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Home , Corticosteroid, Dexamethasone, Glucocorticoid, Glucocorticoid receptor, Progesterone receptor, Transcortin

[ RESEARCH 36, 4602-4609, December 1976] The Effects of and on responsive Human in Long-Term Tissue Culture1

Marc Lippman, Gail Bolan, and Karen Huff

Medicine Branch, National Cancer Institute, Bethesda, Maryland 20014

SUMMARY mon mechanism involving -cyloplasmic in heraction. This is followed by interaction of the steroid Glucocorticoids, at physiological concentration , inhibit receptor complex with the target cell ganome, leading ho call division and thymidina incorporation in three lines of accumulation of new species of mRNA that code for pro human breast cancer maintained in long-term tissue cul hems characteristic of the observed tissue-specific me tune. At steroid concentrations sufficient ho inhibit thymi sponses (4). dine incorporation 50%, little or no affect is seen on Glucocorticoids appear to be essential for the growth and synthesis 48 hr after hormone addition. All three of these differentiation of immature rat in organ lines are shown to have receptors demon culture (30). Similar effects have been noted in normal strable by competitive protein binding assays. Receptors human mammary gland in primary tissue culture (5). Gluco are extensively characterized in one line by sucrose density corticoids have also been reported to increase expression gradient analysis and binding specificity studies. Good con of mouse mammary tumon virus in tissue culture (20). De relation between receptor-binding specificity and biological spite these observations, suggesting a stimulahony role for activity is found except for progesterone, which binds to glucocorticoids in mammary cancer, pharmacological ad but is noninhibitony. Cross-compe ministration of glucocorticoids is eithen without effect or tihion and quantification studies demonstrate a separate leads to tumor regression in patients with breast cancer in receptor for progesterone. This receptor has limited bind about 15% of cases (27). The fact that adrenalectomy is ing specificitias restricted largely to progestahional agents, frequently a satisfactory form of palliation for metastatic whereas the glucocorticoid receptor bound both glucocom breast cancer may not be construed as evidence that gluco hicoids and progesterone. Two other human breast cancer corticoids inhibit the growth of mammary cancer. The lines neither contain glucocorticoid receptor nor are in mechanism of this effect has usually been ascribed hoabla hibiled by glucocorticoids. It is concluded that in some hion of C-19 precursors of andmogen and/or since cases glucocorticoids can directly limit growth in human physiological amounts of glucocorticoids must always be breast cancer in vitro without requiring alterations in other replaced following surgery (17). Thus, whether on not glu trophic . cocorticoids have any direct effect on human breast cancer has remained moot since one might easily imagine that INTRODUCTION pharmacological doses of glucocorticoid could be palliative indirectly, as, for example, through suppression of the The estrogen-dependent nature of some breast cancer hypothalamic pituitary axis, in affect, a medical adrenal has bean amply demonstrated in clinical settings (3), animal ectomy. models (9), and recently in tissue culture (10). Physiological In this work, we describe the affects of glucocorticoids on concentrations of estrogen promote tumor growth, while 5 human breast cancer call lines maintained in tissue cul withdrawal of estrogen leads to either slower tumor growth tune for at leash 1 year. Some of these cell lines show on regression. Recent work has emphasized the importance marked inhibition of call division and precursor incorpo of an initial interaction between estrogen and specific cyto rated by glucocorticoids under conditions that preclude the plasmic receptor as the 1st step in the pathway influence of other steroid on polypeptide hormones. Re through which steroid hormones regulate phanohypic sponsive calls contain high-affinity limited-capacity recap expression. tons for glucocorticoids demonstrable by competitive pro The role of glucocorticoids in human breast cancer is far hem binding analysis and sucrose density gradients. Bind lass clear. Glucocorticoids evoke strikingly diverse me ing is highly specific for biologically active glucocorticoids sponsas in different target tissues (28). Nonetheless, most and correlates reasonably with the dose response of these investigations have landed to support the notion that such cells ho . disparate affects as enhanced RNA and protein synthesis in the (8) or lymphocyholysis (18) amamediated by a com MATERIALSAND METHODS , This is Paper 2 in a series on hormone-responsive human breast cancer cells in long-term tissue culture. Cells and Culture Techniques. Methodsfor establish Received November 4, 1975; accepted September 10, 1976. ment or sources of cell lines and their propagation are as

