Long-Term Hydroxytamoxifen Treatment of an MCF-7-derived ...cancerres.aacrjournals.org/content/canres/60/15/4130.full.pdf · Eric Badia,2 Marie-Jose`phe Duchesne, Abdelhabib Semlali,

[CANCER RESEARCH 60, 4130–4138, August 1, 2000]

Long-Term Hydroxytamoxifen Treatment of an MCF-7-derived Breast Cancer CellLine Irreversibly Inhibits the Expression of Estrogenic Genes throughChromatin Remodeling1

Eric Badia,2 Marie-Josephe Duchesne, Abdelhabib Semlali, Maryse Fuentes, Claire Giamarchi, He´lene Richard-Foy,Jean-Claude Nicolas, and Michel PonsUnite 439, Institut National de la Sante et de la Recherche Medicale, 34090 Montpellier [E. B., M. J. D., A. S., M. F., J-C. N., M. P.], and Laboratoire de Biologie MoleculaireEucaryote, Centre National de la Recherche Scientifique, 31062 Toulouse Cedex [C. G., H. R-F.], France

ABSTRACT

Antiestrogen resistance is frequently observed in patients after long-term treatment with tamoxifen, a nonsteroidal antiestrogen widely usedfor endocrine therapy of breast cancer.In vitro studies in resistant cellsshowed that the expression of natural estrogen-responsive genes is fre-quently altered. Using MVLN cells, an MCF-7-derived cell model, wepreviously demonstrated that 4-hydroxytamoxifen (OHT) treatment irre-versibly inactivated an estrogen-regulated chimeric luciferase response bya direct effect of the drug and not through a cell selection process (E.Badia et al., Cancer Res.,54: 5860–5866, 1994). In the present study, wepresent tamoxifen-resistant but still estrogen-dependent clones isolatedafter long-term treatment of MVLN cells with OHT and show that pro-gesterone receptor (PR) expression was irreversibly decreased in some ofthese clones, whereas the PRA:PRB ratio of residual PR remained un-changed. The irreversible inactivation of both chimeric luciferase geneand PR gene expression was associated with the disappearance of DNaseI-hypersensitive sites. In the case of the chimeric gene, at least one of thesesites was close to the estrogen responsive element. Genomic sequencinganalysis of a clone with very low PR content did not reveal any methyl-ation on CpG dinucleotides or any mutation in the PR gene promoterregion. In all of the resistant clones tested and independently of their PRcontent, estrogen receptor expression was only lowered by half and re-mained functional, whereaspS2 expression was not modified. We alsoobserved that the residual luciferase activity level (1–2%) of the MVLNclones, the luciferase expression of which had been irreversibly inacti-vated, was raised 4-fold by trichostatin A treatment. We conclude thatlong-term OHT treatment may modify the chromatin structure and thuscould contribute to differentially silencing natural target genes.

INTRODUCTION

Long-term tamoxifen therapy is widely used in the management ofhormone-responsive breast cancer and is also being evaluated as apreventive treatment for breast cancer in healthy women at risk forbreast cancer (1). Although curative tamoxifen treatment is efficient inmany patients, most of them experience further tumor progressionafter several years of treatment,i.e., so-called acquired tamoxifenresistance. The exact molecular mechanisms that account for acquiredtamoxifen resistance remain unclear, but there is an emerging body ofevidence that multiple mechanisms could be involved, concerning, formost of them, modifications intrans-elements at different pointsalong the ER3 pathway: (a) alterations in the pharmacology andmetabolism of the AE (2); (b) ER mutations (3); and (c) alterations in

cross-talk between signal transduction pathways (4, 5) or in thebalance of nuclear receptor coeffectors (6, 7). Although each of thesemechanisms has been invoked to explain the tamoxifen resistanceprocess, none of them could account for the triggering of resistanceacquisition.

Acquired resistance to tamoxifen may be due either to a simpleselection process involving cells from an already heterogeneous pop-ulation or to a two-stage process first implying cell alteration by thedrug (here termed “direct effect”) followed by the selection of the newphenotype, provided that cell growth is no longer inhibited by tamox-ifen. In previous works (8, 9) on an MCF-7-derived cell line,i.e.,MVLN cells that express the luciferase gene under the control of anERE (10), we showed that OHT was able to induce rapid and irre-versible silencing of the luciferase gene. The subsequent appearanceof cell heterogeneity (cells expressing or not expressing luciferase)was clearly incompatible with a selection process, because the time ofhalf inactivation was as short as 7 days. We hypothesize that irrevers-ibly affecting natural estrogen-responsive gene(s) by an AE directeffect could be a primordial event that triggers the resistance process;it then would lead to altered expression of other genes involved in cellproliferation.In vitro studies on tamoxifen- or OHT-resistant variantcell lines showed that expressions of some natural estrogen-respon-sive genes are frequently altered in these cells. The process leading tosuch alterations has not yet been elucidated (11–13).

