(CANCER RESEARCH 49. 6670-6674. December 1. 1989]

Mechanisms of Estrogen Action on the Proliferation of MCF-7 Human Breast

Cancer Cells in an Improved Culture MediumYoshihiko Furuya,1 Norio Kohno, Yoshisada Fujiwara, and Yoichi SaitohDepartment of Surger); flyogo Medical Center for Adults. Akashi. Ityogo 673 [Y. Fur., \. A'./, and Department of Radiation Biophysics [Y. Fuj./ and the FirstDepartment of Surgery/Y. S.J, Kobe L'nirersity School of Me die ine, Kusunoki-cho 7-5-/. Chuo-ku, Kobe 650. Japan

ABSTRACr

The effects of 17/3-estradiol and tamoxifen (TAM) on the proliferationof responsive MCF-7 and unresponsive 11Id -I human breast cancer cells

were studied in a defined culture medium containing insulin (2 ¿ig/ml),transferrin (2 >ig/ml), ethanolamine (2 JIM), and selenite (25 UM). MCF-7 cells grew at a population-doubling rate of 2.0 days in serum-free

medium and at a rate of 1.7 days in the medium containing 1 mg/ml ofthe 55-70% ammonium sulfate fraction of bovine serum or 1% dextran-coated charcoal-treated fetal bovine serum. Increasing concentrations ofthe ammonium sulfate fraction and/or dextran-coated charcoal-treatedfetal bovine serum increasingly inhibited the growth of MCF-7 cells but

did not inhibit III« 4 cell growth, indicating that such serum preparations contain some growth inhibitor specific for estradiol-responsiveMCF-7 cells. A sufficiently high concentration of exogenous estradiol

(100 ¡IM) had the dual action of neutralizing the growth inhibition by the55-70% ammonium sulfate fraction of bovine serum and dextran-treatedcharcoal-treated fetal bovine serum and enhancing directly the MCF-7cell growth maximally 2-fold. Bovine serum albumin fraction V containingglobulin remnants also inhibited growth, but globulin-free bovine serumalbumin did not. Eliminating growth inhibition by the use of globulin-

free bovine scrum albumin enabled us to develop an ideal medium forassaying the direct effects of estradiol and TAM on MCF-7 cells. With

this medium, we clearly identified (a) a direct mitogenic effect of exogenous estradiol on MCF-7 cells which was initiated at 3 pM and maximized

at 0.2 to 10 MM.(/>)an acute lethal effect of l /IM TAM and its preventionby 100 pM estradiol, and (<•)a nearly 50-fold increase in the concentration

of exogenous estradiol (10 HM) required for maximum growth enhancement in the presence of 1 JIMTAM than without TAM (0.2-0.3 HM).

INTRODUCTION

Estrogen plays an important role in the regulation of mammary tumor growth. Established human breast cancer cell linessuch as MCF-7 (1), ZR-75-1 (2), and T47D (3) have been usedto study the mechanisms of action of exogenous estrogens andantiestrogens on cell proliferation in various culture media.Lippman et al. (4, 5) have shown evidence of direct growthstimulation by E22 and inhibition by TAM (1 UM) of MCF-7

cells. Such an estrogen enhancement may arise from the estrogen receptor (ER)-mediated process, since steroid receptorsconstitute a family of regulatory proteins such that the specificbinding of the hormone-receptor complex to enhancer elementsof target genes seems to activate transcription (6, 7). However,there have been interlaboratory controversies about the degreeof estrogen responsiveness in terms of macromolecular synthesis and the growth of MCF-7 cells (4, 5, 8-13). It often has

Received 1/19/88; revised 12/27/88. 7/6/89; accepted 9/6/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.2The abbreviations used are: E2, estradiol; ER. estrogen receptor; E,, estrone;

