affinity for estrogens and antiestrogens. Results of a similar experiment are shown in Chart 6, but [3H]-5a-DHT was used as the trace. The ability +lOOx DHT of various steroids to compete with [3HJ-5a-DHT for recap . +1000 x Cyproterone ton sites was examined . 5a-DHT and testosterone as well as Acetate antiandrogans R2956 and cyproterone acetate completely Bottom 5 10 15 20 25 Top 20% 5% prevent [3H]-5a-DHT from binding to specific binding sites. Fraction Number The slightly excessive competition of a 2-fold molar excess Chart 3. Sucrose density gradients of androgen receptor in MCF-7 human of unlabeled 5a-DHT probably is due to the order of addi breast cancer. Cytosolextracts were incubated with 5 x 1O@M [3H]-5cr-DHT alone, plus 5 x 10-. Mtamoxifen, plus 5 x 10-v M 5a-DHT, or plus 5 x 10-. M lion used. Unlabeled steroid is added to cytoplasmic extract cyproterone acetate. BSA, â€œC-labeledbovineserum albumin marker. followed immediately by labeled ligand. The use of some

DECEMBER1976 4613

40 0

C 4) 0 a 0 0) E in 4) 0 0.08 E r0.9824 I- Kd=8.7xlOlO M I 0.06 Chart 4. Binding of [3H]-5a-DHT to cytoplasmic extracts from MCF-7 human breast cancer. The data are replotted z B/F according to the Scatchard method (20) (inset). B/F, bound/ 0.04 free. 0

>- -J @.1 0.02 0 U- 0 w a- 10 50 U) BOUND (fmoles/mI)

5x1O@9 DHT CONCENTRATION (M)

.@ â€˜C'o x a. 0

Chart 5. Ability of various steroids to compete with [3H]estradiol for specific binding sites in MCF-7 human breast cancer. DES, diethylstilbestrol. Shaded area, Â±2 S.D. of uncompeted binding.

2 200 MOLAR RA'flO COMPETITOR/13H3ESTRADIOL what lass than saturating concentrations of labeled ligand Because of the ability of unlabeled testosterone to com will also contribute to the excessive apparent competition pete with [3H1-5a-DHT for androgan receptor sites, we ax by unlabeled 5a-DHT. While 17f3-estmadiolcompetes with amined the binding of [3H]testosterone to receptor directly. [3H]-5a-DHT for receptor sites, it is about 100 limes lass Since themeis evidence that testosterone and not 5a-DHT is affective as a competitor than are the 2 androgans. The the â€œactiveâ€•andmogenin some tissues (13), we hoped that antiastrogans nafoxidina and lamoxifen amalass effective large differences in the affinity of testosterone and 5a-DHT competitors than either antiandrogen at equivalent molar for receptor might suggest which was the biologically male ratios. The reduced C-19 mahabolita, ehiocholanolone, does vant hormone. The binding data are shown in Chart 7. The not compete with [3H]-5a-DHT for receptor sites. Although binding data are replotted in the inset according to the the results amanot shown in this chart, we ware unable to Scatchard technique (20). The straight line that is obtained demonstrate significant competition for the receptor site by (r = 0.939) suggests that testosterone is binding to a single 5/3-DHTeven when it was present in 2000-fold molar excess. class of receptors of uniform affinity. The dissociation con We conclude that these calls contain 2 receptors: an and no slant for testosterone, 1.3 x 10@ M, is about 2-fold greater gen receptor with a much lower binding affinity for estro than that obtained for 5a-DHT. This small and possibly gen, and an estrogen receptor with virtually no affinity for insignificant difference in binding affinity does not allow androgens. h7p-Estradiol and the antiandrogan cyprohen conclusion as to whether 5a-DHT and/or testosterone is the one acetate appear ho have similar affinities for the andro active androgen. For this particular cytosol preparation, we gen receptor. found total 5a-DHT- and testosterone-binding capacity to

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0 x 0

0 Chart 6. Ability of various steroids to compete with [3H]-5a-DHT for specific binding sites in MCF-7 human 0 breast cancer. Shaded area , Â± S.D. of uncompeted I binding.

