1998;58:871-876. Cancer Res   Carmen Pacilio, Domenico Germano, Raffaele Addeo, et al.   Growth by AntiestrogensInhibition of Hormone-responsive Human Breast Cancer Cell Constitutive Overexpression of Cyclin D1 Does Not Prevent

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[CANCER RESEARCH 58. 871-876. March 1. 1998]

Advances in Brief

Constitutive Overexpression of Cyclin Dl Does Not Prevent Inhibition of Hormone-responsive Human Breast Cancer Cell Growth by Antiestrogens1

Carmen Pacilio, Domenico Germano, Raffaele Addeo, Lucia Altucci, Valeria Belsito Petrizzi, Massimo Cancemi,Luigi Cicatiello, Salvatore Salzano, FrançoisLallemand, Rob J. A. M. Michalides, Francesco Bresciani, andAlessandro Weisz2

Istituti! ili Patologia Generale e Oncologia, Facoltà di Medicina e Chinirgia, Seconda Università di Napoli, I-80I3S Naples, Italy ¡C.P., D. C.. R. A., L. A., V. B. P., M. C„L C„F. B„A. W.I; Centro di Endocrinologia e Oncologia Sperimentale del Consiglio Nazionale delle Ricerche and Dipartimento di Biologia e PatologìaCellulare e Molecolare 'L.Cali/ano', Università 'Federico II', Naples, Italy ¡S.S.]; and Department of Tumor Biolog\, the Netherlands Cancer Institute. Amsterdam, the Netherlands ¡F.L, R. J. A. M. M.]

Abstract

Cyclin Dl is a target for positive regulation by estrogens in growth-

responsive cells, in which it mediates their mitogenic effects. Amplificationand overexpression of the cyclin DI gene (CCNDl) might thus represent agenetic lesion inducing hormone-independent growth of transformed cells.

Indeed, cyclin Dl overexpression has been found in up to 50% of primarybreast cancers, and in about one-third of these cases, this is linked to

amplification of the Ilql3 chromosomal region, which also includes theCCNDl gene. These tumors are predominantly estrogen receptor-positive,and for this reason, these patients are often selected for adjuvant anties-

trogen therapy. No information is available, however, as to whether cyclinDl overexpression due to gene amplification might interfere with andreduce antiestrogen efficacy. This was investigated here by taking advantage of an experimental model that reproduces cyclin Dl overexpressionresulting from increased CCNDl gene dosage in hormone-responsivehuman breast cancer cells. For this, MCF-7 cells stably transfected with atef-inducible cyclin Dl expression vector were tested for their in vitroresponse to steroidal (ICI 182,780) and nonsteroidal (/r«//.v-4-liydr<ixyl;i-

moxifen) antiestrogens under condition of low (endogenous only) or high(exogenous) cyclin Dl levels. Results show that although cyclin Dl over-

expression seems to interfere with the early cell cycle effects of antiestrogens, it does not prevent their cytostatic actions, so that growth of cyclin-overexpressing MCF-7 cells is still efficiently inhibited in vitro by these

drugs.

Introduction

The quest for genetic lesions that might help explain the biologicalproperties of breast cancer cells and improve the available means forthe clinical management of these diseases led to the identification ofmultiple chromosomal aberrations and gene mutations in mammarycancers. Among these lesions, one of the best characterized and morefrequently found in primary breast cancers is the amplification of the1Iql3 chromosomal region that, in the vast majority of the cases, alsoinvolves the CCNDl gene, encoding cyclin Dl (1). This gene, whoseproduct carries out a central role in cell cycle regulation (2), is ofparticular interest, because it is under direct transcriptional regulationby estrogens and progestins in breast cancer cells in culture (3-5),

where its constitutive expression induces significant acceleration ofcell cycle kinetics and triggers autonomous growth in vitro (6-7).

Received 9/26/97; accepted 1/13/98.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.