4602 CANCERRESEARCHVOL. 36

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1976 American Association for Cancer Research. Glucocorticoids and Breast Cancer in Vitro previously described (10). The human and mammary na pension, 0.8 ml, was added to 12- x 75-mm tubas. [3H]Dexa tunes of these cells have been substantiated by chromo ,0.2 ml, with or without an excess of unlabeled somal analysis, morphological features, and a- competitor was added to each tube and mixed. Tubes were synthesis (11).All lines havebeen maintained in culture for incubated at 0°for3 hr on37°for1 hr. Cells ware collected at least 1 year without loss of hormone responsiveness. by cantnifugation in a Senofuge (Clay-Adams, Parsippany, Precursor Incorporation and Growth. Methods for the N. J.), for 30 sec and washed twice in Dulbacco's phos measurementof [3Hjthymidineand ‘4C-labeledaminoacid phata-buffered ; pH 7.4, supplemented with 5 mM incorporation including incubation times, labeling tech . Cell buttons were suspended in water and son niques, and precursor specific activities havebeandetailed ically dispensed for 4 sec in a Branson sonicator at the previously (10, 11). wane determined using the lowest setting; aliquots were counted in Aquasol (NewEng technique of Lowry at a!. (14). All hormonal additions are land Nuclear, Boston, Mass.) in a Packard liquid scintilla done with cells maintained in 1% serum from which endog lion counter (efficiency for [3H], —35%). enous steroid and polypeptide hormones have been ma moved by treatment with activated charcoal (1). Removalof endogenous steroid is monitored by the addition of a small RESULTS amount of 3H-labeledsteroid to the serum and following removal of radioactivity by charcoal ho less than 1% (see The inhibitory effect of 10@ M on call also Ref. 11). As previously noted (10), these experiments growth of MCF-7human breast cancer is shown in Chart 1. can be performed with identical results using serum-free Dexamathasona inhibition is apparent after the 1st 24 hr in conditions. hormone as compared to cells in non-hormone-containing Steroid Receptor Assays. Sucrosedensity gradient anal medium. Despite diminished thymidine incorporation, glu ysis (7), dextran-coated charcoal assays,and multiple com cocorticoid-treahedcells multiply very slowly and, by phase petition studies (16)followed previously published methods microscopy, appearmorphologically indistinguishable from except as noted . were incubated with Initiated control cells. steroid with or without an excess of unlabeled competitor The effect of dexamethasone on thymidine incorporation as described in the figure legend. Following a 2-hr incuba into DNA is shown in Chart 2. As indicated, thymidine incor lion at 0°,the cytosols were treated with dextran-coated poration is inhibited ho55% of control levels when inhibition charcoal (16) and layered onto sucrose gradients (7). Gra is measured after 48 hr of exposure to 5 x 10_6M dexameth dients were centrifuged at 275,000 x g for 18 hr at 4°in a asone. No concentration of dexamathasone is stip'iulahory. Beckman Model L5-65ultracentnifuge.[3H]Dexamethasona, Inhibition is apparent with as little as 5 x 10@M dexametha 22 Ci/mmole, and [3H]progestemone,86 Ci/mmole, were sona and is greater than 50% at 5 x 10_aM. The total obtained from Amensham/SeanleCorp.(Evanston, Ill.) and decrease in thymidina incorporation is lass than that in used without repunificahion.Unlabeled steroids were ob duced by anlieshnogens in the same cells for which pro tamed from Steraloids (Plattsburg, N. Y.). For competition longed incubation in 5 x 10@Mhamoxifan(ICI46474;trans studies, cytoplasmic extracts were added to 12- x 75-mm of 1-[4-(2-dimatrylammnoethoxy)phenyI@-1,2-diphen chilled glass tubes containing unlabeled steroids as 1000- ylbuh-1-enacitrate) is lethal (10).Interestingly, there is insig fold concentrates. Immediately thereafter, [3H]dexamatha nificant inhibition by glucocorticoid of laucine inconpona sone (5 x 10@M)was added to each tuba. Following over lion into protein when protein synthesis is measured at 48 night incubation, specifically bound dexamethasone was assessedusing dextran-coated charcoal to separatebound 40 and free steroid. For competitive binding studies, competed and uncom peted tubes were generally nun in triplicate at each steroid concentration. Following overnight incubation at 4°,specif Control ically bound steroid was measuredby daxtran-coaled char 30 coal assay.Binding data ware preparedfor Scatchard anal a ysis (24) using a programmable Hewlett-Packard 9830A K .@ desktop computer. Individual binding components for the 0 progesterone binding experiments were estimated using the methodology of Buller at a!. (2). In all binding studies .5 12 cells were preincubatad for at least 24 hr in serum-free, 0 + iO@ M Dexamethasone hormone-free medium ho allow for dissociation of steroid from maximal cytoplasmic localization of receptor sites. On occasion, 2 changes of serum-free medium spanning 48 to 72 hr were used (see “Results―). Whole-CellBindingStudies. Cellsgrowinglogarithmic ally were changed to serum and hormone-free Eagle'smini mal essentialmedium. After 24 hn,cells were harvestedwith 6 7 8 Day trypsin-EDTA, washed, and then suspended in Dulbecco's Chart 1. Effect of dexamethasone on cell multiplication in MCF-7 human phosphate-buffered saline, pH 7.4, supplemented with 5 breast cancer. Dexamethasone is added at 0 time, dishes are counted in mM glucose at 0°and a density of 10 calls/mi. Cell sus triplicate, and values shown are means ±S.D.

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60 This peak of dexamathasone binding is not , since R5020 does not compete for transcortin sites (22) and 0 0 I transcortin sediments at about 4 5 on sucrose density gradi C a, . ants (29) as compared to the 8 S binding peak shown in 0 a. Chart 3. Thus, this peak of [3H]dexamethasona binding . . orSO does not represent contamination by plasma protein.

