Estrogen Conjugates and Serum Factors Mediating the Estrogenic Trophic Effect on MCF-7 Cell Growth1

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[CANCER RESEARCH 47, 5883-5887, November 15, 1987] Estrogen Conjugates and Serum Factors Mediating the Estrogenic Trophic Effect on MCF-7 Cell Growth1

N. Devleeschouwer, N. Legros, N. Olea-Serrano,2 R. Paridaens, and G. Leclercq3 Sen-ice de MÃ©decineet Laboratoire d'Investigation, Clinique H. J. Tagnon, Laboratoire de CancÃ©rologieMammaire, Institut Jules Bordel, Centre des Tumeurs de l'UniversitÃ©Librede Bruxelles, Brussels, Belgium

ABSTRACT other investigators found an antagonistic activity towards estra Estrogens stimulate growth of MCF-7 breast cancer cells in monolayer diol in the serum (4). culture. Possible interference of serum factors leading to an estrogen- Our recent data are in agreement with this concept that serum insensitive cell growth was analyzed in various experiments carried out factors modulate the trophic effect of estradiol. Initially, upon receipt of MCF-7 cells, we were unable to detect any estrogenic on serum batches producing no estradici stimulation. Out of five estrogen conjugates, only 3-glucurono-estradiol partly suppressed the inhibition of growth stimulation. Meanwhile, the sensitivity of our cells was hydroxytamoxifen; the conjugate also reduced the estrogen receptor demonstrated by the ability of estradiol to reverse the inhibitory content of the cells, probably by a down regulation process ("processing"). effect of several antiestrogens (7) as well as by its capacity to Moreover, prolonged subcultures in dextran-coated charcoal-treated increase the synthesis of proteins (8). Subsequent experiments serum attempting to remove possible intracellular estrogens produced no carried out on fresh serum batches from various origins (9) growth stimulation. Interference by hormone carriers of the serum was reproduced the estradiol-stimulated growth originally described ruled out by the fact that two strong synthetic estrogens, tnoxestrol and by Lippman. Passage from "inactive" (unstimulatory) to "ac diethylstilbestrol with weak binding affinity for these carriers, were tive" (stimulatory) serum conferred a permanent sensitivity to' unstimulatory. Reduction of the carrier concentration also failed to confer any estrogen sensitivity. This lack of effect of most estrogen conjugates our cells. and serum carriers seems to contradict the hypothesis of their interference The reason for the appearance in our laboratory of a perma leading to an estrogen-insensitive growth. nent estrogenic growth stimulation is under investigation to Presence in the serum of potential inhibitors towards estrogen action provide information on the mechanism by which serum factors was also examined. Dilution of sera inducing an estrogenic stimulated modulate the in vitro estrogen sensitivity of mammary tumors. growth failed to show any growth increase, either in the absence or Several experiments are being conducted on both active and presence of estradiol, thus excluding the possibility of a major influence inactive sera. of an antagonism on growth control. Moreover, clonogenic assays in soft agar eliminated the hypothesis that a difference between "active" (stim The present paper describes several of our studies; in partic ulatory with estradiol) and "inactive" serum batches may result from ular, it analyzes the influence of estrogen conjugates, serum distinct adherence properties rather than from real growth stimulation. concentration, and cell adherence. All of these data are consistent with the concept that serum factors which are not of estrogenic nature mediate the trophic effect of estradiol; their absence in some serum batches may lead to an estrogen-insensitive MATERIALS AND METHODS cell growth. Steroids