' Supported by Associazione Italiana per la Ricerca sul Cancro. Consiglio Nazionaledelle Ricerche 97.04417.CT14, Ministero per l'Università e la Ricerca Scientifica e

Tecnologica (fondo 40% e 60%) of Italy, and Grant 97-143 from the Netherlands Cancer

Foundation.2 To whom requests for reprints should he addressed, at Istituto di Patologia Generale

e Oncologia, Facoltàdi Medicina e Chinirgia, Seconda Università di Napoli. Larghetto S.Aniello a Caponapoli, 2, 1-80138 Naples. Italy. Phone: 39-81-566-5671; Fax: 39-81-566-5695; E-mail: a.weisz@area.ba.cnr.it.

Cyclin Dl overexpression is found in up to 50% of primary breastcancers (8-20), in which it shows a strong correlation with thehystological grade of the tumor, going up to 83-87% of the cases inmore malignant lesions (21-23). The vast majority of tumors with

1Iql3 amplification also show high cyclin Dl levels, suggesting thatCCNDl gene amplification might result in constitutive and/or dis-

regulated expression of this cyclin in tumor cells, and are most oftenBR+ (11-13, 17-18, 24-27). Because the presence of steroid recep

tors is considered to indicate that tumor cells are still responsive toestrogens, patients with receptor-positive neoplasms are often subject

to adjuvant therapy with antiestrogens. However, it is conceivable thatoverexpression of this estrogen-regulated gene, as a consequence of

its amplification, might be a condition that renders the growth oftumor cells autonomous from the estrogenic stimulus and, as a consequence, less responsive to the growth-inhibitory effects of anties

trogens. This could thus result in failure of the antiestrogen therapy,which is indeed clinically observed in about one-third of the cases in

ER+ tumors (28).Using estrogen-responsive human breast cancer MCF-7 cells trans

fected with a /f/-inducible human cyclin Dl cDNA expression vector

(6), we tested whether overexpression of this cyclin, due to artificialgene amplification, interferes with the cell cycle and cytostatic effectsof the nonsteroidal antiestrogen Tarn, a mixed agonist-antagonist, or

of the pure antiestrogen ICI 182,780. Results show that althoughMCF-7 cells expressing elevated levels of this cyclin are less readily

arrested in G, by both drugs during the first 72 h of treatment, whencompared to control cells, their growth rate is still efficiently inhibitedby antiestrogens. In all cases, ICI was found to be far more potent thanTarn in inducing cell cycle arrest in G, and in inhibiting cell proliferation in vitro.

These data demonstrate for the first time that ER+ breast tumorcells overexpressing cyclin Dl as a consequence of a genetic mutationthat renders the CCNDl gene estrogen independent, such as, forexample, gene amplification, are likely to still be responsive in vivo tothe cytostatic and cytotoxic effects of antiestrogens.

Materials and Methods

Materials. ICI 182,780 and Tarn were a kind gift of Dr. A. De Pasqua(Zeneca S.p.A., Milan, Italy) and D. Salin-Drouin (Laboratoires Besins Isco-vesco, Paris, France), respectively. Tet (chlorotetracycline-HCI) was from Life

Technologies, Inc. (Milan. Italy). All other reagents were of analytical gradeand were provided by major suppliers.

Cell Culture Conditions. MCF-7/cl3 cells, stably transfected with a tet-responsive human cyclin Dl expression vector (6). were propagated as mono-layer cultures in DMEM with phenol red. supplemented with i.-glutamine (2

3 The abbreviations used are: ER, estrogen receptor; ICI. ICI 182,780; Tarn, trans-4-hydroxylamoxifen; têt.letracycline; ERE, estrogen response element; ß-gal,ß-galacto-

sidase.

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CYCLIN Dl OVEREXPRESSION AND ANTIESTROGEN ACTION

mM), penicillin (100 units/ml), streptomycin ( 100 jig/ml), amphotericin B (250ng/ml), and 10% FCS. Where indicated (+tet), 10 /j.g/ml let were also presentin the medium. Before the use described here, stable transfectants werereselected in the presence of 400 (¿g/mlneomycin (G418) and 100 ng/mlhygromycin for 15 days, and surviving clones were pooled for further use.Estrogen-free medium (CTS) included phenol red-free DMEM and dextran-

coated charcoal-treated FCS.