Ej, estriol; TAM, tamoxifen; DC, dextran-coated charcoal; FBS. fetal bovineserum; DCFBS. dextran-coated charcoal treated fetal bovine serum; BSA, bovineserum albumin; BSA-V, BSA fraction V; sGF, 55-70% ammonium sulfatefraction of bovine serum containing growth factor; RIA, radioimmunoassay;ITES-T medium, serumlcss basal medium supplemented with 2 /xg/ml of insulin.2 «ig/mlof transferrin. 2 /IM ethanolamine. and 25 ITIMselenite; AIT medium.ITES-T medium supplemented with 3 mg/ml of globulin-free BSA; TGF-fi,transforming growth factor /}.

been reported that the growth of MCF-7 cells in serum-free orDC-treated serum-containing media is not stimulated by E2 (8-13), although estrogen-ER complex formation, translocation tothe nucleus, and processing occur sequentially (12, 13). However, another cell line, ZR-75-1, consistently shows direct E2stimulation of growth and TAM inhibition, even in serumlessmedia (14). The complex responses of MCF-7 cells in vitro mayarise from phenotypic changes in E2 responsiveness and bychanges in the serum concentration and different degrees ofsteroid contamination (4, 12, 15). Soto et al. (16-18) recentlyhave indicated that DC-treated human serum and FBS containa specific growth inhibitor for E2-responsive T47D and MCF-7 cells and E2 neutralizes it resulting in indirect promotion ofcell growth (the indirect negative hypothesis). Thus, it seemsthat serum plays a key role in discriminating between thegenuine effects of E2 and TAM on the proliferation of ER-possessing MCF-7 cells and artifact.

Here, we attempted to study specific growth inhibition byserum-born factor and then to develop the ideal serum-freeculture medium for determining the genuine effects of exogenous E2 and/or TAM on MCF-7 cell growth. We will demonstrate a dual effect of E2 in the presence of serum growthinhibitor and a direct mitogenic effect, as well as potent growthinhibition by the antiestrogen TAM in its absence.

MATERIALS AND METHODS

Chemicals. 17tf-Estradiol, BSA-V. globulin-free BSA, and Norit Awere purchased from Sigma Chemical Co. (St. Louis, MO). The antiestrogen TAM was supplied by ICI Pharmaceutical Division (Mac-clesfield, Cheshire. United Kingdom). Dextran T70 was obtained fromPharmacia Fine Chemicals (Uppsala, Sweden). Waymouth's MB752/

1 and FBS were purchased from Gibco (Grand Island, NY). A growthfactor-containing bovine serum fraction (sGF) was prepared from precipitates between 55 and 70% ammonium sulfate. The basal T medium(with or without phenol red) was a 1:1 mixture of Iscove's and Ham's

F-12 media (19). The 100-fold concentrates of insulin, transferrin,ethanolamine, and selenite was a gift of Dr. S. Sasai (Takeda ChemicalInd., Osaka, Japan).

Preparation of DC-treated Fetal Bovine Serum (DCFBS). DC wasprepared as follows (20). Norit A was washed three times with sterilizeddouble-distilled water to remove fine charcoal. Then, 0.5 g of dextranT70 and 5 g of Norit A were suspended in 100 ml of sterilized water,followed by adjustment to pH 7.4 with I M Tris-HCl. To strip endogenous estrogens, 0.1 volume of DC was added to heat-inactivated FBS,followed by slow stirring for 2 h at 20°Cand centrifugation at 1600 x

g for 20 min. After two cycles, we obtained DCFBS. By the sameprocedure more than 99% of [3H]E2 (Amersham. United Kingdom)

added to FBS was removed.Radioimmunoassay of Estrogens. The contents of estrone (I !I- E2,

and unconjugated estriol (E3) in FBS, DCFBS, sGF, and globulin-freeBSA were measured by RIA (Table 1). The concentrations of estrone(E,) and estriol (E3) were measured by an estrone test set and an estrioltest set, respectively (Wien Laboratories, Inc., Succasunna, NJ). Theconcentration of E2 was measured by an E2 assay kit (Daiichi Radioisotope Laboratories Inc., Tokyo, Japan).