INTRODUCTION [3H]Estradiol and [3H]estrone [Â±100Ci/mmol] were purchased from The MCF-7 human mammary breast cancer cell line has been Amersham, United Kingdom; diethylstilbestrol and all steroids were obtained from Sigma Chemical Co., St. Louis, MO; 4-hydroxytamox- established in tissue culture from a metastatic pleural effusion ifen was kindly provided by ICI and moxestrol (R-28S8) by Roussel- (1). It retains properties of a differentiated mammary epithe Uclaf, Paris, France. lium and expresses specific receptors for all steroid hormones (2). It is therefore widely used to study growth regulation of Sera hormone responsive breast cancers. Estrogens belong to the most important category of hor FBS4 was purchased from Gibco; HSs, freshly prepared from blood mones involved in breast tumor induction and growth modu collected without anticoagulants, were obtained from healthy donors; lation. With regard to MCF-7 cells, review of the literature sheep serum was received at the time of death in the slaughterhouse. reveals that the extent of estradiol-induced growth stimulation After heat inactivation (l h at S6Â°C),all sera were depleted of steroids largely varies among laboratories. Early observations by Lipp- by overnight incubation with DCC (0.005 and 0.5%, respectively) and sterilized by passage through a 0.22-^m Millipore filter. Addition of man et al. (3) suggested a strong mitogenic effect. Several [3H]estradiol and [3H]estrone sulfate at IO"7 and 10~" M to a serum investigators confirmed their findings while others did not (4, batch revealed that this treatment removed 98% of both steroids. 5). Recent studies indicated that differences between the serum batches used in tissue culture might be at the origin of this discrepancy. Thus, Page et al. (6) detected a number of factors Cell Line mediating the trophic effect of estradiol in some serum batches; MCF-7 cells were kindly provided by Dr. M. Rich (Michigan Cancer Received 10/29/86; revised 8/6/87; accepted 8/12/87. Foundation). They were maintained at 37*C in our laboratory as The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in monolayer cultures in closed Falcon plastic flasks (75 cm2) containing accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Earle's MEM (Gibco) supplemented with 10% FBS and the usual 1This work was supported by a grant from the Fonds CancÃ©rologiquede la Caisse GÃ©nÃ©raled'Epargneet de Retraite de Belgique and from the Fonds concentrations of L-glutamine, gentamycin, penicillin, and streptomy National de la Recherche Scientifique (Belgium). cin (Gibco) (10). 2 Recipient of a Spanish Fellowship (Servicio de FormaciÃ³n de Personnal Investigador). Present address: HUC Depto de Radiologia Fac. Medicina, Gran 'The abbreviations used are: FBS, fetal bovine serum; HS, human serum; ada, Spain. DCC, dextran-coated charcoal; ER, estrogen receptor SHBG, sex hormone- 1To whom requests for reprints should be addressed. binding globulin; MEM, minimal essential medium. 5883