Cell Cycle Analysis. Flow cytometry was performed with a FACS Analyzer (Becton Dickinson, Inc.) according to standard protocols suggested bythe manufacturer, as described previously (29). Analysis of the data wasperformed with a CellFIT cell cycle analysis program (Version 2.0.2; Becton

Dickinson, Inc.).Preparation of Cell Extracts and Immunoblotting. Whole cell protein

extracts were prepared and analyzed (20 /ng of proteins) by Western blottingas described previously (3). p36DI was immunodetected in the blots with a

mouse monoclonal antibody raised against full-length human cyclin Dl (sc-

6281; Santa Cruz Biotechnology, Inc.).Growth Rate Analysis. Cells were seeded at a density of about 5-6 x IO4

cells/35-mm-diameter dish in medium with or without tet. After 12 h, the

medium was changed to include the indicated compounds; every 48 h, themedium was replaced with fresh medium. Cells were collected at the indicatedtimes by trypsinization, stained with trypan blue, and counted with a hemo-

cytometer. Each experiment was performed in triplicate, and the count wasrepeated twice for each sample. Data represent the average of results obtainedin two to three triplicate experiments (±SE).

Transient Transfections. Triplicate plates were transfected by liposome-mediated (A'-[l-(2,3-dioleoyloxyl)propyl]-MW.A'-trimethylammoniummethylsulfate) gene transfer with 5 fig of ERE-luc (from P. Chambón, Strasbourg,France) and 1 fig of pSV-nlsLacZ (30) DNAs according to the manufacturer's

instructions (Boehringer Mannheim). Six h after the addition of the liposome-

DNA mixture, cells were washed twice with PBS and further incubated for40 h in complete medium with or without 10 fig/ml tet and antiestrogens asindicated. Cells were washed and harvested by scraping in PBS before lysis in0.25 MTris-HCl by three cycles of rapid freeze-thawing. After clearing the cell

lysates with a brief centrifugation, the protein concentration was determinedwith a colorimetrie assay (Bio-Rad); luciferase and ß-galactivities were

assayed in 100 /ig of protein extract as described previously (3, 30). Luciferaseactivity data are reported as Relative Light Units (RLU)/U ß-gal/100 /xg of

proteins.

Results and Discussion

Effects of Antiestrogens on the Transcriptional Activity of theER in Cyclin Dl-overexpressing MCF-7/cl3 Cells. Expression ofthe tet-responsive hybrid gene encoding human cyclin Dl present inMCF-7/cl3 cells is activated on removal of tet from the culturemedium, resulting in an overexpression of p36DI that reaches values

up to 5-fold higher than those of the endogenous protein (6). Over-

expression of the exogenous gene (Fig. IA) in the absence of tet(—tet),already shown to be independent from the presence of serum

mitogens (6), is also maintained in the absence of estrogen, becausep36m levels were still relatively elevated, when compared to control

cells, in cells cultivated in estrogen-depleted medium (CTS) for 3

days. When cells were exposed to 50 nM ICI 182,780 ( + ICI) or 500nM Tam (+Tam) for up to 72 h, endogenous p36DI expression rapidly

dropped to undetectable levels (Fig. IA, +tet), whereas expression ofthe exogenous protein (—¿�tet) remained elevated. It is worth noting that

the additional protein band marked with asterisks in Fig. IA wasdetectable only with the monoclonal antibody used in this study, butnot with other anti-Dl monoclonal or polyclonal antibodies (data not

shown), and thus represents a nonspecific protein species unrelated toP36D1.

The ER complex is a transcription enhancer factor that binds toci'i-acting DNA elements (EREs) located in estrogen target genes and

thereby modulates the activity of the corresponding gene promoters(31). Antiestrogens inhibit the transcriptional effects of ER by interfering at multiple steps in the cascade that lead to receptor activation

+tet

ex-Di

O A 12h 24h 48h 72h 12h 24h 48h 72tiO . | | -

«ICI +Tam

-tet

121- 24h 46« 72h 24n 4Sh 721.

