[CANCERRESEARCH57. 3071-3078, August 1. 1997]

Perspectives in Cancer Research

MCF-7: The First Hormone-responsiveBreast Cancer Cell Line'

Anait S. Levenson and V. Craig Jordan2

Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611

!ntroduction

On Thursday,January2, 1997, Dr. HerbertSoule, the scientist whodeveloped the MCF-7 breast cancer cell line, died. At the time, wewere in the process of writingthis tributeto markthe 25th anniversaryof Dr. Soule's remarkableaccomplishment.The cells, derived from abreast cancer patient in the Detroit area and developed at the MichiganCancerFoundation,Detroit,became a standardmodel in hundredsoflaboratories around the world. In retrospect, the story of the diverseuses of these cells is really the history of our developing knowledgeof hormone-regulatedcell replication, and they provided a uniqueinsight into the endocrine therapyof breastcancer.

Our article is offered as a tributeand memorial to Dr. Soule. Wewill trace researchwith MCF-7 cells to illustratethe change in ourideas about cell replication and to highlight the advances in ourunderstanding of the signal transduction pathway of estrogen and themolecular biology of estrogen action. All of these advances dependedon the uniquepropertiesof MCF-7 cells. Additionally,it is importantto appreciate that the cell system has now found applications inexperimental therapeutics, and the results from these studies are beingtranslated to the clinic for the treatment of patients. None of thiswould have been possible withoutDr. Soule's skill as a cell biologist.

Characterization

The MCF-7 breast cancer cell line was derived from a pleuraleffusion taken from a patient with metastatic breast cancer (1). The69-year-old patient had undergone a mastectomy of her right breastfor a benign tumorand a radicalmastectomyof her left breast for amalignant adenocarcinoma 7 and 3 years, respectively, before primaryculture of cells was started.Interestingly,the Soule et aL article (1)notes that local recurrences were controlled for 3 years with radiotherapy and hormone therapy. In the days before tamoxifen, thepatientwas probablytreatedwith high doses of the syntheticestrogendiethylstilbestrol.Clearly, the disease was very hormoneresponsive,because it was controlled for three times longer than the 1-yearaverage to be expected. Two months after widespread nodular recurrences occurred, in June of 1970, samples were taken from a pleuraleffusion for laboratory studies. The cells were proven to be of humanorigin, and cytogenetic studies indicated a distinct stem line of 88chromosomes. Dr. Sam Brooks, working with Dr. Soule (2), firstdescribed the ER3 in MCF-7 cells by both Scatchard and sucrosedensity gradient analysis. This was a pivotal discovery.

From this point onward,researchacceleratedrapidly.In 1975, Dr.

Received 4/10/97; accepted 5/30/97.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 accordance with18U.S.C.Section1734solelyto indicatethisfact.

1 These studies were supported by NIH Grant CA-56143, Breast Cancer Program

Development Grant P11 CA-657634, and the Lynn Sage Breast Cancer Foundation ofNorthwestern Memorial Hospital, Chicago.

2 To whom requests for reprints should be addressed, at the Robert H. Lutie CancerCenter, Northwestern University Medical School, 303 East Chicago Avenue, OlsonPavilion8258,Chicago,IL60611.Phone:(312)908-4148;Fax:(312)908-1372.

3 The abbreviations used are: ER, estrogen receptor, PGR, progesterone receptor; TGF,

transforming growth factor; [OF, insulin-like growth factor.