0 A high-affinity glucocorticoid binding site is also demon strablabya competitiveproteinbindingassayusingdex a. 0 Iran-coated charcoal. Results are shown in Chart 4. A Scat .2C 40 a 0 . C —. 4, C 430

BSA

@ 20 I -... I I J@ C 0 “ -12 @11 -10 -9 -8 -7 -6 -5 5.OxlO8M Dexamethasone Concentration (Log Ml [3H]Dexamethasone Chart 2. Effect of dexamethasone on thymidine incorporation in MCF-7 a human breast cancer in tissue culture. Mean values of quadruplicate deter minations are shown ±S.D.Cells are incubated in 1O@Mdexamethasone for a. 48 hr and then pulsed for 1 hr with [3H]thymidine and acid-insoluble radioac 0 tivity determined as described in “MaterialsandMethods.― I

1 Effects of dexamethasone or cortiso! on (‘4C]!eucineincorporation radioactivityConcentrationintoTableacid-insoluble

of steroid(M)DexamethasoneCortisol0 ±0.688 10-i 39.1 ±2.3 ±2.8 10@8 46.6 ±6.8 43 ±3.4 10@ 37.3 ±2.9 40.7 ±2.0 10 20 30 10-639.6 37.3 ±3.335.4 38.6 ±1.2 Bottom (20%) Top (5%)

a Values that are [“C]Ieucine dpm x 10_2 per mg protein per hr ± Fraction Number S.D. were determined in triplicate. Incubations in steroid were for 48 hr followed by a 1-hr pulsewith [‘4C]leucine.Precursorincorpo Chart 3. Sucrose density gradients of glucocorticoid receptor from MCF-7 ration was linear with respect to time for incubations of this length. human breast cancer. Gradients are 5 to 20% sucrose made up in 10% . BSA, “C-labeledbovine serum marker run with each gradient. hr (Table 1), implying a specific block at some locus that greatly diminishes the number of cells entering the DNA

0 specific phase of the call cycle. a If the response of the calls to glucocorticoids is mediated E

through an initial interaction with receptor, it should be 0 possibletodemonstratehigh-affinity-bindingmoleculesin E 2 the of these cells. As shown in Chart 3, sucrose 0 U) Kd 2.4 x 10' M density gradients of cyloplasmic extracts reveal a peak of I [3H]daxamathasone that sediments at about 8 S. This peak 050 • r0992 x of binding is not seen when matched extracts amaincubated U, 0 with a 100-fold excess of unlabeled daxamathasona, sug 0 2 gasting that the binding site is saturable. Daxamathasona 0

does not bind to -binding (transcor >. 0.10 tin) (12). R5020 (@‘‘4-pregnadiena,17,21-dimethyl-1 9-nor 0 200 1000 3,20-dione) (kindly supplied by Dr. J. P. Raynaud of the B (fmoles/mf) 0 a. Roussal Corp. , Romainvilla, France) is a synthetic progesta U) lional agent that has bean reported to bind to progesterone 1x10' 5x10' receptor but not plasma-binding components (22). As DEXAMETHASONE CONCENTRATION (M) shown, this compound in 100-fold excess also obliterates Chart 4. Binding of [3H]dexamethasone to glucocorticoid receptor from MCF-7 human breast cancer. The data shown are replotted in the inset using the peak of daxamathasona binding, providing evidence the Scatchard technique (24). Binding was determined using a dextran thatR5020 can compete forglucocorticoidreceptorsalso. coated charcoal competitive binding assay. B/F, bound/free.

4604 CANCER RESEARCH VOL. 36

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1976 American Association for Cancer Research. G!ucocorticoids and Breast Cancer in Vitro chard analysis (24) of the binding data is shown in the inset. The straight line obtained (r = 0.992) is consistent with a single class of necephonsites of uniform affinity being mea sured by this assay. The receptor has a dissociation con slant of 2.4 x 10@@Mat 4°.Waestimate there to be about 764 fmolas of [3Hjdexamethasone bound par mg of cytoplasmic protein in this experiment. In another experiment the disso ciation constant was 5.9 x 10@ M (r = 0.998). Both the Kd and quantity of the receptor are within the range of values found for glucocorticoid receptors in other systems (28). Additional characterization of the cytoplasmic receptor in these calls is provided by the cross-competition studies shown in Chart 5. The ability of various concentrations of unlabeled steroids ho compete with [3H]daxamethasone for binding sites is shown. The concentration of [3H]dexa mathasona used, 5 x 10@ M, is such that >75% of all pro lain bound steroid is associated with high-affinity receptor sites whereasnonspecific binding is minimal. As expected, unlabeled dexamethasone readily competes with [3H]daxa Moist Ratio COInpSdSO,/[3HJD.X.methSSOn. methasona for the receptor. also competes for Chart 5. Specificity of the glucocorticoid receptor in MCF-7 human breast the same binding site but is somewhat less potent than cancer. 5haded area at the top, maximal specific binding ±S.D.The concen tration of [3H]dexamethasone used as “trace―was5 x 1O@M. DHT, dihydro dexamethasona. 11a-Cortisol (apicortisol), a biologically . inactive staraoisomer, does not compete for binding sites even when present in 2000-fold excess. Similarly, the ma ducad mehabolite, hetrahydmocortisol, is also unable to com pate [3H]daxamathasona from receptor. 17f3- and 5a-dihydrolastosterona amaunable to compete with dexa mathasone for receptor when present in 200-fold molar excess,although some competition is apparent at 2000-fold I excess. Cortaxolona, an (19), does not signifi cantly compete with dexamathasone unless present in 200- fold excess. However, since the apparent binding affinity of dexamethasone to receptor is higher than that for cortisol (Chart 5), it is likely that cortexolona would co@@etemore I effectively with cortisol for binding sites than would dexa methasona. R5020, a progashational agent (22), also can compete with