B

2503

tg.60PX

Wi

40èi120

0+tet-rI

1llT1'l-tet1iT\-t--¡O 5 50 500 5 50 500

ICI[nM] Tam[nM]

O 5 50 500 5 50 500ICI [nM] Tam [nM]

Fig. 1. Effects of antiestrogens on p36DI levels and ER transcriptional activity inMCF-7/cB cells grown in the presence or absence of tet. A. Western blot analysis of p36D'

levels in cells treated with 50 nM ICI 182,780 (+/CY) or 500 nM Tam ( + Tam) for theindicated times in the presence (+tel) or absence ( —¿�let) of 10 ng/ml tet in the culturemedium. CTS, estrogen-depleted medium. The position of p36DI in the autoradiograms is

indicated by arrows; asterisks mark the position of a nonspecific band. B, transcription ofa transiently transfected ER-responsive reporter gene (ERE-luc) in cells treated with theindicated concentrations of antiestrogens in the presence ( + tei) or absence (—tet)of tet in

the culture medium. Data were corrected for changes in transfection efficiency based onthe expression levels of a cotransfected internal control (pSV-nlsLacZ:units /3-gal) and

represent the average of results obtained from two independent experiments performed intriplicate; bars indicate the range of the results obtained.

by its cognate hormones, including dimerization, DNA binding, andinteraction with transcriptional coregulators (32). To determinewhether cyclin Dl overexpression interferes with antiestrogen inhibition of ER-mediated transcription under the conditions selected forthis study, MCF-7/cB cells overexpressing (-tet) or not (+tet) ex

ogenous cyclin Dl were transiently transfected with an ER-responsive

reporter gene comprising a perfectly palyndromic ERE sequencecloned upstream of a rabbit ß-globinpromoter-firefly luciferase reporter gene (ERE-luc). Luciferase expression in the cells was thenmeasured in the absence (0) or after a 40-h exposure to various

concentrations of antiestrogens, all within or above the expectedaffinity of ER for each of these molecules. Results, reported in Fig.In, show that although cyclin Dl overexpression induces a significantincrease of reporter gene expression per se (compare 0 in +tet and—¿�tetconditions), probably reflecting activation of the ER complex by

this cyclin as reported previously (33), both antiestrogens can significantly reduce transcription of this ER-responsive hybrid gene under

all conditions tested, with ICI being more effective than Tam. Because

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CYCLIN Dl OVEREXPRESSION AND ANTIKSTROGEN ACTION

no significant changes in ER levels could be detected during these fewhours of treatment (data not shown), this result indicates that anties-

trogens are capable of inhibiting the transcriptional activity of EReven in the presence of high levels of cyclin DI. A similar result wasobtained by Neuman et al. (34) that showed how stimulation of thetranscriptional activity of the ER by ectopie expression of cyclin Dlis inhibited by both ICI 182,780 and Tam, in apparent contradictionwith results reported earlier by Zwijsen et al. (33). It is worth mentioning, however, that in the presence of Dl overexpression (-letcells), ERE-luc activity in Tarn-treated cells was still relatively high,

so that luciferase activity under these conditions was about 50% ofthat detectable in the absence of antiestrogens in cells with normalcyclin Dl levels (0 +tet). This is comparable to what was observed byZwijsen et al. (33) in T47D cells, with the exception that in thepresence of very high intracellular levels of p36DI, such as those

achieved in that study after transient transfection of a Dl cDNAexpression vector, ER was able to activate transcription of ERE-

containing reporter genes even in the complete absence of hormone,and this was Tarn-resistant.