The aim of this paper is to show that long-term OHT treatment caninduce irreversible inhibition of the expression of estrogen-responsivegenes along with having a direct effect on chromatin structure. Byanalyzing several OHT-resistant clones issued from OHT-treatedMVLN cells, we show that the AE irreversibly inactivated the ex-pression of the natural estrogen-controlled PR gene. This inactivationrequired a longer treatment period than for the luciferase gene. Con-versely, thepS2gene, another estrogen-regulated gene, was not inac-tivated by such treatment. Inactivations of luciferase and PR geneswere associated with chromatin remodeling as revealed by the disap-pearance of DHSSs, one of which was localized close to the ERE inthe chimeric gene.

Our results strongly suggest that (a) OHT can induce direct andstable silencing of gene expression; and (b) this inactivation process isassociated with stable modification of the chromatin structure.Whether a similar mechanism might be involved in the silencing ofgenes leading to resistance acquisition of our resistant cells and, moregenerally, of tumors primarily responding to AE therapy is an openquestion.

MATERIALS AND METHODS

Materials

Materials for cell culture came from Life Technologies (Cergy-Pontoise,France). Luciferin was synthesized by G. Auzou (INSERM, Montpellier,France) according to the method of L. J. Bowie (14). OHT was a gift from Dr.A. E. Wakeling (Imperial Chemical Industries, Macclesfield, England). Estra-diol, sodium bisulfite, hydroquinone, spermine, spermidine, proteinase K, and

Received 12/13/99; accepted 5/30/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Institut National de la Sante et de la RechercheMedicale and Association pour la Recherche sur le Cancer Grant 5002.

2 To whom requests for reprints should be addressed, at Unite 439, Institut National dela Sante et de la Recherche Medicale, 70 rue de Navacelles, 34090 Montpellier, France.

3 The abbreviations used are: ER, estrogen receptor; AE, antiestrogen; ERE, estrogenresponse element; OHT, 4-hydroxytamoxifen Z; PR, progesterone receptor; DHSS,DNase I-hypersensitive site; tk, thymidine kinase; NL, nonluminous; H, homogenization;C, centrifugation cushion; W, washing.

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NP40 were purchased from Sigma Chimie (Saint Quentin Fallavier, France).DNase I (reference 6330) was from Worthington Biochemicals (Freehold, NJ).Restriction endonucleases were from New England Biolabs (Ozyme, Montignyle Bretonneux, France). The T-vector ligation kit, Wizard DNA Clean-UpSystem, and competent JM 109 cells used for cloning PCR products were fromPromega (Charbonnieres, France). The sequencing kit, random labeling kits,nylon Hybond-N1membrane, and the ECL system were from AmershamPharmacia Biotech (Les Ulis, France). Detection of luciferase activity oncell-free extracts was performed with an LKB-Wallac (Sundyberg, Sweden)1251 luminometer. A photon-counting camera (Argus 100) from HamamatsuPhotonics (Hamamatsu, Japan) was used to detect luciferase activity in intactcells and to analyze Western blots. The monoclonal anti-PR antibody (MA1–410) was from Affinity-BioReagents (Interchim, Montlucon, France). A FujixBAS1000 PhosphorImager was used to analyze Northern blots.

Cell Line, Culture Conditions, and Luciferase Assay

The stable MVLN transfectant expressing the luciferase reporter gene underestrogen control has been described elsewhere (10). These MCF-7-derivedcells express the firefly luciferase geneLuc under control of the 59flankingregion of theXenopusvitellogenin A2 geneVit (15), inserted in front of theherpes simplex virus promoter fortk. The stability of this expression waschecked over 70 passages in DMEM with phenol red, supplemented with 5%FCS (FCS medium). For long-term treatments, cells were cultured in DMEMwithout phenol red supplemented with 3% of a steroid-free, dextran-coatedcharcoal-treated FCS (DCC medium). Medium was replaced every other day.Luciferase activity was determined as described by Badiaet al. (8).

