[CANCER RESEARCH 55, 258ÃŽ-2590, June 15, 1995]

An Estrogen-Independent MCF-7 Breast Cancer Cell Line Which Contains a Novel80-Kilodalton Estrogen Receptor-related Protein1

John J. Pink, Shun-Yuan Jiang, Michael Fritsch, and V. Craig Jordan2

Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin 53792 [J. J. P., S. Y. J., M. F., V. C. J.], and Roben H. LurieCancer Center, Northwestern University Medical School, Chicago, Illinois 60611 fV. C. J.J

ABSTRACT

Long-term growth of estrogen-responsive human breast cancer celllines in estrogen-free media leads inevitably to the development of estrogen-independent growth. We have identified and characterized a uniquesubclone of the MCF-7 human breast cancer cell line, named MCF-7:2A,

which grows maximally in the absence of endogenous estrogens but whosegrowth is inhibited by the antiestrogens 4-hydroxytamoxifen and ICI164,384. The MCF-7:2A cells express high levels of estrogen receptor (ER;477 fmol/mg protein), which can be reduced by growth in 10 n\i 17/i-

estradiol (201 fmol/mg protein). Basal progesterone receptor synthesis isvery low in the 2A cells «I fmol/mg protein) but can be dramaticallyincreased by 10 UM 17ß-estradiol (384 fmol/mg protein). Clearly, thepathways that control growth and estrogen-regulated genes such as theprogesterone receptor are now dissociated in these cells. MCF-7:2A cellsalso possess two unique characteristics. First, the MCF-7:2A cells consti-tutively activate an ER-responsive luciferase reporter construct in the

absence of any estrogens, and this activation can be blocked by either4-hydroxytamoxifen or ICI 164,384. This constitutive activity is not observed in the parental MCF-7 cells. Second, they express an 80-kDaprotein that cross-reacts with three distinct antibodies to the ER.

The MCF-7:2A ceils were subjected to an additional round of limiting

dilution subcloning, and 10 independent clones were all shown to expressboth the 66- and 80-kDa ERs as observed in the MCF-7:2A line. This

confirms that both ERs are being expressed in each cell and are not theresult of a mixed population of cells. While numerous ER variants havebeen reported previously, no ER has until now been described that islarger than the wild-type 66-kDa ER. The MCF-7:2A cells provide a

unique model to use in the study of ER action and the development ofestrogen-independent growth in human breast cancer cells.

INTRODUCTION

The prevailing paradigm of estrogen action in ER3-positive cells

suggests that the ER is located in the nucleus loosely associated withthe chromatin. Following binding to its ligand, the ER undergoes aconformational transformation that causes the ER to become moretightly bound to specific DNA sequences referred to as EREs. Thisactivated ER interacts with other members of the preinitiation complex, causing stimulation of transcription from genes in the vicinity ofthe EREs (1, 2). In responsive cells the transcription of these genesleads to the initiation of a cascade of events resulting in mitogenesisor differentiation (3).

Estrogens are also involved in many steps of the development andfunction of normal mammary epithelium (4). However, the functionof the ER in the natural history of breast cancer is difficult to discern.

Received 1/13/95; accepted 4/18/95.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grant CA32713. J. J. P. was supported in part by

NIH Training Grant 5T32-CA09471. S. Y. J. was supported by a scholarship from theNational Science Council and National Defense Medical Center (Taiwan, Republic ofChina).

2 To whom correspondence should be addressed, at Robert H. Lurie Cancer Center,

Northwestern University Medical School, 303 East Chicago Avenue, Olsen Pavilion 8258,Chicago, IL 60611.

3 The abbreviations used are: ER, estrogen receptor; PR, progesterone receptor; E2,

17/3-estradiol; 4-OHT, 4-hydroxytamoxifen; ERE, estrogen response element; PBST,PBS + 0.1% Tween 20; EIA, enzyme-linked immunoassay.

While only ~7% of the epithelial cells in the normal breast contain

measurable ER (5) up to 70% of primary breast cancers express ER(6). This would suggest that expression of the ER may be linked to thedevelopment of breast cancer. Further support for the involvement ofER in the etiology of breast cancer is provided by the very lowincidence of breast cancer observed in males or in women withcongenitally dysfunctional ovaries (7). However, the loss of ERand/or the development of estrogen-independent growth is observedin most breast cancers following long-term treatment with antiestro

gens. While it is clear that the loss of the ER leads invariably to anantiestrogen-resistant phenotype, the presence of ER does not always

correlate with antiestrogen sensitivity. The strong selective pressure toescape the growth inhibition exerted by antiestrogens in both laboratory and clinical tumors gives rise to numerous forms of antiestrogenresistance. Various mutations in the ER have been observed which cangive rise to antiestrogen resistant tumors (8). Even cells which expresswild-type ER can display an antiestrogen-resistant phenotype (9). The

interplay of other growth regulatory factors is most likely the cause ofthis unusual phenotype.

The development of estrogen-independent cell lines has been used

as a laboratory model to study the more clinically relevant problem ofantiestrogen resistance. It is believed that the development of estrogen-independent growth is a step in the pathway leading to antiestro

gen resistance (10). The discovery of the estrogenic action of acontaminant present in commercial preparations of the pH indicatorphenol red, and its subsequent removal from tissue culture mediaallowed the demonstration of growth stimulation by estrogens inER-positive breast cancer cell lines (11). This opened the door for thestudy of the mechanism of estrogen-mediated growth stimulation and,conversely, investigation of the development of estrogen-independentgrowth. Our laboratory and others immediately began the long-term culture of the ER-positive cell line MCF-7 in media devoid of

any estrogens (12, 13). Since that time additional studies havefocused on the phenomenon of estrogen-independent growth inother cell lines (9, 14-16).

When cultured in estrogen-free media, the growth of MCF-7 cells

slows for a number of months followed by an increase in the growthrate and the establishment of stable clones. These clones consistentlymanifest an unusual phenotype. While the cells continue to expresshigh levels of ER their growth cannot be increased by estrogens butcan be inhibited by antiestrogens. The expression of estrogen-respon

sive genes such as the PR is often shown to remain dependent onexogenous estrogens. This demonstrates that the pathways controllinggrowth and the expression of other estrogen-regulated genes have

been uncoupled. The mechanism responsible for this phenomena hasyet to be unraveled; however, an hypothesis (17) has been proposedwhich suggests nonestrogen-mediated stimulation of the ER.

Following growth for 8 months in the absence of estrogen and tworounds of limiting dilution cloning, we isolated a clone named MCF-

7:2A. These cells grow maximally in the absence of estrogens, andtheir growth can be inhibited by antiestrogens as seen in previousstudies of MCF-7 cells adapted to these growth conditions (12, 13). In

these studies we describe characteristics of this particular clone whichhave until now not been observed.