Growth Experiments Table 1 Effect of estrone conjugates on growth inhibition by 4-hydroxytamoxifen of MCF-7 in monolayer cultures Growth of Cells in Monolayer Culture. Fifty thousand cells were removed by trypsinization (trypsin, 0.05%-EDTA, 0.025%) and plated in MEM with 10% DCC-treated FBS. After 24 h, FBS was maintained or replaced by HS for 120 additional h of culture in the absence or presence of IO"8M estradiol (intercurrent medium change after 72 h of culture) (7). Growth was estimated by measuring the amount of DNA of collected cells by the method of Burton (11). Each experiment was ng DNA Â±SD conducted in quadruplicate. Compound(R)Controls IO"7 M 4-hy Growth of Cells in Soft Agar. Soft agar cultures were run according (M)io-7 alone15.3 droxytamoxifen5.5 to a modified form of the method of Hamburger and Salmon (12). Â±1.5(100)Â° Â±0.8 (36)* 11.7 Â±0.8 (76)' Upper and under layers contained MEM supplemented with 10% DCC- HSOjNaGlucuronoConcentrationio-Â«io-713.4Â± 1.5(88) treated serum and the usual antibiotics; estradiol was added to both 15.4 Â±0.9(101)14.7 4.6 Â±0.9(30)4.7 layers of agar at a final concentration ranging from IO"8 to 10~* M. Cells were plated in agar at the level of 10s cells/dish. Cultures were Â±1.8(96) Â±0.6 (31) io-'io-7 14.5 Â±0.7(95)12.8Â± 5.4 Â±0.4(35)5.4 kept for 2 weeks. Colonies were counted and data expressed as a percentage of treated to control plates. Each experiment was conducted 10-'Compound1.0(84) Â±0.7 (35) in triplicate. 18.2 Â±1.0(119)With 4.4 Â±1.0(29) " Numbers in parentheses, percentage of control values. * P < 0.001; one-way analysis of variance. Receptor Assays ' P < 0.001; interaction between estrone and 4-hydroxytamoxifen by two-way Biochemical Assay (13). Cytosolic ERs were measured by the stan analysis of variance. dard DCC assay established by the European Organization for Research on Treatment of Cancer Breast Cancer Cooperative Group. Table 2 Effect of estradiol conjugates on growth inhibition by 4- Whole Cell Assay (14). Cells from a confluent monolayer were removed by trypsinization and suspended at ~2 x 10s cells/ml in the hydroxytamoxifen on MCF-7 in monolayer cultures growth medium supplemented with 10% DCC-treated FBS. Cellular suspension was then cultured in multiwell dishes (six wells/dish; 1.5 ml/well) in a humidified 95% air/5% COÂ¡atmosphere. Four days after plating, the growth medium was replaced by a serum-free medium containing increasing amounts of [3H]estradiol with or without a 200- fold excess of unlabeled estradiol. After 50 min incubation, the medium was removed and the monolayer washed three times with ice-cold saline. ng DNA Â±SD Compound Concentration IO"7 M 4-hy Steroids were extracted in ethanol for radioactivity counting. All meas aloneControls(R/R') (M) Compound urements were performed in triplicate. In each experiment an additional droxytamoxifen5.5 Â±1.5(100)Â° Â±0.8 (36)* multiwell was run in parallel for DNA measurement by the method of IO-7 16.6 Â±1.5(108) H,HH. 16.0 Â±1.1 (105)f Burton (11). Specific [3H]estradiol uptake was calculated from the (94)10-10-10-IO'' 14.4 Â±1.3 14.0 Â±0.5(92)5.3 difference of incorporated radioactivity after incubation in the absence or presence of an excess of unlabeled steroid. ER concentration was glucuronoGlucurono, 1.5(84) Â±0.4 (35) determined according to the procedure of Scatchard and expressed in 14.4 Â±0.8(94)15.1 6.0 Â±0.6(39)8.6 fmol (10~15 mol)/^g DNA content. This experimental protocol was Â±0.9 (56)"' slightly modified to assess the effect of 3-sulfo-estrone and 3-glucurono- HSOjNa, 10-10-10-12.9Â±Â±1.4(99) estradiol on the ER level, i.e., these conjugates were added at IO"7 M 14.8 Â±0.7(97)13.8Â± 4.7 Â±0.5(31)4.7 either during incubation with [3H]estradiol or 4 days before the assay H15.3 1.2(90) Â±1.3(31) (at the plating time). 14.1 Â±1.8(92)With 4.8 Â±0.7 (31) " Numbers in parentheses, percentage of control values. * P < 0.001 ; one-way analysis of variance. c P < 0.001; two-way analysis of variance; estradiol suppressed 4-hydroxyta RESULTS moxifen effect. *P< 0.05; the 3-glucurono derivative also significantly influenced 4-hydroxy Experiments Conducted on Inactive Sera tamoxifen inhibition. Influence of Estrogen Conjugates on Growth and Estradiol Receptivity. Hydrolysis of residual estrogen conjugates not re Table 3 ER content of MCF-7 in presence of conjugated estrogens at IO''1 M moved during the DCC treatment of the serum has been pro ER content (fmol/Vg DNA) posed as an explanation for the absence of estrogenic growth stimulation of MCF-7 cells (15). This hypothesis was analyzed Addition of compound during incubation by testing the antagonistic potency of a series of glucuronates (classical whole-cell Addition of compound and sulfates of estradiol and estrone towards the growth inhi CompoundNoneWith the platingtime4.0 bition of the strong antiestrogen 4-hydroxytamoxifen (Tables 1 and 2). Only one conjugate, 3-glucurono-estradiol, was able to 3-sulfo-estroneâ€¢None 4.94.0 4.74.01.0 slightly reduce the inhibition of the compound (20%) (P < 0.05; two-way analysis of variance). On the contrary, and as expected, With 3-glucurono-estradiolassay)4.0Â° 5.2at free estradiol and estrone abolished this effect (P < 0.001; two- " Values correspond to the ER binding capacity measured by whole-cell assay way analysis of variance) indicating that most conjugates did (13). not hydrolyze during the experiment. Prolonged exposure of MCF-7 cells to free estrogens pro in presence of 3-sulfo-estrone and 3-glucurono-estradiol before duces a nearly total disappearance of their ER content ("proc being assessed for their capacity to specifically concentrate essing") (16). To analyze the influence of potent estrogen con pHJestradiol (ER whole-cell assay) (Table 3). 3-Sulfo-estrone jugates on the receptor levels, cells were incubated for 4 days did not reduce this capacity while 3-glucurono-estradiol was 5884