Marc Lippman (3) demonstrated that the antiestrogen tamoxifen inhibited the growth of MCF-7 cells, but the inhibition could be reversed by estrogen. The drug was classified as a competitive inhibitorof estrogen action, but at high concentrationstamoxifen killed cells.Because these results appeared to parallel the emerging clinical ohservations (4), Lippman (3) went on to predict a future path forendocrine research: “Thepotential value of a hormone-dependenthuman breast cancer in long term tissue culture for the study ofmechanism(s) by which steroid hormones exert their trophic effects issignificant,particularlyin view of the likelihood of obtainingregulatory variantsor mutantswhich are hormone independent.―At aboutthe same time, KathrynHorwitz,who was completingherPh.D. in thelate Dr. Bill McGuire's laboratory, identified the receptors for glucocorticoids, progestins, and androgens, as well as the ER (5). Sheconcluded, “MCF-7may be an excellent in vitro model for studyingthe mechanismof tumorresponse to endocrinetherapyas well as thecomplex relationshipsbetweenbindingandbiological actionsof thesehormones―(5). The predictionsfrom both researchprogramswere tobe proved correctover the next 2 decades.

Horwitz and McGuire focused initially on the regulation of PgRsynthesis in MCF-7 cells by estrogens and antiestrogens. They developed a largebody of evidence to associate processing(destruction)ofnuclearreceptorcomplexes with the initiationof PgR synthesis (6, 7).This work paralleled their suggestion of using PgR assays as apredictive test for hormone-dependent breast cancer (8). Turning tothe effects of antiestrogens,Horwitzet a!. (9) found thattamoxifen issufficiently estrogenic to initiate PgR synthesis by itself. The estrogenic propertiesof tamoxifen would subsequently be importanttoexplain the additionalbenefits of tamoxifen on bones and lipids andthe possible development of drug resistance (10). However, at thattime in the late l970s, work on the antiproliferative actions of anticstrogens was of paramountimportance. Tamoxifen and other cornpounds were shown to inhibit replication (11). Drs. Rob Sutherland(12, 13) and Kent Osborne (14, 15) would demonstrate subsequently,using MCF-7 cells, that tamoxifen produces a reversible block in theG1 phase of the cell cycle.

Despite the interesting findings with antiestrogens, the central focusof laboratoryresearchin the 1970s and early 1980s was to prove thatestrogen actually stimulated tumor growth directly. Dogma predictedthat the MCF-7 ER-positive cell line should grow faster with exogenous estrogen. The experiment could be accomplished routinely ifcells first were treated with antiestrogens for a few days. Estrogencould “rescue―antiestrogen-blockedcells (3), but the effects of estrogen alone were less dramatic.Here was a paradox.

Cells Grown in Estrogen

Although Lipprnan'sgroup consistently demonstratedstimulatoryeffects with estrogen using [3H]thymidineincorporationas a methodof monitoringDNA replication(3, 11, 16), otherswere unableto showprofound effects on cell proliferation (17—19).This led to intensedebate (20) and also to the suggestion that estrogen really produced

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growth by an indirect method (21). This latter conclusion was basedon the observation that estrogen did not stimulate MCF-7 cells toreplicate in vitro, but if the same cells were grown in athymic mice,then estrogen stimulated growth. It was reasoned that estrogen mustbe triggeringa second messengerin vivo. At the time (1980), this wasnot an unreasonable conclusion, because it was accepted that carcinogen-induced rat mammary tumors require both estrogen and estrogen-stimulated prolactin for growth (22). Subsequently, Huseby et a!.(23), at the Michigan Cancer Foundation, provided evidence, in athymid mice, to demonstrate that estrogen had a direct growth-stimulating effect on MCF-7 tumor cells in vivo. Indirect growth stimulationthrough a second messenger was improbable.

The breakthrough in our understanding of direct estrogen actioncame with the discovery that MCF-7 cells had been grown in estrogen-containing media from the start. Indeed, if the occult estrogen hadnot been there, the MCF-7 cell model would not have been possible.