2583

Research. on February 5, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

AN MCF-7 CELL LINE EXPRESSING AN 80-kDa ESTROGEN RECEPTOR

MATERIALS AND METHODS

Tissue Culture. MCF-7 cells were obtained from Dean Edwards (San

Antonio, TX) (originally obtained from the Michigan Cancer FoundationDetroit, MI). All tissue culture components were purchased from GIBCOLaboratories (Grand Island, NY) unless otherwise stated. Cells were originallygrown in MEM supplemented with 5% calf serum, 6 ng/ml bovine insulin, 25min HEPES, 2 HIM L-glutamine, 100 units/ml penicillin, and 100 (xg/mlstreptomycin (fully estrogenized media). Estrogen-free media substitutes phenol red-free MEM and 3X dextran-coated charcoal-treated calf serum. Cellswere routinely passed at 1:5-1:20 dilutions once per week using 0.1% trypsin.All cells were grown in a 37°Chumidified incubator with 5% CO,. MCF-7:2A

cells were regularly grown in estrogen-free media, and MCF-7:WS8 cells were

grown in fully estrogenized media. Experiments were also performed usingRPMI 1640 (minus phenol red) and 10% heat-inactivated 3X charcoal-stripped

fetal bovine serum (Bioproducts for Science, Inc., Indianapolis, IN), and nodifferences were seen as compared with cells grown in MEM and calf serum.

Limiting dilution cloning was performed by diluting cells to 25 cells/200 julmedia and seeding 200 /j.1 into the first row of a 96-weII plate. Using aneight-channel micropipette (Oxford Labware, St. Louis, MO) 100 /id of this

solution were then added to 100 ju.1of media in the second row with mixing,these 1:2 dilutions were then repeated for the remaining rows. After 2 weeksall wells were inspected under low magnification, and wells containing singlecolonies were noted and harvested ~3 weeks later.

Growth Assays. All cells were grown in estrogen-free media for at least 2

days prior to the beginning of each experiment. Cells were seeded into eachwell of a 24-well plate (20,000 cells/well) in 1 ml of estrogen-free media on

day 0. The following day (day 1) this media was removed, and 1 ml of mediacontaining the appropriate compound(s) was added. All compounds weredissolved in 100% ethanol and added to media at a 1:1000 dilution. Media waschanged on day 4 and experiments were ended on day 6. For time courseexperiments, cells were harvested daily and media was changed on days 4 and6. DNA content was determined according to the method of LaBarca andPaigen (18) using an fluorocolorimeter II (SLM Aminco, Urbana, IL). E2 waspurchased from Sigma Chemical, 4-OHT was a generous gift from Zeneca

Pharmaceuticals (Macclesfield, England), and ICI 164,384 was a generous giftfrom Alan Wakeling, Zeneca Pharmaceuticals.

EIAs (ER and PR EIAs). For EIA cells were grown to near confluenceand scraped into homogenization buffer containing 1 mM monothioglycerol,10 min Tris, 1.5 mM EDTA, 5 mM Na2MoO4, and 0.4 M KC1. Cytosols werecollected following centrifugation at 100,000 X g for 45 min at 4°C.Assayswere performed as to the manufacturer's specifications. ER and PR EIA kits

were obtained from Abbott Laboratories (Abbott Park, IL).Scatchard Analysis. For ligand binding analysis, whole-cell uptake of

[3H]estradiol (New England Nuclear Co., Boston, MA) was carried out inHBSS containing a range of 0.01-5 nM [3H]estradiol with or without a200-fold molar excess of diethylstilbestrol for 30 min at 37°Cin a humidified

incubator. The cells were then washed using HBSS containing 0.1% BSA threetimes and using HBSS alone three times. The cells were then sonicated in asolution of calcium- and magnesium-free HBSS, and aliquots of the lysate

were counted in a liquid scintillation counter. Scatchard analysis of the sampleswas then performed as described previously (19).

Western Blotting. Whole-cell extracts were prepared by direct lysis ofPBS-washed cells in sample buffer (10% glycerol, 150 mM Tris-HCl, pH 6.8,0.5 mM EDTA, 0.125% SDS, 1% ß-mercaptoethanol, and 5 ng/ml bromphenol

blue) followed by immersion in a boiling water bath for 5 to 10 min. Equalamounts of protein were added to each lane of a 8.75% polyacrylamide gelwith a 3% stacking gel. For some studies cytosols were prepared, and ERcontent was determined using a hydroxylapatite binding assay (20) prior toWestern blotting. Following electrophoresis proteins were transferred to Hy-bond-C (Amersham Corp., Arlington Heights, IL) using a Multiplier II semi-

dry electroblotting device (Pharmacia Biotech, Inc., Piscataway, NJ) accordingto the manufacturer's directions. Loading equivalence and transfer efficiency

were monitored by Ponceau S (Sigma Chemical Co., St. Louis, MO) stainingof the membrane. The membrane was then destained using IX PBST andblocked overnight using 50% calf serum in IX PBST at 4°Cwith gentle

shaking. The membrane was then incubated with a 1:500 dilution of antibodyin 10% calf serum/lX PBST for 2 h at 20°Cwith gentle shaking. Antibody

H222 was a generous gift from Abbott Laboratories, and antibodies 986 and

987 were a generous gift from Helga Ahrens, Tim Schuh, Gerald Mueller, andJack Gorski (21). The antibody was removed and the membrane was rinsedfive times with IX PBST followed by a 10-min wash; this sequence is repeatedthree times. The alkaline phosphatase-conjugated secondary antibody was

diluted 1:2500 in IX PBST + 10% calf serum and incubated with themembrane for 2 h at 20°Cwith gentle shaking. For H222 an alkaline phos

phatase-conjugated goat anti-rat IgG was used as the secondary antibody. For986 and 987 an alkaline phosphatase-conjugated goat anti-rabbit IgG was used

(Hyclone Laboratories, Inc., Logan, UT). Following washes as describedabove, the proteins of interest were visualized by incubation with 0.33 mg/mlnitro blue tetrazolium, 0.166 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate(Promega, Madison, WI) in alkaline phosphatase buffer (100 mM Tris-HCl, pH9.5, 100 mM NaCl, 5 mM MgO2> 25 mM ZnCU) at 20°Cfor 5 to 20 min. This

reaction was stopped by adding 7% acetic acid followed by three washes indouble-distilled water. The membrane was then air dried and photographed.