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1987 American Association for Cancer Research. ESTROGEN CONJUGATES AND MCF-7 HUMAN MAMMARY CELL LINE largely effective (75% reduction). Hydrolysis of the latter con Molanty jugate was most probably responsible for this property since it cioVo-6 was unable to produce any reduction of [3H]estradiol uptake when added at the time of receptor assay, exactly as did 3-sulfo- 20, estrone. Effect of Prolonged Exposure of Cells to Steroid-depleted Serum on Growth and Estradiol Receptivity. MCF-7 cells are D â€¢H maintained in culture with 10% FBS. On the contrary, experi ments with estrogens and/or antiestrogens are always run in 10% DCC-treated serum to avoid possible interference by serum estrogens. Literature data suggested that this procedure might not be sufficiently effective. Thus, after estradiol stimulation, receptor-bound hormone has been reported to remain within OJ E2 DES R-2858 the cell nucleus for several days (17). Moreover, serum lipoidal Fig. 1. Absence of effect of three estrogens [17/j-estradiol (/â€¢..):diethylstilbes derivatives of estradiol, i.e., 17/3-stearyl-and 17/3-oleyl-estradiol trol (DES); moxestrol (R-2858)] on MCF-7 cell growth. The compounds were were shown to accumulate in the membrane of human and tested at concentrations ranging from 10"' to in' M.One-way analysis of variance murine mammary cells (18). As esters of estradiol are potent revealed no difference between control (C) and treated groups. long-acting estrogens we analyzed whether such a prolonged cellular incorporation of estrogens might be at the origin of the [Â¡Control estradiol insensitivity of our cells. Before exposure to estradiol, JJ.IO-'ME MCF-7 cells were cultured for up to 10 weeks with DCC- ug DNAÃŽSD treated serum. Table 4 shows that this approach did not confer IO, any sensitivity nor did it modify the ER content of the cells. The lack of increase in ER concentration after this long period is consistent with an absence of major cellular incorporation of 5. serum estrogens, since the latter would normally have produced a dramatic reduction of receptor concentration (processing) (16). Influence of Carriers of Serum on Growth Stimulation. Natural estrogens are known to bind to hormone carriers of the serum I 5 IO 20 such as a-fetoprotein, SHBG, and albumin (19). The binding SERUM CONCENTRATION CM Fig. 2. Influence of increasing concentrations of inactive FBS on MCF-7 cell affinity to such carriers being only 10- to 1000-fold less than to growth in the absence or presence of 10~* M estradiol (/ ..) One-way analysis of ER, one may speculate that it may interfere in the inter/extra variance revealed no difference between groups. cellular ratio of the estrogens as shown by Pavlik and Katze- nellenbogen (20) in human primary cultured endometrial cells. [] Control When present in high concentrations, carriers may reduce the normal cellular uptake of estradiol thereby producing growth DNAÃ®SD insensitivity to the hormone. Such an interference was ruled out by the fact that two synthetic estrogens with very weak affinity for SHBG failed to produce any MCF-7 cell growth stimulation. Thus, similar to estradiol, both diethylstilbestrol and moxestrol (l lÃŸ-methoxyethinylestradiol; R-2858) were in 5. effective at concentrations ranging from 10~8 to IO"6 M (Fig. 1). Moreover, reduction of the carriers' concentration by dilu tion of the serum did not confer any estrogenic sensitivity (experiments conducted in 1,5, 10 and 20% inactive FBS; Fig. l S 10 20 2). SERUM CONCENTRATION CM Fig. 3. Influence of increasing concentrations of active HS on MCF-7 cell Experiments Conducted on both Active and Inactive Sera growth in the absence or presence of Id " M estradiol (/ .â€¢>.One-wayanalysis of variance revealed no statistical difference between control groups but a very Influence of Nature of Serum on Growth Stimulation. An significant i/' < 0.01) difference between control and estradiol-treated groups. antagonism between estradiol and one or several growth inhib-