Human Breast Cancer Cell Lines and Routine Culture. The MCF-7cell line (Mason Research Institute, Rockville, MD), originally estab-

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Table 1 Concentrations of estrogens in serum preparations and in mediumEstrogen concentrations in the original solutions were measured by RIA, from

which concentrations in culture medium were calculated. Detection limits were85, 55, and 37 prvifor EI, E2, and E3, respectively.

pM

SerumpreparationsFBSOriginal10%

inmediumDCFBSOriginal1%

inmedium10%inmediumsGFOriginal

(35 mgprotein/ml)1mg/ml inmedium3mg/ml inmedium8mg/ml inmediumGlobulin-free

BSAOriginal(50 mgprotein/ml)3

mg/ml inmedium8mg/ml in mediumE,440441701.717280824641681027E223023<55<0.55<5.51203.41027<55<3.3<8.8E3<37<3.7<37<0.37<3.7<37<1.1<3.2<8.5<37<2.2<6.0

lished by Soule et al. (1), was supplied through Dr. M. Nomura(National Kyushu Cancer Centre Hospital, Fukuoka, Japan). The HBC-4 cell line was a gift from Drs. T. Kawaguchi and H. Sugano (CancerInstitute, Tokyo, Japan). The HBC-4 cells originally possessed ER (21),but the ER were lost during long-term culture in our laboratory. Thecells were maintained routinely by growth in Waymouth's MB752/1

medium supplemented with 10% heat-inactivated FBS, 2.5 mg/ml ofsodium bicarbonate, 100 Mg/ml of penicillin, and 100 units/ml Linamycin in a humidified atmosphere of 5% CO2-95% air at 37°C.For

regular transfer, nearly confluent cultures were passaged at a 1:16 splitratio after trypsinization with 0.5% trypsin-0.02% EDTA (Gibco).

Cell Proliferation Media and Addition of Kstradiol and/or Tamoxifen.To assay cell growth in the presence or absence of exogenous E2 and/or TAM, first, we constructed a serumless basal T medium supplemented with 2 Mg/ml of insulin, 2 Mg/ml of transferrin, 2 uM ethanol-amine, and 25 nM selenite, designated ITES-T medium. To assaygrowth in the presence or absence of exogenous E2 and/or TAM, weused various modifications of the ITES-T medium. A serum growthinhibitor-free medium was made by supplementing ITES-T mediumwith 3 mg/ml of globulin-free BSA and was designated AIT medium.

MCF-7 and HBC-4 cells were plated in Corning 24-well plates at aninitial density of 1 x IO4 cells/well containing 1.0 ml of the seedingmedium (ITES-T medium with 1 mg/ml sGF for Fig. 1 and WaymouthMB752/1 medium plus 10% FBS for Figs. 2-6) and incubated for 24h. The medium was removed, and the cells were washed gently 3 timeswith phosphate-buffered saline and incubated in the above assay media.To the assay media a 5-^1 of stock ethanol solution of either E2 orTAM was added by Eppendorf pipe! to yield the desired final concentrations described in the text. The final ethanol concentration wasdecreased to less than 0.2%. which had no effect on cell growth. Duringthe incubation period, medium with or without E2 and/or TAM waschanged every 48 h. The cells grown in the wells were trypsinized ondesired days and single cells were counted by a model ZB-1 CoulterCounter (Coulter Electronics, Inc., Hialeah, FL). Each data pointrepresents the mean ±SE of 4 wells. Phenol red-containing ITES-Tmedium was used, since the growth cune of MCF-7 cells was notsignificantly different between its presence and absence.

RESULTS

Estrogen Content in Serum Preparations. Table 1 lists theconcentrations of estrogens (E,, E2, and E}) in the FBS andserum preparations used, which were measured by RIA. Theconcentrations of E, in the original solutions of FBS, DCFBS,sGF (35 mg protein/ml), and globulin-free BSA (50 mg protein/ml) were between 168 and 440 pM. The E, content (170 p.M)in