The story unfolded through a series of fortuitous accidents. Dr.John Katzenellenbogen was particularlyinterested in developing afluorescent-tagged estrogen so that ER could be detected easily inbreast cancer specimens under the fluorescence microscope. Naturally, he was using MCF-7 cells as his laboratory model, but thefinding that unwashed cells fluoresced led to an examination of thechemical constituents in media. Phenol red (Fig. 1) is the indicatorused routinely in commercial media to monitor the oxidative state ofthe cells. The indicator is, however, present not as a few drops but in@.LMconcentrations, and its structure is reminiscent of nonsteroidalestrogens first synthesized by Sir Charles Dodds in the l930s (24)prior to his landmark discovery of diethylstilbestrol (25). Drs. Johnand Benita Katzenellenbogen established, in a seminal publication,that removal of phenol red from media resulted in the exquisitesensitivity of MCF-7 cells to the growth-promoting effects of estrogen(26).Aspredictedfromdata,in vivoantiestrogenswerepartialagonists and were antagonists of estradiol action.

As an aside, it was found that different preparations of phenol redhad different potencies as estrogens (27), and it was a dimerizationproduct, which occurred during manufacture, that was responsible forestrogenicity (28, 29). The similarity of the structure of the contaminant with the most potent synthetic estrogen, diethylstilbestrol, isillustrated in Fig. 1.

The discovery of an occult estrogen that is ubiquitously present inthe culture media is an important lesson, obvious in hindsight, but

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Fig. 1. Structures of estrogenic components identified in tissue culture media and themost potent synthetic nonsteroidal estrogen, diethyistilbestrol.

pivotal to all of the subsequent progress in the understanding andinterpretation of data obtained with MCF-7 cells in culture.

Monoclonal Antibodies to the ER

The discovery and detection of the ER protein (30—33)in the ratuterus was of fundamental importance to progress in breast cancertherapy. However, studies on the biochemistry of the proteininvolved expensive experiments using rats or the collection of calfuteri from slaughterhouses. Clearly, the development of antibodiesto the human receptor would be extremely important in researchand for the convenient detection of ER by clinical laboratories.Progress in the detection of the ER using antibodies primarilycentered on Jensen's group at the Ben May Laboratory in Chicago.Polyclonal antibodies against ER protein were obtained initiallyusing highly purified ER as an antigen to immunize rabbits and agoat (34, 35). To avoid the heterogeneity in the antibodies, thehybridoma techniques of Kohler and Milstein (36) were usedsubsequently to obtain monoclonal antibodies to the calf uterinenuclear ER (37). Unfortunately, there was no absolute proof thatmonoclonal antibodies to animal ER would be of value for humanstudies. Almost immediately, but working in collaboration withscientists at Abbott Laboratories, Dr. Geof Greene, who played theleading experimental role in the antibody research at the Ben MayLaboratories, described the preparation of monoclonal antibodiesto the human ER from MCF-7 breast cancer cells (38). Theproperty of the cells to replicate indefinitely under carefully controlled conditions provided a constant supply of receptor to makeantibodies.

Although the development of monoclonal antibodies to the ERwould ultimately revolutionize the detection of ER in clinical breastcancer samples, the new laboratory tool would also change the subcellular model of estrogen action. The original model for estrogenaction placed the ER in the cytoplasmic compartment. Estrogen boundto the receptorandcausedtranslocationof the complex to the nucleus,where the events associated with hormone action would be triggered.This model had been challenged in 1977 by Zava and McGuire, whoused careful fractionation methods and found unoccupied ER in thenuclear compartment of MCF-7 cells (39). However, the ability to seereceptor without cell disruption would change the textbook model thathad stood for nearly 2 decades.