[3Hjdexamethasona for binding activity. This agent does not Log Steroid Concentration bind to tnanscortin, but its affinity for glucocorticoid cyto Chart 6. Effects of various steroids on thymidine incorporation into DNA plasmic receptor sites is unknown. Unlabeled progesterone in MCF-7 human breast cancer. Shaded area, thymidine incorporation in is effective in competing with [3H]dexamalhasonafor bind control cells ±2 S.D. ing sites. Progesterone can bind to glucocorticoid recap tons in hapaloma cells (23). The observation that progester the MCF-7 cell line. The apparent higher potency of dexa one can inhibit [3H]dexamathasone binding raises the pos methasone may in part be related ho binding of cortisol to sibilihy, however, that the receptor might be a progesterone the transcortin binding sites in the hormone-stripped― 1% rather than a glucocorticoid receptor. This question is ax serum used. Since >99% andoganous serum cortisol has amined extensively in experiments described later. Progas bean removed , an appreciable fraction of the added cortisol temonecompetes for as many [3H]daxamehhasone-binding would be bound to this transcortin since its dissociation sites as do biologically active glucocorticoids when maxi constant for cortisol at 37°hasbeen estimated to be about mally effective doses of each are used (Chart 5). This sup 3.3 x 10_8 M (31). lla-Hydnoxy contisol (epicortisol) does ports the results of the Scatchard analysis shown in Chart 4 not inhibit thymidina incorporation into DNA at any concan which suggested that [3Hjdaxamathasone was bound ho a tration used. As shown in Chart 5, the ha compound does single class of receptors of uniform affinity. The compeli not compete with [3H]dexamathasona for receptor binding; lion data suggest that the affinity of progesterone for the therefore biological activity would not be anticipated. Simi glucocorticoid receptor is between 10- and 100-fold lower Ianresults are obtained with the reduced cortisol matabolita than that for daxamethasona. tatrahydrocortisol. This compound neither binds to recap The effects of various steroids on thymidina incorporation ton (Chart 5) nor inhibits DNA synthesis. are shown in Chart 6. Active glucocorticoids such as daxa Cortexolone, an antiglucocorticoid (19), is able to com mathasone and cortisol affectively inhibit DNA synthesis in pale with [3Hjdaxamahhasone for receptor when present in