DCFBS was 40% ofthat of unstripped FBS (440 pM), indicatinga considerable resistance of E, to the two-cycle DC treatments.However, E2 was removed from FBS by the DC treatmenteffectively, so that its level in DCFBS was below the RIAdetection limit of 55 pM using manual procedures. UnstrippedFBS and sGF (35 mg/ml) contained higher concentrations ofE2, 230 and 120 pM, respectively. E2 was not detected in a 50-mg/ml solution of globulin-free BSA. In all of the above originalserum preparations, the E., concentration was below the RIAdetection limit of 37 pM. The concentrations of estrogens inmedium when the serum preparations were added at the indicated final concentration also were calculated (Table 1). Wefirst selected 1% DCFBS or 1 mg/ml sGF as a basic supplementto the ITES-T medium, since both were expected to give riseto picomolar concentrations of estrogens in the medium (1%DCFBS, 1.7 pM EI and 1 p\i E2; 1 mg/ml sGF, 8 p\i E, and3.4 pM E2). The growth of MCF-7 cells in ITES-T mediumwith 1 mg/ml sGF was exponential with a doubling time of 1.7days, which was shortened by 100 p\i exogenous E2 (1.1 days)(Fig. 1). A similar growth curve was obtained with 1% DCFBS(curve not shown). Without serum preparations the growth ofMCF-7 cells became exponential within 5 days (2.0 days) butit could not be maintained thereafter.

Effects of DCFBS and sGF on MCF-7 Cell Growth (Fig. 2).MCF-7 cell growth was changed as a function of the DCFBSor sGF concentration in ITES-T medium. Although the increase from 2.2 x 10" cells/well on day 0 to 6.0 x IO4 cells/

well on day 4 was low, the addition of 1% DCFBS (Fig. 1Õ)or1 mg/ml sGF (Fig. IB), giving <2.3 piùor 11.4 p\i concentrations of estrogens (E, + E2), respectively (Fig. 2, bottom),allowed the cells to proliferate maximally to a day 4 yield of2.0-2.3 x IO5 cells/well. However, concentrations of DCFBS

higher than 1% or sGF higher than 1 mg/ml inhibited cellgrowth in a dose-dependent manner, despite increasing concentrations of serum-born estrogens. For example, 8 mg/ml ofsGF, giving 64 p\i E, and 27.4 pM E2, inhibited maximalgrowth by 65%. Ten % DCFBS, 17 pM E, and <5.5 pM E2,caused a 45% inhibition of growth. Thus, proliferation stimulation was greatest at 1% DCFBS and 1 mg/ml of sGF, and

0 1 567234

Days

Fig. I. Proliferation of MCF-7 cells in ITES-T medium with or without Img/ml sGF and the effect of exogenous E2. Exponential!) growing cells wereplated at a fixed initial density of I x K)4cells/well in the seeding medium (ITES-T plus I mg/ml sGF) on day —1.Continuous E2 treatment started on day 0. A,ITES-T medium: O. ITES-T medium containing I mg/ml sGF; •.ITES-Tmedium containing I mg/ml sGF and 100 pM E¡.Bars. SEM.

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E2 I0 <0.6 <2.7 <5.5pM 0 3.4

8 ma/rii

20.6 27.4pM

Fig. 2. Modulation of MCF-7 cell growth as a function of the concentrationof DCFBS or sGF. The basic growth experiment was the same as in Fig. I exceptthe plating medium was Waymouth MB 752/1 supplemented with 10% FBS.ITES-T medium was supplemented with increasing amounts of DCFBS (A) orsGF (B). Cell yields were measured on Day 4. Bars, SEM. The calculated finalconcentrations of E, and E2 are shown below the graphs (Table 1).

A1A2A3A4

B1B2B3B4

C2Ç3C4D1D2D3

DCFBS%

E2pM

100100100100

TAM O

iM

Cells/well x 10"'

1 2 3

MCF-7

-Hi,

HBC-4

n»

Fig. 3. Effects of exogenous E¡or TAM on the day 4 yields of MCF-7 andHBC-4 cells in the presence of increasing concentrations of sGF and/or DCFBS.The basic growth experiment was the same as in Fig. 2. Cells were treatedcontinuously with E¡or TAM from day 0 to day 4. MCF-7 cells; Al to A4,growth-inhibitory effects of increasing amounts of sGF or sGF plus DCFBS; Blto B4, growth-stimulating effect of 100 pM E; at increasing sGF or sGF plusDCFBS concentrations; C1 to C4, effects of increasing sGF or sGF plus DCFBSconcentrations on TAM-induced growth inhibition. HBC-4 cells: Dl to D3,effects of sGF or DCFBS on growth; E. effect of 100 pM E2: f. effect of I ¿iMTAM. Bars, SEM.