Transient Transfection Assays. Cells were seeded into a 6-well plate(500,000 cells/well) in phenol red-free RPMI 1640 + 10% 3X charcoal-

stripped fetal bovine serum. The following day media was removed andreplaced with fresh estrogen-free media. A solution containing 1 ju,gluciferasereporter construct pVIT3-luc (22) and 0.5 /xg ß-galactosidase reporter pCMVß

(23) in 0.25 M CaCl2 was mixed dropwise with an equal volume of 2X HBS(0.28 MNaCI, 0.05 MHEPES, and 1.5 mM NaPO4, pH 7.05) by gently bubblingair through the solutions. This solution was then incubated at room temperaturefor 20 min to allow a DNA/CaP04 precipitate to form. This solution wasslowly added to the cells and incubated at 37°Cin a humidified incubator with

5% C02 for 6 h. At that time the DNA solution was removed, and media withor without compounds were added to the wells and incubated at 37°Cin a

humidified 5% CO, incubator for an additional 18—48h. The media were thenremoved, and the cells were washed once with ice-cold PBS. The cells werethen scraped in extraction buffer (0.1 M KHPO4, pH 7.5, 1% Triton X-100, 100

/xg/ml BSA, 2.5 mM phenylmethylsulfonyl fluoride, and 1 mM DTT) andpipetted vigorously to ensure complete cell lysis. Debris was then pelleted byspinning in a microfuge for 1 min, and the lysate was stored on ice untilluciferase activity was assayed. Luciferase activity was assayed by mixing 50/xi each lysate with 350 ;xl reaction buffer (160 mM MgCl2, 75 mM glycyl-glycine, pH 7.8, 0.5 mg/ml BSA, 19 mg/ml ATP, and 15 mM Tris-HCl, pH

7.5). To begin each assay 100 /il substrate (0.4 mg/ml luciferin, potassium saltin 10 mM NaH2CO„pH 6.0) were automatically injected into the lysatemixture. Each point was monitored for 10 s using a Monolight 2010B lumi-

nometer (Analytical Luminescence Laboratory, San Diego, CA) and relativeluciferase units were then reported. All points were corrected for transfectionefficiency by dividing relative luciferase units by ß-galactosidase activity.

ß-Galactosidase activity was measured using a ß-methlumbelliferone assay

(24). Briefly, an aliquot on the cell extract is mixed with 1.3 ml reaction buffercontaining 0.1 M NaPO4, 10 mM KC1, 1 mM MgSO4 (pH 7.0), and 2.2 (10~5)

g/ml ß-methylumbelliferone (Molecular Probes, Inc., Eugene, OR). The sam

ple is incubated at room temperature for 1 h and 750 ;u,l stop buffer (15 mMEDTA and 0.3 M glycine, pH 11.2) were added. The samples are then read ina LS-5 fluorescence spectrophotometer (Perkin Elmer/Cetus, Foster City, CA)

with excitation at 350 nm and absorption at 450 nm. All samples are correlatedto a standard curve using purified ß-galactosidase (Boehringer Mannheim

Biochemicals, Indianapolis, IN).

RESULTS

Selection of a Variant of the MCF-7 Human Breast Cancer CellLine. In order to study the development of estrogen-independentgrowth in human breast cancer cell lines we cultured MCF-7 cells in

media devoid of exogenous estrogens. Following 8 months of culturethe cells were subjected to two rounds of limiting dilution cloning inestrogen-free media. A number of clones were selected for furtherstudy including the MCF-7:5C cell line, which has been describedpreviously (9). Due to its unique properties the MCF-7:2A cell linewas chosen for more detailed study. Wild-type MCF-7 cells were also

subjected to two rounds of limiting dilution cloning in media containing phenol red and whole calf serum. The line MCF-7:WS8 waschosen as a representative clone that expressed the typical estrogen-responsive growth characteristics seen in the parental MCF-7 cell line.

2584

Research. on February 5, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

AN MCF-7 CELL LINE EXPRESSING AN 80-kDa ESTROGEN RECEPTOR

The WS8 cells were used as a clonal estrogen-responsive line. In all

studies completed thus far no significant differences have been observed between the parental MCF-7 and MCF-7:WS8 lines.

The 2A cells display more rapid growth in the absence of estrogensthan the WS8 cells, 2.9 days versus 5.4 days doubling time, respectively (Fig. 1). The growth rate of the 2A cells is unaffected by theaddition of estrogens. As expected, the growth of the WS8 cells isgreatly enhanced by 1 nM E2, showing a decrease in doubling timefrom 5.4 to 2.5 days.

Growth-Response Studies. Growth-response studies were initi

ated to determine the effects of estrogens and antiestrogens on the 2Acells. WS8 cells were included as a control to ensure that the mediawas truly devoid of any estrogens. 2A cells were grown continuouslyin estrogen-free media, and the WS8 cells were grown for 4 days inestrogen-free media prior to seeding in order to eliminate residual

effects of the estrogen present in the standard media. Media with orwithout compounds were added 1 day later and replaced with freshmedia on day 4. The experiment was ended on day 6.

As seen in Fig. 2A, the growth of the WS8 cells is stimulated 7-foldover control levels by E, during the course of this 6-day assay, withmaximum growth observed at concentrations of greater than 10~" M.

4-OHT demonstrates typical partial agonist activity causing a 2-foldstimulation of growth at 10"'" M. The pure antiestrogen ICI 164,384

showed no effect on the growth of the WS8 cells. These are typicalresponses for estrogen-dependent cell lines and demonstrates that theestrogen-free media does not contain any measurable estrogenic ac

tivity. This WS8 response is consistent with that reported earlier forMCF-7 cells (25).

TIME (days)

Fig. 1. Growth rate of WS8 und 2A cells ¡nmedia ±E2. Cells were seeded into eachwell (20.000/well) (if a 24-well plate ±l nM ET. Each day cells were harvested and storeduntil DNA was measured at the end of the experiment. Each [Kfint, mean of triplicatedeterminations ±SEM. D, WS8 cells in estrogen-free media; •¿�.WS8 + l nM E2; O, 2A

cells in estrogen free media; •¿�,2A cells + 1 nM Ej.

A competition experiment was also performed (Fig. 2B) with0.1 nM E2 present in all groups along with increasing concentrationsof the antiestrogens. The WS8 cells show a typical dose-dependentinhibition of the E2-stimulated growth with both antiestrogens.

Fig. 2. Growth response of WS8 (A and fl) and 2A (Cand D) cells to E2, 4-OHT, and ICI 164,384 (/C/). WS8cells were grown in estrogen-free media for 4 days priorto seeding. On day 0, WS8 or 2A cells were seeded intoa 24-well plate (20.IXX) cells/well). The following day(day I) media containing compound were added andmedia were changed every other day. In B and D, 0.1 nME2 was included in all wells, and increasing concentrations of antiestrogens were then added to demonstratecompetition of the E2-stimulated growth. On day 6 cellswere harvested and DNA measured. Each point, mean oftriplicate determinations ±SEM.

et

Zo

35

30

25

20

15

m-

ta'•T.

0-13 -12 -11 -10 -9 -8 -7 -6

LOG MOLAR CONCENTRATION

25-

20-

IS'

•¿�5»Hici.