Influence of Cyclin Dl Overexpression on the Cell Cycle Effectsof Antiestrogens in MCF-7/cl3 Cells. Estrogens show dramatic effects on cell cycle kinetics of MCF-7 and other hormone growth-

responsive cells, in which they induce recruitment of quiescent cellsin cycle and accelerate cell cycle kinetics in cycling cells, mostly byshortening G, transition time and by facilitating cell entry into S phase(35). These effects of estrogens are all inhibited by estrogen antagonists, in particular, pure antiestrogens such as ICI 182,780.4 A corre

lation between the activity of ER on gene transcription and its abilityto stimulate cell proliferation has been postulated and is sustained byseveral lines of evidence (3, 29, 35), although at present, no univoca!demonstration of a direct link between these two actions of ER isavailable. Because a substantial cytostatic action is one of the expected prerequisites for the efficacy of estrogen antagonists againsthormone-dependent breast cancers, lack of cell cycle effects of an

tiestrogens in cells overexpressing cyclin Dl would indicate thatgenetic lesions inducing constitutive expression of this protein shouldbe considered as a potential mechanism for resistance of ER+ breastcancer cells to adjuvant antiestrogen therapies. This question wasaddressed using MCF-7/cl3 cells as follows: cells were maintained in

complete medium with or without tet to reproduce conditions of basalor high expression of p36D1 in an identical cellular background; and

they were then exposed to increasing concentrations of antiestrogensfor various times (up to 72 h) before analysis of cell cycle parametersby flow cytometry. As shown in Fig. 2A, cyclin Dl overexpressionwas accompanied by reduction of G() + G, (61.1% in +tet versus47.9% in -tet) and G, + M-phase (15.5 versus 11.6%) cells in the

cultures in favor of S-phase cells that increased from 23.4 (in +tet) to40.5% (in -tet). Because it has been shown that cyclin Dl overex

pression does not affect the length of S phase and G2-M phases inMCF-7/cl3 cells (6), these data indicate that the increase in S-phase

cells observed here results from an increase of the number of cyclingcells in the cultures, due to a reduction of G0-arrested cells and/or a

shortening of the G, transition time. The effects of antiestrogens oncell cycle parameters differed substantially between the two compounds tested. In +tet cultures, ICI 182,780 very rapidly inducedaccumulation of G0 + G, cells even at the lowest concentration tested(5 nM), so that 87.4-90% of cells accumulated in these cell cyclecompartments after 24-48 h. On the contrary, in —¿�tet,this antiestro

gen was much less efficient in inducing the redistribution of cells incycle (only 70.3-75.8% of the cells were in G0 + G, after 24-48 h

treatment with up to 500 nM ICI). Cells overexpressing cyclin Dl thusseem to be less readily responsive to the cell cycle effects of the ICIcompound, even when the data are analyzed after normali/.ation forthe initial differences in cell cycle phase distribution due to theelevated intracellular levels of cyclin (Fig. 2B, +ICf). It is worthnoting, however, that because G,, + G, cells increased significantly inthe cultures between 48 and 72 h of treatment with ICI under bothculture conditions (+tet and —¿�tet),—¿�tetcells are indeed not fully

resistant to the cell cycle effects of this antiestrogen. Because ICI182,780 induces cell cycle arrest in G,, or early G, in MCF-7 cells (5,

36), and it has been shown that cyclin Dl overexpression inhibits cellcycle exit in MCF-7/cl3 cells (6), slower G,, + G, cell accumulationin the -tet cultures probably indicates that in the presence of cyclin

Dl, overexpression ICI-induced cell cycle exit occurs less efficiently.

Indeed, Wilcken et al. (37) reported that expression of cyclin Dl froman inducible transfected vector reverses the growth-inhibitory effectsof steroidal and nonsteroidal antiestrogens on hormone-responsive

human breast cancer cells in culture when analyzed during the first24-48 h of cyclin overexpression.

Contrary to that observed for ICI 182,780, Tam is much lesseffective in interfering with cell cycle kinetics in MCF-7/cl3 cells

(Fig. 3A). In +tet, only 79.3% of the cells after 48 h or 83.5% of thecells after 72 h can be found in G0 + G,, in accordance with the lowerefficiency of this molecule as a growth inhibitor for MCF-7 cells,which is probably related to its mixed agonist-antagonist action inbreast cancer cells (32). This was even less marked in —¿�tetcells

(67-67.7% of GO + G, cells after 48 h and 79.6-80.3% of G() + G,

cells after 72 h). Little differences could be observed in cell cyclephase redistribution efficiency between control (+tet) and Dl-over-expressing (—tet)cells exposed to Tarn (+Tam in Fig. 2ß).with the

latter being slightly more responsive to cell cycle inhibition by thisantiestrogen. This was observed in multiple experiments and at different time points and antiestrogen concentrations and might be related to the mechanism of action of nonsteroidal antiestrogens, whichin estrogen-responsive human breast cancer cells also show pleiotro-pic inhibitory effects on ER-independent pathways (32), which thuscould also include cyclin D l-dependent regulatory cascades.