Table 4 Influence of various estrogen-depriving periods on ER level and cell itors of the serum has been reported (21, 22). Accordingly, growth estradiol insensitivity of MCF-7 cells may result from an ab SDER (/ig DNA) Â± sence of such inhibitors) in some serum batches. This hypoth Depriving period (culture with DCC- content Mestradiol13.110-Â« esis was analyzed by diluting an active HS in which the stimu treatedserum)3 (fmol/MgDNA")4.6 latory effect of estradiol was highly significant. Fig. 3 shows days that dilution did not increase growth in either the absence or Â±1.1 (100)* 5 days 3.7 Â±0.8(94) presence of estradiol (the estradiol/control ratio was approxi 3wk 2.44.14.5 4wk mately the same at 1, 5, 10 or 20% serum). Although this 6wk observation did not at all conflict with the possibility of growth 10 wkGrowth 4.7Controls13.99.3 Â±2.7 (100)With8.3 Â±1.6 (89) inhibitors being present in the serum (see below), it seems to " ER binding capacity (13). '' Numbers in parentheses, percentage of control values (one-way analysis of exclude a major antagonism between such inhibitor(s) and variance shows that there is no difference between controls and estradiol-treated estrogens under our standard experimental procedures (10% groups). serum). Several other experiments confirmed this assumption: 5885

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1987 American Association for Cancer Research. ESTROGEN CONJUGATES AND MCF-7 HUMAN MAMMARY CELL LINE addition of estradiol to various cultures conducted at lower factors mediate the trophic effect of estradiol. The nature of (5%) serum concentrations (and thereby lower concentration of such factors is unknown and merits investigation. Preliminary putative inhibitors) always failed to show a growth stimulation. studies from this laboratory suggest a high thermic stability Influence of Culture System on Growth Stimulation. The major (serum treated at 90Â°C),a property ascribed notably to growth difference in estrogen sensitivity between active and inactive factors. On the other hand, the presence of such a factor is not serum batches observed in monolayer culture may result from limited to mammals since the estradiol trophic effect was found distinct adherence properties rather than from a real growth with an avian serum (chicken) (24). As growth stimulation was stimulation. Clonogenic assays in soft agar in which cell adher equally detected with HS from women and men (9), it is clear ence must not be taken into account were performed to examine that these factors are not sex dependent. Moreover, the obser this hypothesis (Fig. 4). Remarkably, with the latter method vation revealed the same trophic effect at various serum con ology an estradiol-induced stimulation was observed in the centrations (from 1 up to 20%) indicating that they are always presence of two active serum batches (sheep and fetal bovine). in sufficient amounts to produce an optimal effect. Finally, an It is noteworthy that growth inhibition was found at unusually estrogenic nature should be rejected in view of our present high serum concentrations (sheep, 15 and 20%; fetal bovine, investigations. 20%); this effect was partly suppressed by estradiol. Vignon et al. (15) first suggested that estradiol insensitivity might result from residual estrogen sulfates that are not ex DISCUSSION tracted during the DCC treatment of the serum. According to these authors, MCF-7 cells are able to incorporate and almost Whether or not estrogens are direct mitogens of MCF-7 totally hydrolyze 3-sulfo-estrone within 24 h, giving a substan mammary tumor growth remains an unsolved question. Al tial concentration of active free hormone. Our observations do though results vary among laboratories, there is a clear trend not support such a major interference of residual estrogen in favor of an increase of growth rate in the presence of the conjugates. As a matter of fact, according to our measurement hormone (for review, see Ref. 23). Differences in experimental of residual [3H]estradiol and ('H)estrone sulfates, an effective conditions are most likely responsible for the lack of consensus DCC treatment should remove up to 98% of free and conjugate in this regard. Data from our laboratory indicate that the nature estrogens. In FBS, the following concentrations were reported of the serum is an important factor of variability; with some for free estrogens: estrone, 10~9 M; estradiol 17a, 10"' M; batches estradiol stimulates growth while with others it does estradiol 17'ÃŸ,IO"10M. Concentrations of conjugate estrogens not. Since it occurs at low as well as at high growth rates, were found to be about 50 to 100 times higher: estrone, IO"7 stimulation does not seem to result from a compensation of an M, estradiol 17a, IO"7 M; and estradiol 17/3, 5 x 10~9 M (25, unsatisfactory growth (i.e., suppression of growth inhibition). 26). Therefore, under our growth conditions (10% serum) non- Thus, DNA contents of cells cultured in active and inactive sera extracted estrogens should amount at the maximum to 2 X ranged at the end of the experiments from 3.9 to 21.2 /Â¿g(mean 10~12 and 2 x 10"'Â°M, respectively, for free and conjugate of 40 values) and from 4.2 to 27.2 (mean of 33 values), respec steroids. These concentrations are too low to produce a growth tively. stimulation which may mask the effect of estradiol at 10~8 M. Our observations are consistent with the concept that serum This conclusion is in complete agreement with the observation of Darbre et al. (27) who found that pretreatment of the serum Serum concentration: with sulfatase prior to the DCC procedure did not decrease the basal growth of the estrogen sensitive ZR 75-1 cell line. FETAL BOVINE Stronger evidence of the absence of interference of serum estrogen conjugates was provided by the assessment of MCF-7 cell growth in the presence of 4-hydroxytamoxifen. Only 3- glucurono-estradiol partly antagonized the inhibitory effect of the drug at equimolar concentration (IO"7 M); all other conju gates, 3-sulfo-estrone, 3-glucurono-estrone, 17-glucurono-estradiol, 3-sulfo-estradiol and 3-glucurono-estrone were totally ineffective. 3-Glucurono-estradiol also reduces the cellular in corporation of [3H]estradiol after a prolonged preincubation period. This effect, which corresponds to a reduction of ER concentration (processing), was most likely the consequence of the hydrolysis of the compound during the preincubation since it did not occur at the time of ER assay. In view of all these data, it seems that hydrolysis of residual estrogen conjugates might not be the major source of estrogenic insensitivity of the MCF-7 cells. In particular, the lack of a major influence of sulfates seems to eliminate the importance ascribed to such conjugates by Pasqualini and Gelly (28). To close this question, it should be stressed that two synthetic strong estrogens (moxestrol and diethylstilbestrol) which do not bind to a-fetoprotein and SHBG failed to produce a trophic effect suggesting that interaction between natural estrogens and serum carriers is not C E2i(T8M the factor conferring insensitivity. Fig. 4. Influence of estradiol I/ .â€¢>onMCF-7 cell colony formation in soft The molecular mechanism by which estrogens increase MCF- agar. Cultures were done with two active sera in the absence or presence of 10"* M estradiol. One-way analysis of variance shows that estradiol significantly 7 cell growth remains unknown. Secretion of preexisting and increases the number of colonies. *,P< 0.05; **, P < 0.01; C, control; bars, SD. induced (29) proteins under estradiol stimulation has been 5886