D

0 -13 -12 -11 -10 -9 -8 -7 -6

LOG MOLAR CONCENTRATION

2585

B

u•¿�s.

Za

D

-

W'•f.

•¿�eb

a

35

30-

25-

_ 15

Mi-

5-

"•o

—¿�D— O.lnM E2 t 4-OIIT

--O-- O.lnM E2 * ICI

0 -12 -11 -10 -9 -8 -7 -6

LOG MOLAR CONCENTRATION

20

15

10

>-$.

•¿�•o

-9 -8 -7 -6

LOG MOLAR CONCENTRATION

Research. on February 5, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

AN MCF-7 CELL LINE EXPRESSING AN 80-kDa ESTROGEN RECEPTOR

Table l ER and PR expression in WS8 and 2A cellsCells were plated in 15-cm dishes after 2 days growth in estrogen-free media. Either

control media or media containing 10~8 ME2 were added, and cells were harvested after

5 days. ER and PR levels were then determined using the protocol described in the ER andPR-EIA kits provided by Abbott Laboratories. Binding affinities for the ER were determined using a [~H]estradiol whole-cell uptake assay.

CellsWS8WS82A2ATreatmentControl10

nME2Control10

nM E2ER(fmol/mg)292.1146.0477.5201.6ERKd(nM)0.250.54PR(fmol/mg)2.9561.80.31384.5

The 2A cells clearly show enhanced growth in the absence ofestrogen, with a 5-fold higher DNA amount in the control group as

compared with the WS8 cells (Fig. 2C). E2 at concentrations greaterthan 1CF"' Minhibited the growth of the 2A cells in this 6-day assay.

This is in contrast to the growth rate studies which do not show anydifference in the growth rate of the 2A cells in the presence or absenceof 1 nM E2 (Fig. 1). The significance of this observation is unclear.However, 4-OHT significantly inhibited the growth of the 2A cells ina dose-dependent manner. Maximum inhibition was observed at concentrations greater than 0.1 /XMwith 75-85% inhibition of control cell

growth seen. ICI 164,384 also inhibited growth of the 2A cells withmaximum inhibition at concentrations greater than 0.1 /AM. Thegrowth of the 2A cells is not stimulated by the addition of 0.1 nM E2;however, the growth inhibition caused by the antiestrogens is alteredsuch that —¿�10times more antiestrogen is needed for the same level of

growth inhibition as seen in the absence of E2 (Fig. 2D and data notshown). This growth response observed in the 2A cells is consistentwith that reported previously for MCF-7 cell lines adapted to grow inestrogen-free media (12, 13).

ER and PR Expression in MCF-7:2A Cells. ER and PR expres

sion was determined using EIA kits obtained from Abbott Laboratories. The data presented in Table 1 show that the ER and PR levels inthe WS8 cells are subject to regulation by estradiol. The addition of 10nM estradiol for 5 days caused a 50% reduction in ER expression anda 200-fold induction in PR expression in the WS8 cells. In the 2A cells

the regulation of ER and PR expression was similar, with a 58%reduction in ER and greater than 300-fold increase in PR expression.

The finding that PR levels were not elevated in the 2A cells in theabsence of estrogen demonstrates that the ER in the 2A cells is notconstitutively activating all estrogen-responsive genes in the cell.

The appearance of estrogen-independent growth, which can be

inhibited by antiestrogens, could be the result of a supersensitive ERwith a 10-100-fold enhanced binding affinity for estrogens. This type

of ER could scavenge the very low amounts of estrogens which are

not removed during the serum-stripping procedure and transduceestrogen-responsive signals. In order to investigate this possibility,Scatchard analysis of [3H]estradiol uptake was used to determine the

binding affinity of the ERs in the 2A and WS8 cells. The calculatedKds in the 2A and WS8 cell lines were quite similar, 0.54 nM and 0.25nM, respectively. Therefore, a supersensitive receptor does not appearto be responsible for the constitutive activity in the 2A cells.

Western Blotting Using Specific ER Antibodies. To characterizethe ER in the 2A cells further, Western blot analysis was performedon cytosols from the 2A and WS8 cells following growth in estrogen-

free media for 5 days. The initial Western blot was probed using themAb H222 (26), which is also utilized in the Abbott ER-EIA kit.H222 recognizes an epitope in the steroid-binding domain of the ER.

This analysis revealed a unique protein in the 2A extract that migratedat -80 kDa in addition to the expected 66-kDa ER (Fig. 3).

In order to determine whether this extra band was the result of avariant ER or cross-reactivity of the H222 antibody with an unrelated

protein, the analysis was repeated with the addition of two polyclonalantibodies to the ER (21). Antibodies 986 and 987 were raised inrabbits following immunization with recombinant human ER peptidesthat were overexpressed in Escherichia coli. Two peptides were usedto immunize the rabbits. ER-N is a 38-kDa peptide from the NH2

terminus of the human ER that consists of a portion of the A domainas well as the B, C, and D domains. ER-C is a 41-kDa peptide from

the COOH terminus which consists of the D, E, and F domains of theER (27). Antibody 986 was derived from rabbits immunized with theER-N peptide. Antibody 987 was raised in rabbits against a mixture ofthe ER-N and ER-C peptides which constitutes the majority of the

human ER protein.As seen in Fig. 3, all three antibodies bind to the 80-kDa protein in

the 2A cell extracts as well as the wild-type 66-kDa ER. In thisanalysis equal amounts of ER, as determined by [3H]estradiol binding,

were loaded per lane. This demonstrates that the 80-kDa proteinshares at least three independent epitopes with the 66-kDa ER and isnot the result of nonspecific cross-reactivity.

Subcloning of the 2A Cell Line. The presence of a higher molecular weight ER in the 2A cells was a unique observation. While manyvariant ERs have been described, none has ever been observed that islarger than the wild-type 66-kDa protein. Although the 2A cells were

isolated following two rounds of limiting dilution cloning, there wasconcern that the two ERs were perhaps the result of a mixed population of cells, each expressing only one ER. In order to address thispossibility an additional round of limiting dilution cloning was performed. Western blot analysis was performed on 10 individual clonesof the 2A cell line using the H222 antibody. As seen in Fig. 4, all 10clones expressed both the 66- and 80-kDa ERs. For this analysis equal

Fig. 3. Western blot of WS8 and 2A cytosolsusing three independent antibodies. Each lane wasloaded with equal amounts of ER as determined byhydroxyapatite binding. Following electrophoresisand transfer, the blot was cut into three sections andeach section was probed with a different antibody.The letters below the antibody designation indicatethe domain to which each antibody binds.