King and Greene (40) used monoclonal antibodies to the MCF-7ER to demonstrate that ER was in the nucleus of cells. Although theresults were published before the discovery that MCF-7 cells wereactually growing in the estrogenic phenol red-containing media, thesedata (Fig. 2) altered the scientific concepts. Parallel studies byWeishons et a!. (41), who used cytochalasin B to prepare nucleoplastsfrom the rat pituitary cell line GH3, found that the majority of ER wascontained in the nucleoplasts and not the cytoplasts. Subsequently,Welshons et al. (42) showed that nucleoplasts from MCF-7 cellsgrown in phenol red-freemedia containedER. Thus, it would not beunreasonable to state that the MCF-7 cell line was essential for theunderstanding of the subcellular organization of not only the ER butalso all of the other steroid hormone systems. The importance of thedevelopment of monoclonal antibodies to ER extends to mechanisticstudies of receptor dynamics during estrogen and antiestrogen action(43, 44). Additionally, antibodies can be used as probes to study thedirect effects of antiestrogens on the ER protein (45, 46). Indeed, anadditional epitope has been discovered to occur when ER is ligandedwith 4-hydroxytamoxifen (47), and this may be important to understand antiestrogen action.

Estrogenic Contaminantbis(4-hydroxyphenyl)[2-(phenoxy.

sulfonyl)phenyljmethane

Diethylstilbestrol

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MCF-7 BREASTCANCERCELLS

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Fig. 2. Immunocytochemistry-specific nuclearstaining for ER in MCF-7 cells grown on glassslides and incubated with H226 after fixation inpicric acid/paraformaldehyde. Controlpanel (left),cellsthatwereincubatedwithnonspecificrat IgCi.Specific staining was visualized with diaminobenzidine following incubation with rabbit antirat IgGand rat peroxidase-antiperoxidase complex.

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Cloning and Sequencing of the ER

Without the preparationof monoclonal antibodies to the MCF-7ER, it would not have been possible to make progress in cloning andsequencing the ER from MCF-7 cells (48). The development ofprobes that hybridized in the cDNA library preparedfrom MCF-7cells resulted in two simultaneous reports of the sequencing andexpression of the full-length cDNA for ER (49, 50). However, it wasnoted initially that there was an alteration in the cloned MCF-7 ERcDNA [Gly-400 (GGG) -+ Va1-400(GTG)]. Otherspecies (chicken,mouse, rat,andXenopus)all have a glycine at position400, but initialspeculation discounted the relevance of the finding for the human ER,because the binding affinity for estradiol was unaffected at 4°C(50,51). However, it was concluded eventually that the original cDNAdesignated HEO produced a protein that has decreased affinity forestradiol at 25°C(52), and it is, because of the mutation, unstable andrapidly turned over when expressed in cells. Subsequently, it was alsofound that the mutation increases the estrogen-like activity of tamoxifen (53, 54). The wild-type ER from MCF-7 cells is now referredtoas HEGO, with G denoting the presence of glycine at position 400.

The human ER gene is >140 kb in length and is split into eightexons with the position of the introns highly conserved with otherreceptors (51). The MCF-7 ER mRNA is 6322 nucleotides in lengthand encodes a 595-amino acid protein (50). The functional organization of the ER has been demonstrated by site-directed mutagenesis(55). The human ER comprises six regions (regions A—F),with asmall, centrally located DNA-binding domain and a large, COOHterminal ligand-binding domain (Fig. 3). The development of reverse

transcription-PCR (56) allowed a close examination of the mRNAs

from MCF-7 cells to establish whether variantsof the ER exist andwhether these could have biological relevance. The research strategyis controversial, because there is only clear evidence that the variantsactually produce a protein in one sublime of MCF-7 cells called 2A(57). The protein is a high molecular weight ER variant (Mr 77,000rather than Mr 65,000) that does not bind estradiol or antiestrogens(58). This is because the protein has a duplication of exons 6 and 7 inthe ligand-bindingdomain (58, 59). Otherinvestigatorsusing reversetranscription-PCR have produced variant cDNA from MCF-7 cells(60);however,the biologicalrelevanceof anyof the variantshasrecently been questioned (61—63).