Molar Ratio Competitor/DHT

w z 0 0 U, I- @ U)g .@ U)a)U, C.' r 0.9392 K,j1.3x10'9 M Chart 7. Binding of [3H]testosterone to cyto zo plasmic extracts from MCF-7 human breast cancer. DC The data are replotted according to the Scatchard 0.01 method(20)(inset).B/F, bound/free. >-E B/F -J 5 0 U. U,0 a. 0.002 U)

TESTOSTERONECONCENTRATION (M)

be within 5% of each other. The good agreement of these affinity of the calls for [3H]DHT appeared to be 3 limes numbers with the results shown in Chart 6 suggests that higher at 37Â°thanat 0Â°,andboth dissociation constants are testosterone and 5a-DHTare binding hothe sameandnogan within a factor of 10 of that seen on cytosol preparation at 0Â° receptor site. (Chart 4). While the somewhat higher affinity of binding at Comparison of Charts 2 and 4 reveals that there is about a 37Â°is surprising, this result may be due to temperature 100- to 1000-fold discrepancy between concentrations of dependent transformation of receptor and nuclear Inanslo DHT that half-maximally saturate andnogan receptor and cation that decreasesthe apparent Kd.It is certainly possi concentrations that half-maximally stimulate either leucine ble that a transformed receptor on one bound to chromatin or thymidine incorporation. It would appear that every me sites might have an altered affinity for steroid . Whether or captor site would have to be occupied by androgen before not this increase in binding affinity at 37Â°issignificant, we any induced response is seen. One possible explanation is must conclude that at leash it is not strikingly reduced, the that binding of [3H]DHT to cytosol preparations at 0Â°is of major point of the experiment. Furthermore, andmogan is much higher affinity than that seen in intact cells at physio clearly able to enter and bind to high-affinity sites in these logical temperatures or that andnogen cannot enter intact cells. Thus, alterations in either binding affinity onandrogen cells. To explore this possibility intact cell binding expemi uptake in intact cells at 37Â°do not appear to explain the ments were performed at 0Â°and37Â°.Scatchardanalysesof discrepancy between androgen binding and cell shimula the results are shown in Chart 8. At both 0Â°and37Â°essen lion. The straight lines obtained by Scatchard analysis at 0Â° tially the same number of binding sites amaidentified. The and 37Â°(r = 0.956 and 0.994, respectively) suggest that,