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M. Lippman et a!. large molar excess. As shown in Chart 6, only 10_6M cortex glucocorticoid receptor of rat hepahoma without inducing olone is able to exert inhibitory effects on MCF-7 cells. glucocorticoid effects. Also the results shown in Chart 7 Thus, there is good agreement between relative binding clearly establish that progesterone can compete for dexa affinity and biological effect. methasona-binding sites. However, unlike glucocorticoids, R5020, a synthetic progastational agent (22), inhibits progesterone appears to be biologically inactive in terms of [3H]thymidina incorporation at a concentration of 10@ M. effects on thymidina incorporation (Chart 6). Quantitative Although this compound has been shown to distinguish analysis of the binding data reveals a significant difficulty completely between and transcortin with the interpretation that progesterone is simply binding (21), our results (Charts 5 and 6) suggest that it may also to just the glucocorticoid receptor. In 1 experiment 553 have glucocorticoid activity mediated by an interaction with fmoles of [3H]pmogaslemonewere specifically bound per mg glucocorticoid receptor. In as yet unpublished observa ofcyhoplasmicproteinatsaturatingsteroidconcentrations, lions, we have observed that R5020 will also compete for while the same cytoplasmic extract bound only 280 fmolas glucocorticoid receptor in human peripheral lympho of [3H]dexamethasone per mg protein. In a separate expeni cytes with an apparent affinity equivalent to that of daxa mant on another pnapanation, 410 and 138 fmolas of mathasone. Furthermore R5020 will block phyhohemagglu [3H]pmogestenone and [3H]daxamethasone, respectively, 1mm-provoked milogenasis of these as affac wane bound per mg of protein. Some variation in total lively as does cortisol. numbers of silas is to be expected in tissue culture cells Progesterone binding contrasts sharply with progastar grown and harvested under somewhat varying conditions, one effects on DNA synthesis. Progesterone has no inhibi but reproducible differences in total numbers of binding tony effect on thymidina incorporation (Chart 6) and yet, as sites wane observed in at leash 8 experiments. Generally, shown in Chart 5, progesterone competes effectively for glucocorticoid receptor concentrations were somewhat daxamethasona-binding sites although with a lower affinity. less than those found with progesterone. Whether progas In an attempt to distinghish between the possibility of 1 or 2 hemonereceptor levels vary due to specific hormonal factors receptors for dexamathasone and progesterone, the ability is under investigation. Preliminary results suggest hhahpro of progesterone to compete with [3H]daxamathasona was longed serum-free incubation in the absence of hormones examined in greater detail. In Chart 7 double reciprocal leads ho a loss of progesterone receptor activity. If both plots of specific binding of [3Hjdexamathasone to receptor steroids ware binding to the same site, one would expect performed at various inhibitor (progesterone) concenhma equal numbers of steroid molecules ho be bound at salural lions are shown. The set of lines obtained with a single ing concentrations of steroid. If progesterone is binding ho2 interceptonthey axisatinfiniteextrapolateddexamatha receptors, glucocorticoid and progesterone, one might ax sone concentration is consistent with a true competition pact a difference in total numbers of progesterone and of daxamethasone and progesterone for a common binding glucocorticoid binding sites as already described and a site. different Scatchand plot showing both binding components. [3H]Progesterone binding was initially examined directly The relative closeness of the affinity constants differing by a using relatively low concentrations of progesterone. Lim small factor might make this technically difficult to see. ihed-capacily high-affinity binding was seen . One might as Nonetheless, detailed binding studies of [3H]progestemone suma that this represents [3H]pmogesterone binding to the binding ware carried out across a broader range of concen glucocorticoid receptor, since it has been shown previously trations hhan those previously used. Results are shown in (24) that progesterone can bind with a high affinity to the Chart 8. Scatchard analysis of the binding data (Chart 8, inset), reveals a 2-component curve. Separate elements of

2 x 1O7M

2 0

tfloU,'. Wa C 0 .@

0 z ‘-::@ 0 Control

<.5

a.U, 0.2 0.1 Ui 0.2 0.3 0.4 U)

(nM FREE