Growth Factor Regulation

The MCF-7cell model has been examinedextensively to determinethe mechanism(s) of estrogen-stimulatedgrowth. The early work ofDr. Henri Rochefort identified a secreted glycoprotein from MCF-7cells that was believed originally to be a growth accelerator (64, 65).However, identification of the protein with monoclonal antibodies(66)andthecloningandsequencingof thegeneshowedtheproteintobe the enzyme cathepsin D (67). The clinical community has extensively studied the protein as a potential marker of prognosis innode-positive and node-negative breast cancer. In contrast, a proteinof unknown function, pS2, was identified in MCF-7 cells by Chambon's group (68—70).The protein has, like the PgR, been linked togood prognosis in breast cancer. The estrogen response element forpS2 is used routinely as an analytical system for reporter genes tostudy the molecular biology of ER activation (e.g., see Ref. 71).

The majorconceptualbreakthroughin ourunderstandingof cellularcommunication occurred in the late 1970s with the identification ofpositive and negative growth factors. It was reasoned that a link mustexist between estrogen action and growth factor secretion. Lippman'sgroupused MCF-7 cells extensively throughoutthe l980s to describethe hormonalregulationof the TGF-a/epidermalgrowthfactorreceptor system (72—76).This has been particularlydifficult to accomplish,because, unlike hormone-independent cells that overexpress growthfactors and their receptors, the MCF-7 system has only low levels ofthe relevant components. Studies showed initially that factors secreted

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from MCF-7 cells could support the growth of the same cells inathymic mice (77); however, the transfection of TGF-a cDNA intoMCF-7 was not, by itself, able to provoke estrogen-independentgrowth in animals (78). Nevertheless, transfection of v-ras1' doescause an elevation of stimulatory growth factors that can support cellgrowth in animals (79, 80) and the interaction of MCF-7 cells withbasement membranes (81). Lippman's group also described antiestrogen-induced production of the inhibitory growth factor TGF-@(82). Infact, these studies were performed before the knowledge that MCF-7cells were grown in phenol red and the realization that the estrogeniccontaminant was blocking TGF-@3synthesis. Despite this, the principlc was clear. Antiestrogenscould maintainthe secretion of TGF-@from ER-positive cells, and the growth factor could have a paracrinerole in inhibiting the growth of adjacent ER-negative cells (82).

The MCF-7 cells providedan experimentalsystem for defining therole of insulin and IGFs in the estrogen-induced proliferation of breastcancer cells. Although IGF-I and IGF-ll mRNAs are detected easily inthe majorityof breast tumors,conflicting results have been reportedfor the expression of IGFs in breast cancer cells. Initially, Huff et a!.(83, 84) reported that IGF-I is synthesized under estrogen control inMCF-7 cells. However, Karey and Sirbasku (85), using an immunoassay to measure IGF-I in conditioned medium, reported that MCF-7cells do not produce biologically significant concentrations of IGF-I,and subsequently,Yee et a!. (86), using a sensitive RNase protectionassay, could not detect IGF-I mRNA in breast cancer cell lines,including MCF-7 cells.

The majormitogenic effects of both IGF-I and IGF-IIare thoughtto be mediated via the type I IGF receptor identified in MCF-7 cellsas well as in other breast cancer cells (87—89).The action of IGF-I atits receptor may be modified by IGF-bindingproteins, which havebeen identified in conditioned media from breast cancer cells, including MCF-7 cells (90, 91). It has been shown thattamoxifenresistanceand estrogen independencein humanbreastcancercells is associatedwith complex changes in IGF-binding protein secretion patterns thataccompany changes in IGF receptor expression (92).