DECEMBER1976 4615

active androgan available to the calls. In results not shown 5a-andnostanediol was detectable in call homogenates as early as 1 mm following addition of [3H]-5a-DHT to the medium. Because of the similar dissociation constant of the and no 02 gen receptor in these calls for [3H]-5a-DHT as compared to testosterone, one might surmise that 5a-DHT is also an â€œaffactomâ€•androganin these calls. For this to be true, one would expect the cells to contain 5a-meduchaseactivity ho convert circulating testosterone to 5a-DHT. This question was examined directly by incubating the cells with [3Hjtestostarona for various times. At 10 mm, i hr, and 4 hr Kd : 2.2 x 1O-10M the media, crude cytosol, and crude nuclear pellet were (r: 0.994) examined for radioactivity migrating on thin-layer chnoma B/F tognaphy plates in the position of 5a-DHT. At 1 hr 5.9% of the counts extracted from the nuclear pellet migrated in the 0.1 positions of androstanediol and 5a-DHT. At 4 hm, of the counts that could be recovered in the nuclear fraction 13.9% migrated in the position of 5a-DHT and androstane diol. Obviously, the sum of both of these steroids represents a minimal estimate of 5a-neduchasaactivity.Al 1 hr 6.4% of the radioactivity found in the supemnatant fraction after the nuclei were sedimented migrated in the positions of and ros tanediol and 5a-DHT. This was reduced to 5.16% at 4 hr. Less than 0.2% of the radioactivity in the media migrated with androstanadiol or 5a-DHT at any time studied. Recov any monitored by adding a trace of [14Cjtastoshanoneho the 0 10 20 30 40 50 mathylene chloride cell extracts was 75.6% for the 0 con BOUND (fmoles/mI) Chart 8. Comparison of specific binding of [3H]-5a-DHT to MCF-7 human A. 0 TIMECONTROL B. 1 HOUR breast cancer cells at 0Â°and37Â°analyzedaccording to the Scatchard method (20). In this experiment binding is to intact cells rather than cytoplasmic extracts. B/F, bound/free. 6 even in intact calls, high-affinity binding of [3HJDHTis to a single class of receptor sites. An alternative potential explanation for the discrepancy between binding affinities and optimal inducing concantra lions of 5a-DHT is that the cell line is capable of transform ing 5a-DHT to an inactive metabolita. Metabolism of andro gens was examined directly by incubating the calls in se rum-free medium containing [3H]5a-DHT and examining ma dioactiva steroid products at various time intervals by thin a layer chromatography (Chart 9). Analysis of the incubation x medium reveals that there is very significant conversion of [3H]-5a-DHT to a steroid migrating with an RFsimilar to that ). 8 HOURS of 5a-androstanediol. After 1 hr of incubation almost one half of the 5a-DHT has been converted to androstanediol. 4 As the time of incubation increases, themeis further convem sion of DHT to androstanediol as wall as the appearance of a new peak of radioactivity near the origin of the plate and a 2 large increase in counts not extractable in the methylene Androstanediol chloride phase, suggesting even further metabolism on con jugahion of androgan to more polar products. The extant of conversion is nearly 70% by 8 hr. Thus, it appears likely that @IN@HT@HT the discrepancy between optimal inducing concentrations 0 10 20 ZI 0 10 20 30 of androgan and concentrations of androgan that bind to receptor is a function of metabolism and resultant inactiva Fraction# Fraction# lion of 5a-DHT to androstanadiol or other mehabolihes. Also Chart 9. Metabolism of [3H]-5a-DHT to [3H]androstanediol by MCF-7 hu man breast cancer. A to 0, analyses of the incubation medium at 0, 1, 4, and it is apparent that small changes in incubation conditions or 8 hr, respectively, following [3HJ-5a-DHTaddition. Recovery of steroid and cell density could drastically allan amounts of biologically 5a-DHT concentration are given in â€œMaterialsandMethods.â€•