Chart 7. Double reciprocal plot of [3H]dexamethasone binding to receptor PROGESTERONE CONCENTRATION )M) in MCF-7 human breast cancer in the presence of various concentrations of progesterone. Reciprocals of “bound―and“free,―[3H]dexamethasonecon Chart 8. Binding of [3H]progesterone to receptors in MCF-7 human breast centration; molar concentrations to right of each line, progesterone concen cancer. The binding data are replotted in the inset using the Scatchard trations. Control, binding of [3H]dexamethasone without added progester technique. Methods for quantification of these binding components are one. given in Ref. 2.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1976 American Association for Cancer Research. Glucocorticoids and Breast Cancer in Wtro this curve were quantified as recommended by Bullemat a!. binding site is 2-fold higher than that of [3Hjdexamelhasona (2). A high-affinity binding component is seen (Kd = 2.9 x for its binding site, unlabeled dexamethasone competes 10-8 M; r = 0.993). This presumably represents progestem poorly for the progesterone-binding site while progesterone one binding to progesterone receptor. Also shown is a 2nd shows a relatively higher affinity for the [3H]dexametha component for the binding of progesterone ho cytoplasmic sona-binding site. Cleanly, if progesterone wane binding to sites. Although this line is slightly lass reliably defined (r = the glucocorticoid receptor, one would axpach ready com 0.927), this 2nd class of sites is certainly saturable with an petition by dexamehhasona since its affinity is not much approximate Kd of 9.0 x 10@ M. lower (Chart 4) but, as shown in Chart 9, daxamethasona Three separate experiments in which [3H]progasterone competes lass effectively than either unlabeled progastam binding was measured across a broad range of concentra one or R5020 and cortisol barely competes at all even at lions are summarized in Table 2. While there is considerable 1000-fold molar excess. We conclude hhat the differences variation in total numbers of high affinity and Iowan affinity in quantity of dexamathasone and progesterone binding sites, 2-component curves are seen in each experiment. sites, as wall as the large diffamar.c€@sinsteroid specificity Thus, careful analysis reveals 2 silas to which progesterone shown in Charts 5 and 9, and the 2-component [3H]proges binds wihh differing affinities in these cells. We think that tanone Scatchand ploh (Chart 8) suggest that these cells Component A is a true progesterone receptor while Compo contain separaha receptors for glucocorticoid and pmogas nent B represents the binding of [3H]progaslarona hogluco temona. These results also suggest that [3H]R5020 may noh corticoid receptor. Some of the variation in Component B be relied upon to determine progesterone-binding sites as quantification may result in the lower binding affinity of this distinct from glucocorticoid sites (6, 26), since it can bind site and the greater unreliability determining the quantity of with high affinity to both classes of binding sites. this binding component. It is obviously more difficult to The affects of dexamathasona on several other human quanhitate glucocorticoid receptor using a ligand with breast cancer call lines are shown in Table 3. A range of nearly 20 times Iowan affinity than that for [3H]daxamelha responses is seen among MCF-7, MDA-231 , and G-11 al sona. These experiments also illustrate the dangers in though all show inhibition of thymidine incorporation by extending Scatchard plots beyond the points actually dater added daxamathasone. The 496 and Evsa T lines are unaf mined (sea Chart 8). The near horizontal line of the 2nd faded by daxamathasona when the parameters of thymi binding component not seen until bound/fill values < 0.01 could make it quite easily confused with nonspecific bind ing. To further resolve the question as to whether or not there is a distinct progesterone receptor, additional cross-com petition studies ware performed in which [3H]progesherona was used as the trace and the ability of various unlabeled steroids to compete with progeshemona was examined. These experiments strongly suggest that themeare sapara ble necephors for dexamathasone and progesterone. Re suIts are given in Chart 9. Unlabeled R5020 and progesler one completely suppress [3H]progestemone binding. Inac hive glucocorticoid melabolitas, such as tetrahydnocortisol and 11a-hydroxycortisol, do not bind at all. Daxamethasone does not displace [3H]progasterone unless present in 200- ho 2000-fold excess and cortisol competes only at 2000-fold excess. 5a-Dihydrotastosterone, which failed to compete Molar Ratio Competitor /[3H]Proo_esterone with [3H]dexamethasone (Chart 5), does compete with Chart 9. Specificity of progesterone receptor in MCF-7 human breast can cer. Shaded area at the top, maximal specific progesterone binding ±S.D. [3Hjprogestarona when present in only 20-fold molar ax Five x 1O@M [3H]progesterone was used as the “trace―inthis experiment. cess. Thus, although the affinity of [3H]progastaronefor its DKT, .