Despite the appeal of the autocrine model of growth regulation inbreast cancer by different growth factors, the data are not consistentwith an autocrine model of estrogen-regulated growth involving IGF-Iand IGF-H. In the nude mouse model, antibody-induced blockade ofthe type I IGF receptordoes not affect growth of the MCF-7 tumors,whereas the antibody inhibits growth of estrogen-independent MDAMB-23l tumors (93). However, a stimulatory effect of exogenousIGF-I on MCF-7 tumor growth in nude mice has been reported (94),suggesting that these cells do not lose the type I IGF receptors in vivo.Experiments in vitro with phenol red-free medium demonstrate thatIGF-I alone has very little effect on cell proliferationand only in thepresence of estradiol can these agents act synergistically to stimulategrowth (95—97).The observation that estradiol regulates type I IGFreceptor in MCF-7 cells at the transcriptional level (95) suggests adirect sensitizationof breastcancer cells to IGFs by estradiol.

Hormone and Antiestrogen Resistance in Vitro

It has been a century since the first observation that some humanbreast cancers will regress if estrogen is withdrawn(98). Naturally,the developmentof antiestrogensas a successful breastcancertherapy(99) resulted in the quest to understand antiestrogen resistance and theslip to hormone-independent growth. In the laboratories, MCF-7 cellswere used to develop several antiestrogen-resistantcell lines (100),one of which, LY2 (101), was found subsequentlyto have a wild-typeER (102). The finding of wild-type receptors is a consistent finding inantiestrogen-resistant disease. Early laboratory studies to developantiestrogen-resistant breast cancer cell growth were successful be

cause the antiestrogen completely saturated receptors. In contrast, astudy of the direct effects of estrogen withdrawal on hormone-responsive breast cancer cells was not possible until the discovery of phenolred in the media (26).

The first two published reports of the effects of phenol red-freemedia on the growth of MCF-7 cells came to the same conclusions(103, 104). The cells increase their growth rate as adapted clones growout. There is an increase in the levels of ER, but PgR synthesis ceases.Estrogen can reinduce the PgR; however, there is little effect oncellular growth rate. The cells now appear to grow maximally in theabsence of ligand but remain sensitive to the inhibitory actions ofantiestrogen. In fact, Masamura et a!. (105) have demonstrated subsequently that estrogen-deprived MCF-7 cells become hypersensitiveto estradiol.

Several cell lines have subsequently been cloned from the survivingMCF-7 cells in an estrogen-free medium. The line MCF-7.5C is ERpositive (wild type), but PgR cannot be induced by estrogen (106). Ifthe original MCF-7 could be described as making up the ER-positivePgR-positive tumor that has a functioning receptorwith good prognosis, then the MCF-7.5C cells make up the ER-positive PgR-negative tumorthathas a poorerprognosis (107). Dr. Benita Katzenellenbogen has also developed numerous new cell lines from MCF-7 (108),and their growth characteristicshave been described in athymic animals by Dr. Robert Clark at Georgetown University in Washington,D.C. (109). Additionally, Dr. Katzenellenbogen has recently described a tamoxifen (resistant)-stimulated breast cancer cell line derived from MCF-7 cells in vitro (110). The cells are extremelyvaluable to compare and contrast with wild-type lines by differentialdisplay. Identificationand elucidationof the new growthpathwaysinthe tamoxifen-resistantER-positive cells will have importantimplications for the design of new therapeutic agents.

Tamoxifen-stimulated Tumor Growth

The developmentof immunodeficientathymicmice (111) provideda new dimension for the study of breast cancer growth in vivo.Regrettably, early studies with primary breast tumors were, in themain, unsuccessful in producing hormone-responsive disease, becausetumor takes were poor, and hormone-independentdisease usuallygrew preferentially despite estrogen administration (1 12—114).

The breakthroughcame with the transplantationof the breastcancercell line MCF-7 (1 15). Estrogen is required for the growth of MCF-7breast cancer cells in ovariectomized athymic mice. It should benoted, however, that MCF-7 cells do not grow vigorously in intactathymic mice, because the animals have endocrine deficiencies and donot have estrouscycles. Estrogensupplementationis obligatory(116).However, there are considerablevariationsin MCF-7 cell lines, andthis produces different abilities to grow in athymic mice (117).