Ab 986(ABC)

WS8 2A

(80 kDa) -w.t. ER "

(66 kDa)

Ab H222(E)

WS8 2A

Ab 987(ABCDEF)

WS8 2A

2586

Research. on February 5, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

AN MCF-7 CELL LINE EXPRESSING AN 80-kDa ESTROGEN RECEPTOR

12345 WS8

SOkDa66kDa

6789 10WS8

SOkDa66 kDa

Fig. 4. Western blot of 10 subclones of the 2A cell line. Following limiting dilutionsubcloning whole cell extracts were prepared and subjected to Western blotting. Each lanewas shown by Ponceau S staining to contain equal amounts of total protein. The blot wasthen probed with the antibody H222.

WS8 2A

C E2 OHT ICI I C E2 OHI ICI

w.t. ER(66 kDa)

Fig. 5. Ligand-induccd regulation of the 66- and HO-kDa ERs. Cells were grown for 4

days in the absence of estrogens. Media containing compounds were then added, and thecells were grown for an additional 48 h. Cytosols were then prepared using 0.6 M NaClto isolate occupied ER. Protein concentration was determined using the Lowry assay.Equal amounts of total protein were run in each lane. Following clectrophoresis andtransfer, the hlot was probed with the antibody H222. OHT, 4-hydroxytamoxifen.

amounts of total protein were loaded into each lane. Clearly, the 2Acells express a higher level of ER than the WS8 cells in goodagreement with the previously described ER-EIA assays. This data

definitively establish that both ERs are expressed in all 2A cells.Regulation of the Steady-State Levels of the 66- and 80-kDa ER

by Estrogens and Antiestrogens. Regulation of the ER in MCF-7

cells is influenced by exposure to both estrogens and antiestrogens(28-32). To determine whether the regulation of the steady-statelevels of the 80-kDa ER is under the same control as the 66-kDa ER,

WS8 and 2A cells were exposed to estrogen or antiestrogen for 48 h.Whole-cell extracts were then prepared and subjected to Western blot

analysis using the H222 antibody (Fig. 5). Equal amounts of totalprotein were loaded into each lane. The steady-state level of the66-kDa ER in the WS8 cells was significantly down-regulated by E2and ICI 164,384 while 4-OHT did not significantly alter the amountof ER. The 2A cells express higher levels of the 66-kDa ER but theregulation of the 66-kDa ER is similar to that seen in the WS8 cells.The regulation of the 80-kDa ER is more difficult to discern due to its

relatively low level of expression but E2 does seem to cause arepression in the level of the 80-kDa ER, and 4-OHT does not appearto affect the level of the 80-kDa ER. Notably, exposure to ICI 164,384does not appear to cause a decrease in the level of the 80-kDa ER incontrast to the effect seen with the 66-kDa ER.

It is of interest to note that treatment with estrogens and antiestrogens causes the appearance of a doublet in the 66-kDa ER band. This

is presumably due to the presence of phosphorylated ER as described

by Dentónet al. (33). The 80-kDa ER does not show such alterationfollowing any of the above treatments. This suggests that the phos-phorylation state of the 80-kDa ER is unaltered by treatment with

either estrogens or antiestrogens.Expression of a Luciferase Reporter Gene in the MCF-7:2A

Cells. The demonstration of estrogen-independent growth which

could be repressed by antiestrogens was inconsistent with the observation that the PR in the 2A cells was not constitutively expressed. Toexamine the activity of the ERs in the 2A cells a luciferase reporterconstruct was utilized (23). This construct is derived from ptl091uc (34)which contains a luciferase reporter gene downstream from a minimalherpes simplex virus thymidine kinase promoter. Three consensus EREs,derived from the Xenopus vitellogenin A, gene promoter region werecloned upstream from the herpes simplex virus thymidine kinase promoter to yield the plasmid pVlT3-Luc. This ERE was used because it

provides the most sensitive measure of ER binding and transcription of allof the EREs yet examined (35). This would allow maximum sensitivityin determining the activity of the ER from the 2A cells.

pVIT3-Luc was transfected into both the WS8 and 2A cells alongwith a ß-galactosidase reporter construct (pCMVß)which was used

as an internal standard for transfection efficiency (23). As seen inTable 2, the ligand independent expression of the luciferase reporterwas -10-fold higher in the 2A cells than in the WS8 cells. In the WS8

cells E2 was able to increase the luciferase expression 600-fold overthe basal level, and the 2A cells exhibited an approximate 20-foldincrease in luciferase activity. 4-OHT and ICI 164,384 were able to

compete with E2 and inhibit its stimulatory activity in the both theWS8 and 2A cells (data not shown).

In order to characterize the transactivation activity of the 2A cells,estrogens and antiestrogens were added to the culture media 6 hfollowing DNA transfection. Eighteen h following compound additionluciferase activity in the cell extracts was measured. The 2A cellsconsistently demonstrated a greater ability to take up DNA as measured by Southern blot analysis and ß-galactosidase activity (data not

shown). The use of relative galactosidase activity as a correctioneliminated this possible artifact.

The effects of the antiestrogens 4-OHT and ICI 164,384 on the

expression of the luciferase reporter gene is seen in Fig. 6. WS8 cellsshowed an expected response to the antiestrogens, 4-OHT stimulated

expression at low concentrations, and this expression was reduced athigher concentrations. The pure antiestrogen ICI 164,384 had noeffect on luciferase expression (Fig. 6A).

In contrast, the 2A cells showed a higher constitutive activity, andICI 164,384 was able to repress this activity to a level less than 10%of the constitutive activity. 4-OHT caused an increase in luciferase

activity at low concentrations as seen in the WS8 cells. However, athigher concentrations 4-OHT reduced the expression of the luciferase

reporter to less than 40% of the original expression (Fig. 6B).

DISCUSSION

We have isolated a clone of the MCF-7 human breast cancer cellline that exhibits a number of unique characteristics. This MCF-7:2A

Table 2 Constitutive and E2-induced luciferase activity in WS8 and 2A cells

CellsWS8

WS82A2ATreatmentControl0.1 HME2Control

0.1 nM E2Luciferase

activity(X10"6)0.98

±0.03"

589 ±0.649.22

±0.04165 ±0.13Fold

increase60117.9

a Luciferase activity in all groups was corrected for transfection efficiency by dividing

relative luciferase units by ß-galactosidase units. Each value represents the mean of two

determinations ±SEM.