Table 2 Progesterone-binding components in MCF-7 human breast cancer BK.@(x AComponent

10@ of 1O@ of ExperimentComponentsitesb1 M)r0No. sitesK,,@(x M)r8No.

2 2.9 0.993 596 9.0 0.927 685 32.1 3.10.895 0.974257 6569.0 7.50.862 0.900704 219

a Correlation coefficient. b fmoles/mg cytoplasmic protein (total number of sites minus Component A). Individual components of these complex binding curves were separated using published methods (2).

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@30O dine on laucine incorporation are examined. As a control the OC effects of llcr-hydroxycortisol (epicortisol) are also pre E a' 0 santad. In addition, the maximal inhibitory affect seen with E dexamathasona is compared with the quantity of receptor in 4, each of these cell lines. As shown, cells without glucocorti C 4, coid receptor are unaffected by concentrations of dexa a' E methasona more than sufficient to inhibit cell lines that @5 4@ contain receptor. The specificity of the effect is further 0 @0 C emphasized by the lack of significant inhibition by ha 0 hydroxycontisol in any cell line even when concentrations of 0

4, 10_6M were used. By contrast, daxamethasone effects are U clearly apparent in sensitive cells with 10@ M steroid. Thus, responsiveness correlates well with the presence of specific receptor. None of the other cell lines examined in this study aside from the MCF-7 call line appear to contain progestan 1x108 5x108 10x108 one receptor. Dexamethasone Concentration (Ml The above considerations would strongly tend to impli Chart 10. Binding of [3H]dexamethasone to intact MCF-7 human breast cate specific binding of glucocorticoid to receptor in me cancer cells at 37°.Thebinding data are shown replotted in the inset accord ing to the Scatchard technique (24). Techniques used are given in “Materials dialing the affect of hormones on these cells. One potential and Methods.―B/F, bound/free. difficulty with this interpretation is that comparison of Charts 2 and 6 with Chart 4 reveals that, although the affinity of the receptor for dexamathasona is in relative cancer.The human and breastcancer originofthesecellsis agreement with concentrations of glucocorticoid that in suggested by their estrogen responsiveness (10), a-laclal hibit thymidine incorporation, this value is obtained on cy bumin synthesis (11), chromosomal makeup (10), and mom toplasmic extracts incubated with [3H]dexamethasone at 0°. phology (10, 26). Inhibition studies are performed at 37°on whole cells. In The mechanism(s) by which glucocorticoids have been order to see what differences physiological temperature efficacious in some patients with breast cancer has never and intact cells might have on binding affinity, the binding been clarified. Most studies have suggested that responses experiments shown in Chart 10 were performed. In these may be due to suppression ofthe adrenal pituitary axis (13). experiments with intact cells, at 37°,the dissociation equi This would result in decreased production of weak adrenal libnium constant was about 6.8 x h0@ M, very close to that , primarily androstened ione and dahyd roapian obtained on cytoplasmic extracts (2.4 and 5.9 x 10@ M). drostenone, which may be precursors of either more potent The binding curve of dexamethasone to whole cells at 37° androgens or estrogen (13). Our studies show that some very closely approximates the dose-response curve of these breast cancer, at least in vitro, may be directly inhibited by calls to glucocorticoids thus providing additional support glucocorticoids without the intervention of some more for the idea that glucocorticoids inhibit these calls by a complex interaction of glucocorticoids with other polen mechanism involving initial interaction with receptor. hially trophic hormones. Furthermore, the data suggest that some of these glucocorticoid-sensitive tumors may be de tectable by receptor analyses. In data not presented here we DISCUSSION have found significant amounts of glucocorticoid receptor in occasional human breast cancer tumor samples. Much as The present studies demonstrate that glucocorticoids can the examination of tumor samples for has directly inhibit some cell lines derived from human breast aided clinical selection of cases for hormonal therapy (15), it is possible that a subset of human tumors responsive to Table 3 glucocorticoids may be identifiable in a similar way. Comparisonincorporationinto of maximal inhibition of [3H]thymidine Our studies also suggest the presence of progesterone DNAreceptorMaximal and quantity of glucocorticoid receptor in at least 1 of our cell lines that by quantification, % inhibition of thymi specificity, and affinity is distinguishable from glucocorti dine incorporation coid receptor. We cannot at present exclude the possibility that theme are 2 sites on a single class of receptors with Dexametha-Glucocorticoidsone 11 a-Hydroxy- binding4G-11 cortisol dexamethasone binding to 1 site and progesterone binding to both. We also cannot exclude the possibility of melabo 146MCF-7 76 ±4b 0 ±6― 199MDA-231 46±7 0 ±7 lism by the cells of 1 or more of the steroids, thus altering its 77496 23 ±6 0.6 ±6 biological potency, but would be unlikely to 5.3EvsaT 4±8 4 ±8 alter qualitatively the binding competition studies per 0±6 0 ±8 0 formed on cylosols incubated at 0°.Rousseau at a!. (23) a Values are in fmoles [3H]dexamethasone bound per mg cyto have shown that, although both progesterone and gluco plasmic protein as determined by dextran-coated charcoal compet corticoid can interact with the glucocorticoid receptor site, itive protein-binding assay (16). only glucocorticoid-receptor complexes are capable of b Values are (means of quadruplicate determinations) ± S.D. Cells were incubated in steroid for 48 hr and pulsed with “transformation―ofthe receptor and subsequent nuclear [3Hjthymidine for 1 hr. entry of the slaroid-receptom complex. In their studies, theme