presence of 1 mg/ml sGF (Al versus Cl), while increasingamounts of sGF increasingly antagonized TAM inhibition ofgrowth, l ßMTAM had no effect on unresponsive HBC-4 cellsin the presence of 3 mg/ml sGF. These results suggest thatincreasing concentrations of sGF and DCFBS either may compete more with the effect of TAM in E2-responsive MCF-7 cellsor contain more TAM-binding component, as suggested previously (18).

Growth Inhibitor-neutralizing and Growth-stimulating Effectsof Exogenous E2. In Fig. 3, Bl to B4 show the interactions ofexogenous E2 and growth-inhibitory concentrations of sGFand/or DCFBS in ITES-T medium.

Relative growth enhancement by the presence of 100 p\i E2was increased when cell growth was inhibited by serum factor(s),since the absolute cell yields in the presence of E2 were similar.Concentrations of sGF lower than 1 mg/ml did not increasethe cell yield (data not shown); therefore growth enhancementby 100 pM E2 could not be explained by the neutralization ofthe growth-inhibitory effect of sGF or DCFBS only. However,it was difficult to distinguish whether or not 1-10 p\i exogenousE2 stimulated cell proliferation, because 1 mg/ml sGF or 1%DCFBS contained 2.7 or 11.4 pM endogenous estrogens (E, +E2), respectively (Table 1).

Development of Growth Inhibitor-free Medium. It was necessary to develop a growth inhibitor-free culture system whichsupports vigorous cellular proliferation and allows the detectionof any direct mitogenic effect of 1-10 pM E2. For that purpose,we tested BSA-V and globulin-free BSA as a supplement toITES-T basal medium. The addition of 1 mg/ml BSA-V produced submaximal growth of 1.7 x IO5cells/well, compared tomaximum yields of 2.0-2.3 x IO5 cells/well with 1% DCFBSand 1 mg/ml sGF (Figs. 2 and 4). However, BSA-V concentrations higher than 1 mg/ml caused progressive growth inhibition.This effect of BSA-V is more or less similar to that of DCFBSor sGF, thus demonstrating that BSA-V also contains a growthinhibitor. BSA-V was shown to have a considerable amount ofglobulin contamination,' suggesting that globulin remnants inBSA-V inhibit the growth of MCF-7 cells. Globulin-free BSA(50 mg/ml) in which E2 was negligible was selected. One to 8mg/ml globulin-free BSA added to ITES-T medium allowedMCF-7 cells to proliferate to their maximum day 4 yield of 2.2x IO5 cells/well (Fig. 4B). Thus, we were able to develop anideal growth inhibitor-free assay medium, AIT medium, whichcontained 3 mg/ml of globulin-free BSA in ITES-T medium.The exponential growth of MCF-7 cells in AIT medium (dou-

higher concentrations resulted in growth inhibition, despite thepresence of estrogens in the range of 10 pM. This indicates thatDCFBS and sGF exert a growth-inhibitory effect on MCF-7cells which can overcome the stimulating effect of 1-10 pM

estrogen.Cell-type Specificity of Growth-inhibitory Effect (Fig. 3). The

above serum preparations both stimulated and inhibited MCF-7 cell growth, while excess DCFBS or sGF inhibited it. Al toA4 produced similar results since 3 mg/ml sGF, 6 mg/ml sGF,and 3 mg/ml sGF plus 10% DCFBS caused 40-60% inhibitioncompared to 1 mg/ml sGF. However, higher concentrations ofDCFBS and/or sGF did not inhibit HBC-4 cell growth butenhanced it by 30-45% (Dl to D3). This difference in thegrowth response indicates that the growth inhibition by DCFBSand sGF is specific for E2-responsive MCF-7 cells.