2587

Research. on February 5, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

AN MCF-7 CELL LINE EXPRESSING AN 8()-kDa ESTROGEN RECEPTOR

5.0

4.0

'.> 3.0

2.0

1.0

B

wVi

ï:«J Cflu eses -B

'"

¡IîlJS

0 -12 -11 -10 -9 -8 -7 -6

LOG MOLAR CONCENTRATION

3.0

2.5

2.0

1.5

S 1.0

0.5

•¿�o—o—o

«"-n -11 -10 -9 -8 -7

LOG MOLAR CONCENTRATION

Fig. 6. Anliestrogen effects on luciferase induction in WS8 and 2A cells. Transfectionswere performed as described in "Materials and Methods" with compounds being added 6

h following the transfection. Luciferase and galactosidase assays were performed 18 hlater. A, effects of 4-OHT {•)and ICI 164,384 (O) on luciferase expression in the WS8cells, fi, effects of 4-OHT (•)and ICI 164,384 (O) on luciferase induction in 2A cells.Each point, mean of two determinations ±SEM. RLU, relative luciferase units.

clone was selected simply by growing the MCF-7 cells in estrogen-

free media. The 2A cells display constitutive growth in the absence ofestrogens which can be inhibited by both the pure antiestrogen ICI164,384 and the partial agonist 4-OHT. This growth is not part of aglobal stimulation of all estrogen-responsive genes, as demonstrated

by the lack of PR expression in the absence of estrogen. In these cellsan estrogen-responsive reporter gene is also constitutively stimulated.

This reporter gene as well as PR expression can be further induced byaddition of estrogens, in contrast to growth which is already maximalin estrogen-free media. It has been previously shown that ER activity

is dependent on both the cellular and promoter context in which it acts(36). The promoter(s) responsible for the growth of these cells appearto be maximally activated by the 80-kDa ER alone or in combinationwith the 66-kDa ER in the absence of estrogens. Interaction of the

antiestrogen-bound ER(s) with these critical growth promoters can

inhibit the growth of the cells as well as the expression of the reportergene. The differential regulation of growth, PR, and the luciferaseexpression suggests that growth stimulation and expression of estrogen-responsive genes has become uncoupled in the 2A cells. Thisuncoupling may be an early step in the development of estrogen-independent or ultimately antiestrogen-resistant growth.

Laboratory models of this type of progression have been studied formany years using both in vivo and in vitro systems. Elucidation of thispathway has been most thoroughly investigated in the androgen-

dependent SI 15 mammary carcinoma (10, 37). The loss of hormoneresponsiveness in these cells is thought to occur through multiplesteps. Through a series of changes that are as yet poorly understoodthe S115 cells progress from hormone dependent, to hormone responsive, to reversibly hormone unresponsive, and finally to irreversiblyhormone unresponsive. This final stage is due to methylation ofresponsive genes which renders them insensitive to ER stimulation.All of these changes can occur simply by removing androgens fromthe culture media. Withdrawal of androgens can even be shown tolead to the methylation of transfected genes if the cells are cultured forlong periods of time. While these experiments do not directly addressthe loss of the ER, this progression to autonomous growth follows apathway that mimics many of the salient features of the developmentof estrogen-independent growth.

During the past decade a number of estrogen-independent humanbreast cancer cell lines have been derived from estrogen-sensitivecells. Growth in estrogen-free media for extended periods of time hasbeen sufficient to select for these estrogen-independent cells (12, 13,

15). Many of these cells, including the 2A cells, display the perplexingphenotype of maximum growth in the absence of estrogens, highlevels of active ER, and antiestrogen-inhibited growth. These phenomena seem at odds with our current understanding of estrogen-

stimulated growth pathways. A number of hypotheses have beenproposed to explain this phenotype, including supersensitive receptors, ligand-independent activity of the ER, or adaptation to otherunknown factors present in estrogen-free media (17).

Some investigators have shown that various second messengers canstimulate expression of estrogen-responsive genes in the absence of

estrogens, and this effect can be inhibited by the use of antiestrogens(38, 39). While this suggests that second messengers may be able tostimulate growth of estrogen-responsive cells, it does not directlyaddress the phenomenon of growth in estrogen-free media lacking any

exogenous growth factors or second messenger inducers. This modelof estrogen-independent activity is consistent with the behavior of the

2A cells only if these cells are also constitutively expressing sometype of second messenger or activation pathway. In order to addressthis possibility, we are currently in the process of cloning the cDNAfor the 80-kDa ER. Expression of this protein in other cell lines willallow us to investigate the activity of the ER-related protein.

Early data from transient transfection experiments utilizing thewild-type 66-kDa ER suggested that charcoal-stripped serum is notcompletely devoid of estrogens (40). In these experiments the wild-type 66-kDa ER showed significant activity in a chloramphenicol

acetyltransferase reporter system in the absence of any added estrogens. We feel that this is not the case in our system. Previousexperience has led us to use a procedure which we believe removes allphysiologically relevant steroids from the serum. We have beenunable to detect any estrogen by either immunoassay or a bioassayutilizing growth stimulation of our MCF-7 cells. The fact that as littleas 10~13 M E2 can stimulate the growth of WS8 cells and thatconcentrations of antiestrogens greater than 10~u) Mare necessary to

inhibit growth or luciferase induction in the 2A cells strongly suggeststhat endogenous estrogens are not responsible for the autostimulation

2588

Research. on February 5, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

AN MCF-7 CELL LINE EXPRESSING AN 80-kDa ESTROGEN RECEPTOR

seen in the 2A cells. Additionally, the use of a luciferase reportercontaining three EREs gives us an exquisitely sensitive system. Thefinding that the basal level of luciferase activity detected in the WS8cells could not be reduced by the addition of up to l /J.MICI 164,384suggests that residual estrogens are not responsible for the activityseen in the 2A cells. The presence of a "supersensitive" ER in the 2A

cells can be discounted by the analysis of the binding affinity of theER in either whole-cell uptake or cytosol studies using [3H]estradiol.

No significant difference is observed between the dissociation constants in either of the cell lines, 0.54 nM in the 2A and 0.25 nM in theWS8 cells.

The activity of the ER in the absence of estrogens has beeninvestigated in few studies. Most investigators find that the ER isinactive in the absence of estrogens. However, some data have suggested that the ER is bound to specific EREs in the absence ofestrogen (41 ). Work by Tzukerman et al. (42) suggests that the ER hasconstitutive basal transcriptional activity. Using the wild-type ER they

were able to show basal activity following transient transfections intoCV-1 cells. This activity, while enhanced by E2 could not be repressed by 4-OHT. This mimics the behavior we observed in the WS8

cells which is in contrast to that of the 2A cells.A number of variant ERs have been isolated from breast cancer

samples including a unique ER which constitutively activates ER-

responsive genes (43). This ER contains a deletion of exon 5 and wascloned from an ER-negative, PR-positive tumor (44). These ER andPR determinations were originally made using a ligand-binding assay.Exon 5 is in the steroid-binding domain and loss of this exon would

be expected to either completely eliminate or significantly decreaseestradiol binding in these cells, which explains the original classification of the tumor as ER negative. The loss of exon 5 gives rise toan ER which constitutively activates an ER-responsive ß-galactosid-

ase reporter gene in a yeast test system. However, this activity isunaffected by either estrogens or antiestrogens, in contrast to theactivity in the 2A cells.