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Imol, 100% at 1 hr, and 92% at 4 hr for the nuclear pellets. Values fomthe supemnalants were 92 and 94% at 1 and 4 hr. Effects(3HJthymidineincorporation of 5a-DHT and cyproterone acetate on Recoveries from media wane 92 and 100% at 1 and 4 hr. We in MCF-7 humancancerThymidine breast cannot exclude that in vivo, both testosterone and 5a-DHT incorporation(dpm are biologically active. x 1O-2/@Lgprotein/hr The affects of the anhiandrogen R2956 (2) ware investi Additive 2None (M) Experiment 1 Experiment gated . As previously shown in Chart 1, 10@M R2956 inhibits cell division after 3 days in culture. Additional results are 8.5Cyproterone 22.8 Â±33a 43 Â± 1.4Cyproteroneacetate, 1O@ 23.0 Â±2.7 37.4 Â± shown in Table 2. Thymidina incorporation is stimulated 4.1Cyproteroneacetate, 10' 19.8 Â±3.6 31.8 Â± 120% by 10@ M 5a-DHT when measured at 48 hr. R2956 at a 4.05a-DHT,1O7acetate, 1O@ 27.6 Â± concentration of 10@ M inhibits Ihymidine incorporation to 1.85a-DHT, 22.3Â±4.1 62.4Â± 63% of control values. 5a-DHT, 10@ M, slightly reverses 2.95a-DHT,10-s 37.9 Â±1.1 79.4 Â± 9.9acetate,1O@ + cyproterone 20.9 Â±2.0 57.4 Â± inhibition by 106 M R2956. Although R2956 can compete 10@5a-DHT, with [3H]-5a-DHT for receptor (Chart 6), the exact macha 5.7acetate,1O-@+ cyproterone 35.7 Â±3.4 77.9 Â± nism of action of R2956cannot be assumedto be compehi 1O@5a-DHT, lion for the receptor alone since the process is largely 5.0acetate,10â€”i+ cyproterone 59.1 Â± irreversible by addition of 5a-DHT (Table 2). This may reflect 1O@ a mechanism of action in addition to simple competition for a Means of triplicate determinations Â± S.D. cyhoplasmic receptor. Preliminary results suggest that R2956 may inhibit Ihymidine incorporation but not laucina the cells. Differences between Experiments 1 and 2 shown incorporation in other cell lines that appear ho lack andro in Table 3 are not due to differing incubation times. In gen receptor and are not stimulated by physiological andro Experiments 1 and 2, cells were incubated in hormones for gens. Thus, while the partial reversal of antiandrogen af 36 hr before addition of labeled precursors. However, small facts by 5a-DHT and its known physiological actions (2) differences in total call number plated could easily altar the suggest that R2956 is an antiandrogen, its mechanism of amount of 5a-DHT available during the incubation period. action in tissue culture will require further study. We are We presume that 5a-DHT metabolism as described above is particularly hampered by the rapid metabolism of 5a-DHT likely to be a linear function of cell number, and 2-fold previously described. This may be a sufficient explanation variations between experiments in number of calls plated for our inability to reverse antiandrogen affects. are common. The affects of another antiandrogan, cyprolamona ace The affects of androgans on 4 other human breast call late, are shown in Table 3. Cyprolerone acetate by itself has lines are shown in Table 4. The andnogen-nesponsiva MCF-7 little effect on thymidine incorporation at concentrations cell line is shown for comparison. The other 4 lines all fail to less than 10-6 M. There is some inhibition seen at 10-s and show stimulation by 5a-DHT at 10@ M concentration. Lower 10-s M. 5a-DHT at a concentration of 10@ M nearly doubles concentrations (10@and 10@ M) wane also tested and failed Ihymidina incorporation in 2 experiments. Combination of to stimulate these cell lines. For comparative purposes the 10-s M cyproterona acetate and 10_6 M 5a-DHT results in quantity of androgan receptor is shown in Table 4. The G thymidine incorporation significantly above control but less 1 1 , MCF-7, and Evsa E cell lines have detectable receptor than that seen with 5a-DHT alone. Thus cyproterone acetate activity. Despite this, the Evsa E and G-11 lines do not effects appear to be reversible by andmogan.Theseaxpani respond to androgan under conditions we used. mants would be easier to interpret if it were possible to use molar ratios of cyprotarone acetate and 5a-DHT of about DISCUSSION 200:1 since, as shown in Chart 6, cyproterone acetate com pales lass effectively does than 5a-DHT for androgan recap Our studies strongly support the notion that some human ton sites. Unfortunately, concentrations of 5a-DHT greaten breast cancers, at least in vitro, may be stimulated by and no than 10@ M are required for significant stimulation of the gens. In this work, we demonstrate that androgen receptor cells. Thus, it is difficult to achieve concentrations of cypro temoneacetate that would not be nonspecifically lethal to may be distinguished from estrogen receptor present in the same call line. Furthermona, stimulation of macnomolecular synthesis by androgan is probably mediated by interaction Table 2 with this receptor since 5a-DHT, unlike testosterone, is not Effects of 5a-DHTand P2956on (3HJthymidineincorporationin a substrata for aromatization (5) and shows virtually no MCF-7 h@,manbreast cancer ability to displace [3H]estradiol from estrogen receptor. Addition incorporation Our studies also suggest an interesting mechanism protein/hr)None(M)Thymi (dpm xdine10-2/@Lg whereby tumors may escape stimulation by low levels of Â±2.3a androgen. We have shown that the MCF-7 call line can R2956,1O@ 23.6 Â±1.2 rapidly metabolize 5a-DHT ho andmostanadiol and to as yet R2956,1O-@ 16.2 Â±1.5 5a-DHT, 10-p 44.3 Â±3.0 unidentified malabolilas or possibly conjugates. Previous 5a-DHT, 10-s 57.3 Â±9.2 workers (15, 16) have documented the ability of breast 5a-DHT, 10-i + R2956, 1O@ 17.4 Â±0.45 tumor samples to synthesize active androgen and estrogen 3.2a5a-DHT,106+ R2956,10â€”i25.4 23.2 Â± from androgen precursors such as dahydnoepiandmostem Means of triplicate determinations Â±S.D. one. The metabolic potential of a tumor to either synthesize