It is interesting to note that, although tamoxifen is described asestrogen in the mouse (118), tamoxifen does not initially support thegrowth of MCF-7 cells. Tamoxifen inhibits estrogen-stimulatedMCF-7 tumor growth in vivo (119, 120). This observationparallelsthe observationsin vitro. Tamoxifen does, however, maintainoccultdisease for long periods after inoculation into mice, and tumors can bereactivated by estrogen treatment when the drug is stopped (120).

By the mid-l980s, prolonged(2—5years) adjuvanttamoxifen treatment was being used routinely in clinical practice, and it was clear thatthis strategy produced a survival advantage (121, 122). However, itwas unreasonable to expect that drug resistance would not occureventually. An endometrial carcinoma, EnCa 101, had been shown togrow partiallyduring tamoxifen treatment(123), so it was possiblethattamoxifen-stimulatedbreastcancer would also occur. Osborneeta!. (124) first showed that MCF-7 cells could become tamoxifen

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stimulated for growth in vivo but, paradoxically, in vitro, the tumorcells revert to their original phenotype; i.e., tamoxifen blocks estrogen-stimulated growth. In parallel studies, we showed (125) thattamoxifen-stimulated breast tumors grown from MCF-7 cells could bepassaged routinely and the tumors would grow in vivo in response toeither estradiol or tamoxifen. Furthermore, tamoxifen-stimulated ftimom grow in athymic rats and naturalkiller cell-deficient (beige)mice (126), so the growth effect is neither species nor immunology

specific. In fact, target site-specific actions of tamoxifen were noted inthe animals. The uteri in mice treated with tamoxifen become quiescent and refractoryto estrogen, despite the fact that a tamoxifenstimulated MCF-7 tumor is growing in the host (125).

The model of tamoxifen resistance in breast cancer appears to beuniqueto MCF-7 cells, and thereis intense interestin discoveringthemechanismof tamoxifen-stimulatedtumorgrowth.Earlytheoriesthatthe receptormay be mutatedhave provedto be incorrect.Despite oneexception, with a 35l@@ mutant ER (127) that is observed in onetransplant line, the majority of tumors have wild-type ER (128).Similarly, ideas that tamoxifen might be metabolized peripherally toestrogens (129, 130) or locally (131—133)in the MCF-7 tumor havenot been supportedby experimentation.The growth (134) and development of tamoxifen-stimulated MCF-7 tumors (135) occurs withanalogues that are incapable of being converted to estrogenic metabolites. Clearly, the cells are using the intrinsic efficacy of the anticstrogen-ER complex to drive cell replication by an alternate pathway.

Most importantly,compoundssuch as IC! 164,384 andIC! 182,780(pure antiestrogens), which have no intrinsic estrogenic properties inestrogen target tissues (136), inhibit the growth of tamoxifen-stimulated MCF-7 tumors in vivo (137, 138).

Clinical Advances and the Future Possibilities

It would be inappropriate to finish this story of the laboratorydiscoveries made possible by MCF-7 cells withouta brief mentionofsome of the clinical advances that have occurred. The systematicdissection of hormone action has led to significant progress in thetreatment of breast cancer. The following list of examples is itself

impressive but does not claim to be exhaustive.Abbott Laboratories developed kits for the quantitation of ER

(ER-EIA) and PgR (PgR-EIA) in breast tumor (139—141).Steroidreceptors from MCF-7 cells were key to this innovation. Specificmonoclonal antibodies for ER and PgR are now used routinely todetermine receptors in tumor cells by flow cytometry and to determinethe heterogeneityof cell populations in small biopsy samples (142—144).

The ubiquitoususe of long-termadjuvanttamoxifen therapy(145)originally presented a problem for the determination of receptor statusof recurrencesusing conventional ligand-bindingassays. Tamoxifenand metabolites saturate all of the tissues and produce false-negativeresults in breast cancer biopsies because of receptor occupancy.Monoclonal antibodies changed all of that, and it is now known thatmany recurrences remain receptor positive (146). Additionally, thereis evidence that tamoxifen-stimulated disease occurs in patients (147),which is analogous to the tamoxifen-stimulated MCF-7 tumor in thelaboratories that remains receptor positive (128).