The regulation of the 80-kDa ER in the 2A cells appears to bedifferent from the regulation of the 66-kDa ER in either the 2A or

WS8 cells. While both E2 and ICI 164,384 have been shown to causea decrease in the steady-state level of ER, the mechanism responsible

for these effects are quite distinct. E2 appears to act by inhibitingtranscription in early times points and by posttranscriptional inhibitionlater (31, 45). In contrast, ICI 164,384 has been shown to function bydirectly increasing the turnover of the ER protein (28-30). Theobservation that the 80-kDa ER is not down-regulated to the sameextent as the 66-kDa ER by treatment with ICI 164,384 suggests thatthe alteration responsible for the additional size of the 80-kDa ER may

interfere with the regulation of this protein by ICI 164,384. Futherstudies will be necessary to thoroughly assess the regulation of the80-kDa ER.

The 2A cells represent a new model of estrogen-independentgrowth with two unique characteristics. The expression of an 80-kDa

ER and the autostimulation of a luciferase reporter construct has notbeen previously described. While the data presented here do notdefinitively prove the involvement of the 80-kDa ER in the stimula

tion of either growth or luciferase expression, its presence appears tobe necessary for the growth of these cells. The fact that it has beenmaintained for >100 passages in culture and is present in 10 independently derived subclones suggests that it is not a functionless proteinthat has arisen by random mutation. Five of these subclones demonstrate the same autostimulation of the luciferase reporter as seen in the2A cells (data not shown). Given the high mutation frequency inestablished tissue culture cells a "dead" protein would be expected to

be lost over time. The finding that expression of the 80-kDa ER is

maintained suggests that this receptor is an important factor for theestrogen-independent growth of the 2A cells.

ACKNOWLEDGMENTS

We thank Helga Ahrens, Tim Schuh, Gerald Mueller, and Jack Gorski forthe generous gift of the ER antibodies 986 and 987. We also thank AbbottLaboratories for the gift of the ER antibody H222 as well as the ER and PREIA kits. We are grateful to Alan Wakeling for the ICI 164,384 and to ZenecaPharmaceuticals for the 4-OHT. We also thank William Catherino for thegenerous gift of the plasmid pVIT3-Luc and Matthew Bong and James Holsen

for excellent technical assistance.

REFERENCES

1. Green, S.. and Chambón, P. The oestrogen receptor: from perception to mechanism.In: M. G. Parker (ed.). Nuclear Hormone Receptors: Molecular Mechanisms, CellularFunctions, Clinical Abnormalities, pp. 15-37. London: Academic Press, 1991.

2. Martinez, E., Dusserre, Y., Wahli, W., and Mermod, N. Synergistic transcriptionalactivation by CTF/NF-I and the estrogen receptor involves stabilized interactions witha limiting target factor. Mol. Cell. Biol.. //: 2937-2945, 1991.

3. Clarke, R., Dickson, R. B., and Lippman, M. E. The role of steroid hormones andgrowth factors in the control of normal and malignant breast. In: M. G. Parker (ed.),Nuclear Hormone Receptors: Molecular Mechanisms, Cellular Functions, ClinicalAbnormalities, pp. 297-320. London: Academic Press, 1991.

4. Topper, Y. J., and Freeman, C. S. Multiple hormone interactions in the developmentalbiology of the mammary gland. Physiol. Rev., 60: 1049-1106, 1980.

5. Petersen, O. W., Hoyer, P. E.. and van Deurs, B. Frequency and distribution ofestrogen receptor-positive cells in normal, nonlactating human breast tissue. CancerRes., 47: 5748-5751, 1987.

6. Clark, G. M., Osborne, C. K., and McGuire, W. L. Correlations between estrogenreceptor, progesterone receptor and patient characteristics in human breast cancer.J. Clin. Oncol., 2: 1102-1109, 1984.

7. Dickson, R. B., and Lippman, M. E. Control of human breast cancer by estrogens,growth factors and oncogenes. In: M. E. Lippman and R. B. Dickson (eds.). BreastCancer: Cellular and Molecular Biology, pp. 119-166. Boston: Kluwer Academic

Publishers, 1988.8. McGuire, W. L., Chamness, G. C., and Fuqua, S. A. Abnormal estrogen receptor in

clinical breast cancer. J. Steroid Biochem. Mol. Biol., 43: 243-247, 1992.

9. Jiang, S. Y., Wolf, D. M., Yingling, J. M., Chang, C., and Jordan, V. C. An estrogenreceptor positive MCF-7 clone that is resistant to antiestrogens and estradiol. Mol.Cell. Endocrinol., 90: 77-86, 1992.

10. King, R. J. B. Progression from steroid sensitive to insensitive state in breasttumors. In: M. G. Parker (ed.). Growth Regulation by Nuclear Hormone Receptors, pp. 131-146. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory

Press, 1992.11. Berthois, Y., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. Phenol red in

tissue culture is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc. Nati. Acad. Sci. USA, 83: 2496-2500, 1986.

12. Welshons, W. V., and Jordan, V. C. Adaptation of estrogen-dependent MCF-7 cells tolow estrogen (phenol red-free) culture. Eur. J. Cancer Clin. Oncol., 23: 1935-1939, 1987.

13. Katzenellenbogen, B. S., Kendra, K. L., Norman, M. J., and Berthois, Y. Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breastcancer cells grown in the short-term and long-term absence of estrogens. Cancer Res.,47: 4355-4360, 1987.

14. Daly, R. J., King, R. J., and Darbre, P. D. Interaction of growth factors duringprogression towards steroid independence in T-47-D human breast cancer cells.J. Cell. Biochem., 43: 199-211, 1990.

15. Murphy, C. S., Meisner, L. F., Wu, S. Q., and Jordan, V. C. Short- and long-term

estrogen deprivation of T47D human breast cancer cells in culture. Eur. J. CancerClin. Oncol., 25: 1777-1788, 1989.

16. Murphy, C. S., Pink, J. J., and Jordan, V. C. Characterization of a receptor-negative,hormone-nonresponsive clone derived from a T47D human breast cancer cell linekept under estrogen-free conditions. Cancer Res., 50: 7285-7292, 1990.

17. Jordan, V. C. The control of hormone-dependant breast cancer growth- are we talkingabout estrogen alone? Eur. J. Cancer Clin. Oncol., 24: 1245-1248, 1988.

18. LaBarca, C., and Paigen, K. A simple, rapid, and sensitive DNA assay procedure.Anal. Biochem., 102: 344-352, 1980.

19. Jordan, V. C., Wolf, M., Mirecki, D. M., Whitford, D., and Welshons, W. Hormonereceptor assays: clinical usefullness in the management of carcinoma of the breast.CRC Crit. Rev. Clin. Lab. Sci., 26: 97-152, 1988.

20. Fritsch, M., Anderson, L, and Gorski, J. Structural characterization of the trypsinizedestrogen receptor. Biochemistry, 32: 14000-14008, 1993.