The antiestrogen TAM is known to inhibit the growth of E2-responsive cells (4, 8, 10, 13, 14, 22). The addition of 1 MMTAM produced a 60% inhibition of MCF-7 cell growth in the

Fig. 4. Effect of BSA-V and globulin-free BSA on MCF-7 cell growth. Theexperiment was the same as in Fig. 2 except that ITES-T medium was supplemented with increasing concentrations of BSA-V (A) or globulin-free BSA (B).Bars, SEM.

' Our unpublished results.

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bling time, 1.6 days) was almost the same as that in ITES-Tmedium containing 1 mg/ml sGF (doubling time, 1.7 days)(Fig. 1).

Analysis of the Direct Effects of E2 and/or JAM in AITMedium (Figs. 5 and 6). The addition of 100 pM exogenous E2in AIT medium enhanced MCF-7 cell growth, shortening thedoubling time from 1.6 days to 1.1 days (Fig. 5). This demonstrated the direct mitogenicity of E2. The minimum concentration of exogenous E2 for initiating and the maximum concentration for stimulating growth were 3 and 200 pM, respectively(Fig. 6). E2, 200 pM to 10 HM, produced a maximum 2-foldenhancement in the day 4 cell yield. In the absence of serumgrowth inhibitor, exogenous E2 stimulated the mitogenic activity of MCF-7 cells directly through the ER-mediated process.

Addition of l MMTAM to the AIT medium caused cell deathwithin 2 days. This effect may have arisen mainly from TAMER complex formation (22). The lethal effect of TAM in growthinhibitor-free AIT medium was acute compared to its mildinhibition of growth in sGF-supplemented ITES-T medium(Fig. 3). The dramatic lethal effect of TAM was antagonized

10e

10

-1 0 1 3Days

5 6

Fig. 5. Growth of MCF-7 cells in AIT medium and the effect of 100 pw E2or 1 >iMTAM. O. AIT medium; •.AIT medium containing 100 pM E2; A, AITmedium containing 1 >iMTAM. See Fig. 2 legend for details of the experiment.Bars. SEM.

4 -

3 -

2 -

1 pM 10 pM

E

100 pM

Concentration

1 nM 10 nM

Fig. 6. The direct stimulation by exogenous E2 in AIT medium of MCF-7 cellgrowth as a function of its concentration and the antagonistic effect of E2 onTAM-inhibition of growth. Cell yields were measured on day 4 after continuoustreatment with 1 pM-10 nM E2 in the absence (O) or presence of (•)1 ^M TAM.Bars. SEM. For the experimental procedure, see Fig. 2 legend.

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by 100 pM exogenous E2, and maximum growth stimulationwas obtained with 10 nM E2 in the presence of 1 MMTAM.Thus, a concentration of exogenous E2 approximately 2 ordersof magnitude higher can compete with the effect of TAM.

DISCUSSION

AIT medium is a serum-free and growth inhibitor-free medium for the assay of the direct mitogenic activity of exogenousE2 and for determining the minimum concentration for initiating and the concentration for maximizing growth stimulation.Direct growth stimulation by E2 in serum-free medium has beenobserved with ZR-75-1 human breast cancer cells (14), butrarely with MCF-7 cells (9). Further, contradictory results ofMCF-7 cell growth modulation by exogenous E2 in low concentrations and the usual 10% serum concentrations also have beenreported, because DC-treated and untreated sera contain different amounts of endogenous estrogens, serum-born growth factors, and inhibitors, and the cells may carry over the "memoryeffect" (23). In fact, the concentrations of FBS and sGF usually