12. Hamburger, A. W., and Salmon, S. E. Primary bioassay of human tumor reported by many investigators. According to Lippman et al. stem cells. Science (Wash. DC), 197:461-463, 1977. (30) this secretion should play a key role in growth control. 13. EORTC Breast Cancer Cooperative Group. Revision of the standards for the Data from our laboratory revealed that secretion also occurs assessment of hormone receptors in human breast cancer. Eur. J. Cancer, 16: 1513-1515, 1980. when the cells are cultured in inactive sera confirming the 14. Olea-Serrano, N., Devleeschouwer, N., Leclercq, G., and Heuson, J. C. Assay requirement of extracellular factors (from the serum) for opti for estrogen and progesterone receptors of breast cancer cell lines in mono- mal growth. Serum-free culture systems (31) now commercially layer culture. Eur. J. Cancer Clin. Oncol., 21:965-973. 1985. 15. Vignon, F.. Terqui, M.. Westley, B., Derocq, D., and Rochefort, H. Effects available should be extremely helpful in identifying the nature of plasma estrogen sulfates in mammary cancer cells. Endocrinology. 106: of the latter as well as elucidating how they cooperate with 1079-1086, 1980. 16. Horwitz, K. B., and McGuire, W. L. Nuclear mechanism of estrogen action. some secretion products to modulate growth. J. Biol. Chem., 253: 8185-8191, 1978. 17. Sirnhl, J. S., and Lippman, M. E. Prolonged retention of estradiol by human breast cancer cells in tissue culture. Cancer Res., 39: 3319-3327, 1979. ACKNOWLEDGMENTS 18. Abul-Hajj, Y. J. Formation of estradiol-17(i fatty acyl 17-esters in mammary tumors. Steroids, 40:149-156, 1982. The authors wish to thank F. Grillo for helpful technical assistance 19. Raynaud, J. P., Ojasoo, T.. and Vache, V. Unusual steroids useful in and J. Collette for typing. measuring steroid receptors. In: E. B. Thompson and M. E. Lippman (eds.), Steroid Receptors and the Management of Cancer, Vol. 1, pp. 215-232. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1987 American Association for Cancer Research. Estrogen Conjugates and Serum Factors Mediating the Estrogenic Trophic Effect on MCF-7 Cell Growth

N. Devleeschouwer, N. Legros, N. Olea-Serrano, et al.

Cancer Res 1987;47:5883-5887.

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