21. Schuh, T. J., Ahrens, H., Mogensen, M. D., Gorski, J., and Mueller, G. C. Polyclonalantibodies from rabbits and chickens against the estrogen receptor and relatedpeptides. Use in the affinity isolation of estrogen receptors and the retrieval ofchromatin fragments associating with estrogen receptors. Receptor, 2; 93—107,1992.

22. Catherino, W. H., and Jordan, V. C. Increasing the number of tandem estrogenresponse elements increases the estrogenic activity of a tamoxifen analogue. CancerLett., in press, 1995.

23. MacGrcgor, G. R.. and Caskey, C. T. Construction of plasmids that express E. colibeta-galactosidase in mammalian cells. Nucleic Acids Res., 17: 2365, 1989.

2589

Research. on February 5, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

AN MCF-7 CELL LINE EXPRESSING AN 80-kDa ESTROGEN RECEPTOR

24. Luylen, G. P., Hoogcveen, A. T., and Galjaard, H. A fluorescence staining method forthe demonstration and measurement of lysosomal enzyme activities in single cells.J. Histochem. Cytochem., 33: 965-968, 1985.

25. Jordan, V. C., and Murphy, C. S. Endocrine pharmacology of antiestrogens asantitumor agents. Endocr. Rev., //: 578-610, 1990.

26. Greene, G. L., Fitch, F. W., and Jensen, E. V. Monoclonal antibodies to estrophilin:probes for the study of estrogen receptors. Proc. Nati. Acad. Sci. USA, 77: 157-161,

1980.27. Ahrens, H., Schuh, T. J., Rainish, B. L., Furlow, J. D., Gorski, J., and Mueller, G. C.

Overproduction of full-length and truncated human estrogen receptors in Escherichiacoli. Receptor, 2: 77-92, 1992.

28. Dauvois, S., White, R., and Parker, M. G. The antiestrogen ICI 182780 disruptsestrogen receptor nucleocytoplasmic shuttling. J. Cell Sci., 106: 1377-1388, 1993.

29. Dauvois, S., Danielian, P. S., White, R. ,and Parker, M. G. Antiestrogen ICI 164,384reduces cellular estrogen receptor content by increasing its turnover. Proc. Nati. Acad.Sci. USA, 89: 4037-4041, 1992.

30. Gibson, M. K., Nemmers, L. A., Bcckman, W. J., Davis, V. L., Curtis, S. W., andKorach, K. S. The mechanism of ICI 164,384 antiestrogenicity involves rapid loss ofestrogen receptor in uterine tissue. Endocrinology, 729: 2000-2010, 1991.

31. Martin, M. B., Saceda, M., and Lindsey, R. K. Regulation of estrogen receptorexpression in breast cancer. Adv. Exp. Med. Biol., 330: 143-153, 1993.

32. Saceda, M., Lippman, M. E., Chambón, P., Lindsey, R. L., Ponglikitmongkol, M.,Puente, M., and Martin. M. B. Regulation of the estrogen receptor in MCF-7 cells byestradiol. Mol. Endocrinol., 2: 1157-1162, 1988.

33. Dentón, R. R., Koszewski, N. J., and Notides, A. C. Estrogen receptor phosphoryl-

ation. Hormonal dependence and consequence on specific DNA binding. J. Biol.Chem., 267: 7263-7268, 1992.

34. Nordeen, S. K. Luciferase reporter gene vectors for analysis of promoters andenhancers. Biotechniques, 6: 454-457, 1988.

35. Martinez, E., and Wahli, W. Characterization of hormone response elements. In: M.G. Parker (ed.). Nuclear Hormone Receptors: Molecular Mechanisms, Cellular Functions, Clinical Abnormalities, pp. 125-153. London: Academic Press, 1991.

36. Berry, M., Metzger, D., and Chambón, P. Role of the two activating domains of theoestrogen receptor in the cell-type and promoter-context dependent agonistic activityof the anti-oestrogen 4-hydroxytamoxifen. EMBO J., 9: 2811-2818, 1990.

37. Darbre, P. D., and King, R. J. B. Progression to steroid insensitivity can occurirrespective of the presence of functional steroid receptors. Cell, 51: 521-528, 1987.

38. Aronica, S. M., and Katzenellenbogen, B. S. Stimulation of estrogen receptor-

mediated transcription and alteration in the phosphorylation state of the rat uterineestrogen receptor by estrogen, cyclic adenosine monophosphate, and insulin-likegrowth factor-I. Mol. Endocrinol., 7: 743-752, 1993.

39. Cho, H., and Katzenellenbogen, B. S. Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. Mol. Endocrinol., 7:441-452, 1993.

40. Tora, L., Mullick, A., Metzger, D., Ponglikitmongkol, M., Park, I., and Chambón, P.The cloned human oestrogen receptor contains a mutation which alters its hormonebinding properties. EMBO J., 8: 1981-1986, 1989.

41. Murdoch, F. E., and Gorski, J. The role of ligand in estrogen receptor regulation ofgene expression. Mol. Cell. Endocrinol., 78: C103-C108, 1991.

42. Tzukerman, M., Zhang, X. K., Hermann, T., Wills, K. N., Graupner, G., and Pfahl,M. The human estrogen receptor has transcriptional activator and repressor functionsin the absence of ligand. New Biol., 2: 613-620, 1990.

43. McGuire, W. L., Chamness, G. C., and Fuqua, S. A. W. The importance of normaland abnormal oestrogen receptor in breast cancer. In: M. G. Parker (ed.), GrowthRegulation by Nuclear Hormone Receptors, pp. 131-146. Cold Spring Harbor, NY:

Cold Spring Harbor Laboratory Press, 1992.44. Fuqua, S. A., Fitzgerald, S. D., Chamness, G. C., Tandon, A. K., McDonnell, D. P.,

Nawaz, Z., O'Malley, B. W., and McGuire, W. L. Variant human breast tumor

estrogen receptor with constitutive transcriptional activity. Cancer Res., 5/: 105-109,1991.

45. Saceda, M., Knabbe, C., Dickson, R. B., Lippman, M. E., Bronzert, D., Lindsey,R. K., Gottardis, M. M., and Martin, M. B. Post-transcriptional destabilization ofestrogen receptor mRNA in MCF-7 cells by 12-O-tetradecanoylphorbol-13-acetate.J. Biol. Chem., 266: 17809-17814, 1991.

2590

Research. on February 5, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

1995;55:2583-2590. Cancer Res   John J. Pink, Shun-Yuan Jiang, Michael Fritsch, et al.   ProteinContains a Novel 80-Kilodalton Estrogen Receptor-related An Estrogen-Independent MCF-7 Breast Cancer Cell Line Which

  Updated version

  http://cancerres.aacrjournals.org/content/55/12/2583

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/55/12/2583To request permission to re-use all or part of this article, use this link

Research. on February 5, 2021. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from