Growth of MCF-7/C13 Cells Overexpressing Cyclin Dl Is Inhibited by Antiestrogens. MCF-7 cells respond to the mitogenic stim

ulus exerted by estrogens both in vitro and in vivo. This is also truefor clone 3 cells, because their growth rate is significantly reducedin steroid-depleted medium (Fig. 3A, CTS), and this can be reversed by stimulation with 17ß-estradiol.s Interestingly, cyclin Dl

overexpression reduced this hormone requirement for sustainedcell growth, because in CTS medium, cells seems to grow moreefficiently in the absence of tet than in the presence of tet (Fig. 3A).Because the CCNDI gene is a target for transcriptional regulationby estrogens in breast cancer cells (3), and cyclin Dl has beenshown to mediate the mitogenic action of estrogens in these cells(3, 5, 36), growth of MCF-7/cl3 cells in estrogen-depleted medium

could be related to a condition of hormone independence due toconstitutive expression of the product of a gene located downstream of ER in the estrogen-activated mitogenic cascade. How

ever, this cyclin can induce also ER activation in the absence ofhormone (33-34), and for this reason, increased proliferation of—¿�tetcells in CTS medium could be mediated by constitutive ERactivity. In any case, this result indicates that D l-overexpressing

4 L. Cicaliello. R. Addeo. L. Altucci. V. Belsilo Petrizzi, D. Germano. C. Pacilio. S.

Salzano. F. Bresciani, and A. Weisz. The antiestrogen ICI 182,780 inhibits proliferationof human breast cancer cells by interfering with multiple, sequential estrogen-regulated

processes required for cell cycle progression, submitted for publication.

5 R. J. A. M. Michalides, L. Cicaliello. C. Pacilio, R. Addeo. L. Altucci. V. Belsito

Petrizzi, S. Salzano. F. Bresciani, and A. Weisz.. Simvastatin inhibits growth of cyclinD l-overexpressing human breast cancer cells, manuscript in preparation.

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CYCLIN Dl OVEREXPRESSION AND ANTIESTROGEN ACTION

+tet -tet

100%-

50%

40% -

20% -

10% -

Fig. 2. Influence of cyclin Dl overexpression on short-term anties-trogen effects on cell cycle kinetics of MCF-7/cl3 cells. A, exponentially

growing cells were exposed to the indicated concentrations of ICI182,780 (ICI) or Tarn for up to 72 h; at each time point, cells fromduplicate plates were collected and analyzed by flow cytometry. Thedata represent the average of results obtained in two independent experiments; bars indicate the range of the results obtained. B, directcomparison of antiestrogens effects on S-phase cell concentration incultures maintained in the presence (•and •¿�control) or absence (Oand D, D1 overexpression) of tet. The average of results obtained inthree experiments performed in duplicate is reported ±SE (bars).

B

ESEE5SSSSSSSSSSEE2cccccc cccccc ccccccinooinoo inooinoo inooinoo

in in in in

ICI Tarn ICI Tarn ICI Tarn

EEEEEE SESEEScccccc cccccc cccccc

ICI Tarn ICI Tarn ICI Tarn

24h 4Sh 72h O 24h

time of antiestrogen treatment

48h 72h

100% «h+ICI

+Tam

24h 48h

time of antiestrogen treatment

72h

MCF-7 cells, in analogy to their loss of serum requirement (6), lostpart of their hormone requirement for growth.