The ability of pure antiestrogens to control the growth of tamoxifen-stimulated MCF-7 tumors (137, 138) was strong evidence toprogress toward clinical trials. Currently, IC! 182,780, the first ofseveral pure antiestrogens, is performing as predicted. Preliminary

trials (148) indicate good patient acceptability, a response rate ofabout 30%, and a duration of response that extends for several years.

During the past 25 years, the symbiotic relationshipbetween laboratoryresearchand clinical investigations has resulted in clear ad

vances in our knowledge of hormone action and the standardization oftherapy with antiestrogenic drugs. Dr. Herbert Soule had the visionand good fortune to establish the first hormone-responsive breastcancer cell line that played a fundamental role in advancing knowledge. He was involved in the three key studies: cell characterization(1), receptor identification (2), and estrogen-stimulated growth inathymic mice (115). Together, the results of his labors revolutionizedthe opportunities for breast cancer research. In retrospect, it is remarkable that this single cell line has contributed so much to our basicunderstandingof hormone action and the treatmentpossibilities inbreastcancer. However, the story does not end here. Dr. Soule left asecond legacy, MCF-lO (149, 150).

The research challenge for the 21st century is to prevent breast cancer.MCF-l0 and its variantcells are, surprisingly,naturallyimmortalized,normal epitheial cells of mammary origin that do not produce tumors inathymic animals (151). The key for a future generation of researchworkers is to dissect the molecular events involved in the change from

normalto malignantbreastepitheium (152—155).It is ourbelief thatbydiligent research endeavors, and the example of MCF-7 cells before, theMCF-lO cells can be used effectively to formalate a rational strategy forthe preventionof breastcancer.

Acknowledgments

This history is offered as a tribute and memorial to Dr. Herbert Soule andthe staff of the MichiganCancerFoundation,who made possible the development of the remarkable MCF-7 breast cancer cell line. It is also dedicated toSister Catherine Frances, the patient with breast cancer whose cells ultimatelyextended the lives of thousands of women. We offer this history as a primeexample of the value of laboratory research that translates, over decades, to theaid of patientswithbreastcancer.

We thankDr. GeoffreyGreenefor generouslyprovidingFig. 2 and HenryMuenzner for preparing other figures.

References

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2. Brooks, S. C., Locke, E. R., and Soule, H. D. Estrogen receptors in a human cell line(MCF-7) from breast carcinoma. J. Blot. Chem., 248: 6251—6253, 1973.

3. Lippman, M. E., and Bolan, G. Oestrogen-responsive human breast cancer inlong-term tissue culture. Nature (Lond), 256: 592—593,1975.

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7. Horwitz, K. B., and McGuire W. L Nuclear mechanism of estrogen action: effectsof estradiol and antiestrogens on estrogen receptors and nuclear receptor processing.J.Biol.Chem.,253:8185—8191,1978.

8. Horwitz, K. B., McGuire, W. L., Pearson, 0. H., and Segaloff, A. Predictingresponse to endocrine therapy in human breast cancer a hypothesis. Science (Washington DC), 189: 726—727,1975.

9. Horwitz, K. B., Koseki, Y., and McGuire, W. L. Estrogen control of progesteronereceptor in human breast cancer: role of estradiol and antiestrogen. Endocrinology,103:1742—1751,1978.

10. Jordan, V. C., and Murphy, C. S. Endocrine pharmacology of antiestrogens asantitumor agents. Endocr. Rev., II: 578—610, 1990.

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1997;57:3071-3078. Cancer Res   Anait S. Levenson and V. Craig Jordan  MCF-7: The First Hormone-responsive Breast Cancer Cell Line

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