DECEMBER1976 4617

Table 4 Effects of 5cxâ€”DHTonvariouslines[â€˜4C]Leucine human breast cancer cell incorporationCell x 1O3/@gprotein/hr)Quantity an drogen me MMCF-732.8line(dpm ceptoraControla 5ct-DHT, 1O@ M 5$-DHT, 10@ 1.012.0G-1132.6 Â±35b 63.4 Â±0.95 35.0 Â± 3.47.2HT-3924.9Â±8.6 32.3Â±2.1 29.8Â± 7.00MDA-23149.5 Â±3.4 20.7 Â±5.6 27.6 Â± 4.00EvsaE31.0Â±0.78 Â±0.38 45.4 Â±1.1 46.7 Â± 35.8Â±2.8 28.0Â±6.815.3

a fmoles [3H]-5a-DHT bound per mg cytoplasmic protein. b Values are means of triplicate determinations Â± S.D. or degrade steroids is an attractive explanation for the J. Steroid Biochem., 5: 352, 1974. 3. Engelsman, E., Korsten, C. B., Persign, J. P., and Cleton, F. J. Estrogens failure of receptor studies alone to predict hormone respon and Androgen Receptors in Human Breast Cancer. Brit. J. Cancer, 30: siveness of target tissues including tumor samples. For 177, 1975. example, a tumor containing estrogen receptor may be 4. Fracchia, A. A., Randall, H. T., and Farrow, J. H. The Results of Adrenal ectomy in Advanced Breast Cancer in 500 Consecutive Patients. Surg. hormone responsive but, because of its ability to synthesize Gynecol. Obstet., 125: 747-756, 1967. estrogen from precursors, a response to castration is not 5. Gual, C., Morato, T., Hayano, M., Gut, M., and Dorfman, R. J. Biosyn observed. Similarly, in our studies of androgen responsive thesis of Estrogens. Endocrinology, 71: 920-925, 1962. 6. Lippman, M. E., and Bolan, G. Estrogen Responsive Human Breast ness, the responsiveness of the cells would have bean Cancer in Continuous Tissue Culture. Nature, 256: 592-593, 1975. missed had we chosen concentrations of steroid solely on 7. Uppman, M. E., Bolan, G., and Huff, K. The Effects of Estrogens and Antiestrogens in Hormone-responsive Human Breast Cancer in Long the basis of receptor affinity. II would be of obvious value to Term Tissue Culture. Cancer Res., 36: 4595-4601 , 1976. attempt hocorrelate tumor response to endocrine manipula 8. Lippman, M. E., Bolan, G., and Huff, K. The Effects of Glucocorticoids lion with an evaluation of receptors as well as steroid mala and Progesterone on Hormone-responsive Human Breast Cancer in Long-Term Tissue Culture. Cancer Res., 36: 4602-4609, 1976. bolic potential. 9. Lippman, M. E., and Huff, K. A Demonstration of Androgen and Estrogen We (9) and others (3, 12) have reported the existence of Receptors in Human Breast Cancer Using a New Protamine Sulfate androgan receptor in fresh human breast cancer specimens Assay. Cancer, 38: 868-874, 1976. 10. Ustoa, B. P. Thin Layer Chromatography of Steroids. Methods En which we have shown to be distinct from estrogen receptor. zymol., 15: 3-157, 1969. The usefulness of estrogen receptor in guiding clinical daci 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265- sions involving hormonal manipulations in human breast 275,1951. cancer is already established (14). Conceivably, analysis of 12. Maass, H., Trams, G., Nowakowski, H., and Stolzenbach, G. Steroid tumor material for androgen receptors might similarly be of Hormone Receptors in Human Breast Cancer and the Clinical Signifi cance. J. Steroid Biochem., 6: 743-749, 1975. value. 