Both antiestrogens inhibited hormone-stimulated and -independentgrowth of MCF-7/C13cells either in the presence or in the absence oftet (Fig. 3-4),with Tarn (500 HM)being less effective than ICI 182,780

(50 HM).However, as for its effects on cell cycle kinetics (Fig. 2), thenonsteroidal antiestrogen seemed to be relatively more efficient ininhibiting the growth of Dl-overexpressing cells than that of controlcells (see Fig. 3ß,+Tanï),whereas the steroidal compound wasequally effective under both conditions tested (Fig. 3B, +ICI). Be-

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CYCLIN Dl OVl.klM'RI SSION AND ANTIESTROGEN ACTION

Fig. 3. Effects of cyclin Dl overexpression on the response ofMCF-7/C13 cells to the cytostatic action of antiestrogens. A. triplicatecultures of MCF-7/cl3 cells were cultured in complete (left) or estrogen-depleted (CTS, right) medium with <+(?(, control) or without( —¿�tel. Dl overexpression) 10 /¿g/mllet and in the absence (diiimimtts)

or presence of 50 nM ICI I82.780 (circles) or 500 nM Tarn (squares).At the indicated times, cells were collected and counted as reported in"Materials and Methods." B, direct comparison of antiestrogen effects

on proliferation of cells maintained in the presence (•and •¿�control)or absence (O and 0. Dl overexpression) of tet. For each time point,data are expressed as number of cells in antiestrogen-treated cultures/number of cells in the corresponding (-Met or —¿�tet,respectively)

control cultures X 100. The averages of results obtained in multiple(two to four) independent experiments performed in triplicate arereported ±SE (bars).

24 6 8 10 0 :.' 4 6 8

B100%

fc 50%

'S

¿I 100%Ee

â„¢¿�50%

+ ICI

+Tam

CTS +ICI

CTS +Tam

10 O 2

time (days)

cause tamoxifen and its derivatives can act on multiple moleculartargets in breast cancer cells, it is possible that these pleiotropic effectsmight also include direct interference with cyclin D l actions in thecell.

The growth-inhibitory effects of antiestrogens toward Dl-overex-pressing MCF-7 cells could be due to a combination of one of thefollowing actions: (a) an interference with cyclin D l-independent

intracellular pathways, because it has been shown that ICI interfereswith the cell cycle effects of estrogens in breast cancer cells by actingat multiple regulatory points throughout G, and in S phase;4 (b) a

reduction in ER levels in the cell after prolonged exposure to antiestrogens, as reported in other cases (32), assuming, however, that thepresence of ER is required for full growth of the cells under theseconditions; and (c) cytotoxic effects that have been documented forthese drugs toward human breast cancer cells (32) and could also beclearly detected in clone 3 cells, in both +tet and in -tet conditions,

after prolonged (>9 days) exposure to antiestrogens and were moreevident in estrogen-depleted medium (data not shown). A molecularanalysis of the mechanisms that lead to growth inhibition of Dl-

overexpressing ER+ breast cancer cells by antiestrogens, in particular, identification of cytostatic and/or apoptotic pathways activated bythese compounds that might interfere with cyclin actions, as well as abetter knowledge of the ER-dependent mitogenic pathways that they

inhibit, will be necessary to discriminate between these differentpossibilities.

In conclusion, the results of this study, showing that the growth ofER-positive hormone growth-responsive human breast cancer cells

overexpressing cyclin Dl is still inhibited by antiestrogens in vitro,indicate the possibility that ER+ primary breast neoplasms with the1Iql3 amplification coupled to p36m overexpression are likely to be

responsive to antiestrogens in vivo. This hypothesis now needs to beverified clinically, where it will also be important to correlate theclinical response to antiestrogens to the degree of cyclin Dl overexpression in tumors to further confirm the usefulness of these compounds for treatment of a large spectrum of ER+ neoplasms.

Acknowledgments

We thank A. De Pasqua (Zeneca S.p.A., Milan, Italy) for ICI 182,780, D.Salin-Drouin (Laboratoires Besins Iscovesco, Paris, France) for Tam, and P.Chambón (I.G.B.M.C.. Strasbourg. France) for the ERE-luc reporter plasmid.

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CYCLIN Dl OVIKI \l'Ki:SSION AND ANTlhSTMXJEN ACTION

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