used introduce biologically significant picomolar amounts ofEI and E2 into the culture medium (Table 1). DC treatmentremoves E2 effectively, while it leaves a considerable percentageof the original amount of E,. Although E, may be convertedpartly to E2 in the cells (24), the biological significance ofpicomolar amounts of E, is not clear (5, 11). Furthermore, inMCF-7 cells, serum-free or low-serum culture conditions havebeen shown to produce an E2-unresponsive state, which can beconverted to a responsive state by raising the DC-treated serumconcentration to 15% (12). This suggests that serum growthfactors may influence the MCF-7 growth response to E2. Withserum, therefore, the cellular proliferative response to E2 maybe modified by a variety of endocrine and autocrine growthfactors, so that it is difficult to determine the mitogenicity ofE2 itself. In addition, we have shown clearly that sGF, DCFBS,and BSA-V inhibit E2-responsive MCF-7 cell growth (Figs. 2and 4/4), as observed previously with human serum and FBSfor MCF-7 and T47D cells (16-18). This effect was not seen inunresponsive HBC-4 cells. Different degrees of growth inhibition by BSA-V and globulin-free BSA strongly suggest that aserum factor responsible for growth inhibition exists in theglobulin fraction, which also includes sex hormone-bindingglobulin (data not shown). Soto et al. (16-18) have shown thatE2 stimulation of MCF-7 and T47D cellular proliferation occurs as a result of neutralization of serum growth inhibitoralone. However, 100 p\i exogenous E2 not only neutralized theeffect of serum growth inhibitor present in higher amounts ofsGF and DCFBS but also doubled MCF-7 cell growth. Serumgrowth inhibitor also counteracted growth inhibition by TAM,since (a) TAM-induced proliferative inhibition was mild in thepresence of sGF and DCFBS, in spite of the lethal effect ingrowth inhibitor-free AIT medium and (b) TAM inhibition wasless with increasing concentrations of sGF and DCFBS. Thus,this factor may interact directly or indirectly with E2 and TAM.These interactions should be studied further along with theisolation and characterization of the actual serum growth inhibitor. It is still unknown whether this growth inhibitor is thesame as the sex hormone-binding globulin or the M, 53,000serum inhibitor specific for MCF-7 cells (25).

AIT assay medium can detect direct mitogenic activity ofexogenous E2. AIT medium contains 2 Mg/ml of insulin and 2Mg/ml of transferrin as the major growth factors. Insulin in AITmedium permits 3.1 population doublings in 5 days, which iscomparable to or better than the 3 doublings in 8 days obtained

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in a similar serum-free medium containing 0.2 jug/ml of insulinand 200 ng/m\ of BSA (26). Berthois et al. (27) have reportedthat phenol red acts as a weak estrogen for cultured humanbreast cancer cells. However, it does not affect the proliferationof MCF-7 cells when the initial cell density is high (23).3 AIT

medium enabled us to determine the minimum concentrationof exogenous E2 to initiate mitogenic stimulation (3 pivi) andthe E2 dose needed for doubling the maximum stimulation(0.2-1 OHM).

Low and high doses of JAM paradoxically exert estrogenicand antiestrogenic effects, respectively, on MCF-7 cells (8, 14).Growth inhibition of MCF-7 cells by TAM decreases withincreasing concentrations of sGF or DCFBS (10, 13, 14, 18,22), while growth inhibitor-free AIT medium demonstrated alethal effect of TAM, which was rescued by a 50-fold higherconcentration of E2. The mechanism of this effect is yet unknown. However, Knabbe et al. (28) recently presented evidencethat the growth-inhibitory doses of TAM and its derivativesgreatly increase the autocrine secretion of TGF-ßin MCF-7cells and equimolar E2 can reverse it. The view that TAMinhibition of growth results from TGF-ßinduction is stillproblematic, because the addition of exogenous TGF-ß(<20Mg/ml) does not inhibit MCF-7 cell growth (29) and the additionof serum factor in this study reversed growth inhibition. Specificbinding of the hormone-receptor complex to enhancer sequences of target genes is known to regulate their transcrip-tional activity (6, 7). Therefore AIT medium should proveextremely useful in future investigations of what target genenetwork in MCF-7 cells is activated for growth enhancementby the specific binding of activated E2-ER complex to enhancerelements.

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1989;49:6670-6674. Cancer Res   Yoshihiko Furuya, Norio Kohno, Yoshisada Fujiwara, et al.   Human Breast Cancer Cells in an Improved Culture MediumMechanisms of Estrogen Action on the Proliferation of MCF-7

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