13. Mainwaring, W. I. P., and King, R. J. B. Steroid Cell Interaction, pp. 288- Possibly the most significant aspect of the present work 295. Baltimore: University Park Press, 1974. concerns the availability of a markedly androgen-raspon 14. McGuire, W. L., Carbons, P. P., Sears, M. E., and Escher, G. C. Estrogen Receptors in Human Breast Cancer, an Overview. In: W. L. McGuire, P. sive human cell line in long-term tissue culture. Although P. Carbons, and E. P. Vollmer (ads.), Estrogen Receptors in Human studies on the Shionogi carcinoma (21) and a recently char Breast Cancer, pp. 1-8. New York: Raven Press, 1975. actenized Syrian hamster tumor of the ductus defenens (17) 15. Miller, W. R., McDonald, D., Forrest, A. P. M., and Shivas, A. A. Metabo lism of Androgens by Human Breast Tissue. Lancet, 1: 912-914, 1973. have yielded important data, these cell lines show rather 16. Nimrod, A., and Ryan, K. J. Aromatization of Androgens by Human limitedandnogenresponsivenessandhave not been me Abdominal and Breast Fat Tissue. J. Clin. Endocrinol. Metab., 40: 367- 372, 1975. ported to be killed by anhiandnogans. Thus, the currant 17. Norris, J. S., Gorski, J., and Kohler, P. 0. Androgen Receptors in a system may provide palpable advantages for the study of Syrian Hamster Ductus Deferens Tumour Cell Line. Nature, 248: 422- androgen action. We are currently attempting to develop 424,1974. 18. Paulsen, C. A. The Testes. In: G. H. Williams (ed), Textbook of Endocri cell lines resistant to the effects of antiandrogans in the nology, pp. 323-334. Philadelphia: W. B. Saunders Co., 1974. hope that such cells will offer interesting reagents for the 19. Quadri, K., Kledzik, G. S., and Meites, J. Enhanced Regression of DMBA analysis of the mechanism of androgen action. induced Mammary Cancers in Rats by Combination of Ergocornine with Ovariectomy or High Doss of Estrogen. Cancer Res., 34: 499-501 , 1974. 20. Scatchard, G. The Attractions of Proteins for Small Molecules and Ions. Ann. N. Y. Aced. Sci., 51: 660-672, 1949. REFERENCES 21. Smith, J. A., and King, R. J. Effects of Steroids on Growth of an Andro gen-Dependent Mouse Mammary Carcinoma in Cell Culture. Exptl. Cell Res., 73: 351-359, 1972. 1. AMA Committee on Research. Androgens and Estrogens in the Treat 22. Teller, M. N., Stock, C. C., Stohr, G., Merker, P. C., Kaufman, R. J., ment of Disseminated Mammary Carcinoma. J. Am. Med. Assoc., 172: Escher, G. C., and Bowie, M. Biologic Characteristics and Chemother 1271-1274, 1960. apy of 7,12-Dimethylbenz[ajanthracene-inducedTumors in Rats. Cancer 2. Azadian-Boulanger, G., and Bonne, C. Anti-Androgenic Action of R2956. Res., 26: 245-254, 1966.

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Marc Lippman, Gall Bolan and Karen Huff

Cancer Res 1976;36:4610-4618.

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