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were equal numbers of sites identified by either progestar 9. Leung, B., Sasaki, S., and Leung, J. Estrogen-Prolactin Dependency in 7,12-Dimethylbenz(a)anthracene-induced Tumors. Cancer Res., 35: 621- one or dexamethasone in contrast to our own studies. 627, 1975. Recently “progesteronereceptor―has bean demon 10. Lippman, M. E., and Bolan, G. Hormone Responsive Human Breast strated in some human breast cancer by using the proges Cancer in Continuous Tissue Culture. Nature, 256: 592-593, 1975. 11. Lippman, M. E., Bolan, G., and Huff, K. The Effects of and tenone analog R5020 (6). While exhansivadata have been Antiestrogens on Hormone-responsive Human Breast Cancer in Long published suggesting that, unlike progesterone itself, Term Tissue Culture. Cancer Res., 36: 4595-4601 , 1976. R5020 can distinguish between transcortin binding and pro 12. Lippman, M. E., and Thompson, E. B. The Role of Transcortin in Gluco corticoid Mediated Induction: Tyrosine Aminotransferase Induc geslarone receptor (6), no information has been presented tion in Hepatoma Tissue Culture Cells. J. Steroid Biochem., 5: 461-465, as to whether R5020will bind to glucocorticoid receptor. 1974. 13. Lipton, A., and Santen, R. Proceedings: Medical Adrenalectomy Using Our studies suggest that R5020 has binding affinity for both and Dexamethasone in Advanced Breast Cancer. receptors; therefore distinction between them with this sle Cancer, 33: 503-512, 1974. noid may be difficult, particularly in light of the demonstra 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein Measurement with Folin Reagent. J. Biol. Chem., 193: 265-275, 1951. lion of glucocorticoid receptor in some human breast can 15. McGuire, W. L., Carbons, P. P., Sears, M. E., and Escher, G. C. Estrogen can call lines. Receptors in Human Breast Cancer, an Overview. In: W. L. McGuire, P. We also note no obvious relationship between the preS P. Carbone, and E. P. Vollmer (eds.), Estrogen Receptors in Human Breast Cancer, pp. 1-8. New York: Raven Press, 1975. ences of glucocorticoid and ashnogenreceptorsin the small 16. McGuire, W. L., and DeLaGarza, M. Improved Sensitivity in the Measure number of call lines examined for both. Thus, while MDA ment of Estrogen Receptor in Human Breast Cancer. J. Clin. Endocrinol. Metab., 36: 548-552, 1973. 231, MCF-7, and G-11 contain glucocorticoid receptor and 17. Miller, W. R., Forrest, A. P. M., and Hamilton, T. Steroid Metabolism by 496 and Evsa T do not, MCF-7 alone contains estrogen Human Breast and Rat Mammary Carcinomata. Steroids, 23: 379-395, receptor (11). II will be of great interest to learn whether in 1974. 18. Munck, A. Glucocorticoid Inhibition of Glucose Uptake by Peripheral tissue culture or in vivo hhe presence of 1 or more receptors Tissues: Old and New Evidence, Molecular Mechanisms, and Physiologi are “linked―toeach other. For example, in immature fe cal Significance. Perspect. Biol. Med., 14: 265—289,1971. male rats, estrogen induces the appearance of progastar 19. Munck, A., and Brinck-Johnsen, T. Specific and Nonspecific Physi ochemical Interactions of Glucocorticoids and Related Steroids with Rat one recaphomactivity in uteri (25). Possibly, 1 on more Thymus Cells in Vitro. J. Biol. Chem., 243: p556-5565, 1968. nonestrogan receptors may be important markers for pme 20. Parks, W. , Scohnick, E., and Kuzihowski, E. Dexamethasone Stimula tion of Murine Mammary Tumor Virus Expression: A Tissue Culture diching hormonal response. Source of Virus. Science, 184: 158—160,1974. 21. Philibert, D., and Raynaud, J. P. Progesterone Binding in the Immature Mouse and Rat Uterus. Steroids, 22: 89—98,1973. 22. Philibert, D., and Raynaud, J. P. Progesterone Binding in the Immature REFERENCES Rabbit and Guinea Pig Uterus. , 94: 627-634, 1974. 23. Rousseau, G. G., Baxter, J. D., and Tomkins, G. M. Glucocorticoid 1. Armelin, H. A., Wishihawa, K., and Sato, G. H. Control of Mammalian Receptors: Relations between Steroid Binding and Biological Effects. J. Cell Growth in Culture: The Action of Protein and Steroid Hormones as Mol. Biol., 67: 99-116, 1972. Effector Substances. In: B. Clarkson and R. Baserga (ads.), Control of 24. Scatchard, G. The Attraction of Proteins for Small Molecules and Ions. Proliferation in Animal Cells, pp. 97-104. Cold Spring Harbor, N. Y.: Cold Ann. N. Y. Acad. Sci., 51: 660-672, 1949. Spring Harbor Laboratory, 1974. 25. Sherman, M. R., Corvol, P. L., and O'Malley, B. W. Progesterone-Bind 2. Buller, R. E., Schrader, W. T., and O'MaIley, B. W. Steroids and the ing Components of Chick Oviduct. I. Preliminary Characterization of Practical Aspects of Performing Binding Studies. J. Steroid Biochem., 7: Cytoplasmic Components. J. Biol. Chem., 245: 6085-6096, 1970. 321-326,1976. 26. Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan, M. A Human 3. Burn, I. Endocrine Therapy, Ablative Surgery. In: H. Atkins (ed), The Cell Line from a Pleural Effusion Derived from a Breast Carcinoma. J. Treatment of Breast Cancer, pp. 87-1 11. Baltimore: University Park NatI. Cancer Inst., 51: 1409-1413, 1973. Press, 1974. 27. StoIl, B. A. , Corticosteroid and Progestin Therapy. In: B. A. 4. Feigelson, P., and Schutz, G. Studies in the Control of the Level of the Stoll (ad.), Endocrine Therapyof Malignant , pp. 176-182. Phila Messenger RNA for Hepatic Tryptophan Oxygenase during Hormonal delphia: W. B. Saunders Co., 1972. Induction of the Enzyme. J. Steroid Biochem., 5:356, 1974. 28. Thompson, E. B., and Lippman, M. E. Mechanism of Action of Glucocor 5. Flaxman, B. A. In Vitro Studies of the Human Mammary Gland: Effect of ticoids. Metab. Clin. Exptl., 23: 159-202, 1974. Hormones on Proliferation in Primary Cell Culture. J. Invest. Dermatol., 29. Toft, D. 0., and Sherman, M. R. Receptor Identification by Density 61: 67-71, 1973. Gradient Centrifugation. Methods Enzymol., 36: 156-166, 1975. 6. Horwitz, K. B. , and McGuire, W. L. Progesterone Receptors in Human 30. Vonderhaar, B. K., and Topper, Y. J. Critical Cell Proliferation as a Breast Cancer. Clin. Rca., 23: 237A, 1975. Prerequisite for Differentiation of Mammary Epithelial Cells. Enzyme, 15: 7. Jensen, E. V., and DeSombre, E. R. Mechanism of Action of the Female 340-350, 1973. Sex Hormones. Ann. Rev. Biochem., 41: 203-230, 1972. 31. Westphal, U. Assay and Properties of Corticosteroid-Binding Globulin 8. Lee, K., and Kenney, F. T. Regulation of Tyrosine-Ketoglutarate Trans and Other Steroid-Binding Serum Proteins. Methods Enzymol., 15: 761- aminase in Rat . J. Biol. 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Marc Lippman, Gail Bolan and Karen Huff

Cancer Res 1976;36:4602-4609.

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