Isolation of Clones from OHT-treated MVLN Cells

After treatment with 10-7 M OHT in DCC medium for various times,MVLN cells were dispersed at a density of 1 cell/cm2 to obtain separate clonesin tissue culture flasks. They were grown for;1 month in FCS medium untilthe clones were visible without magnification. Their NL or luminous pheno-type was determined using a photon-counting camera (after supplementing themedium with 0.3 mM sterile luciferin). Clones were individually harvested andgrown for 1 extra month in FCS medium. Clones NL 6 and NL 10 are NLclones obtained after a 12-day OHT treatment, whereas clones CL 3.1–CL 3.35and clones CL 6.1–CL 6.35 represent two NL clone series obtained after 3 and6 months of treatment, respectively. Clone CL 32inf was obtained by culturingclone CL 6.32 in the presence of 1027 M OHT for 12 extra months.

Cell Growth Assay

Each MVLN population or clone assayed was cultured for 5 days in DCCmedium. Cells were harvested, and 23 104 cells per well were seeded in24-well tissue culture cluster plates in the same medium. One day later, themedium was replaced by fresh medium containing estradiol (0.1 nM), OHT(100 nM), ICI 164.384 (100 nM), or vehicle alone (0.1% ethanol). These mediawere renewed every 2 days. After an 8-day growth period with the differenteffectors, triplicate wells were assayed for DNA content using 49,6-diamidino-2-phenylindole (16).

PR Binding Assay and PR Isoform Analysis

Each MVLN population or clone was cultured in DCC medium for 5 daysand then in DCC medium containing either 1 nM estradiol or 100 nM OHT in0.1% ethanol. After a 4-day incubation, cells were harvested in PBS-EDTA,washed twice with PBS, pelleted, and frozen at220°C until assay. The pelletswere thawed in 1 ml of buffer (10 mM Tris-HCl, 1.5 mM EDTA, and 1 mM

DTT, pH 7.4), sonicated for 5 s at 0°C, and then centrifuged at 180,0003 gfor 40 min. Cytosol aliquots (200ml) were incubated at 0°C for 4 h in thepresence of either 10 nM [3H]promegestone or both 10 nM [3H]promegestoneand 2mM [1H]promegestone. Binding activity was measured by liquid scin-tillation counting after dextran-coated charcoal treatment. Specific stimulated(with estradiol) or unstimulated (with OHT) binding was expressed as thenumber of femtomoles of [3H]promegestone bound per milligram of cytosolproteins, measured by the Bradford technique. Relative expression of PRA andPRB isoforms was examined by Western blotting on a 15–25-ml cytosolaliquot (i.e.,20 mg of protein), using the ECL system after incubation with

monoclonal mouse anti-PR. The chemiluminescence level was quantifiedusing a photon-counting camera (Argus 100; Hamamatsu).

ER Binding Assay

Each tested clone was cultured in DCC medium for 5 days. Cells were thenharvested in PBS-EDTA, washed twice with PBS, pelleted, and frozen at220°C until assay. The pellets were treated as described above for PR binding;cytosol aliquots (200ml) were incubated at 0°C for 2 h with either 5 nM

[3H]estradiol or both 5 nM [3H]estradiol and 1mM [1H]estradiol.

Northern Blot Analysis of pS2Expression

MVLN cells that had grown for 3, 6, and 9 months in DCC medium aloneor in DCC medium containing 1027 M OHT and clones isolated from MVLNcells treated with OHT for 1, 3, and 6 months were cultured in 25-cm2 dishesand grown for 5 days in DCC medium and then in DCC medium supplementedwith 1029 M estradiol or 1027 M OHT for 4 days. Total RNA was isolatedaccording to the method of Chomczynski and Sacchi (17) using RNAzol Breagent. Tenmg of total RNA were electrophoretically separated on a 1%agarose denaturing gel and transferred to a nylon membrane. The membranewas hybridized overnight with32P-labeled pS2 and 36B4 probes at 42°C in50% formamide, as described previously (8). After stringency washes, filterswere first exposed to the PhosphorImager screen to evaluatepS2 versus 36B4expression and autoradiographed.

DHSS Analysis

The methodology was adapted from that of Richard-Foyet al. (18) asfollows.

Nucleus Isolation. Nuclei were isolated either from untreated MVLN cellsor from clones CL 6.32, CL 6.32inf, and NL 6. Cells contained in 10 T150flasks were brought near confluence; five flasks were then incubated for 2 h inthe presence of 1029 M estradiol, and the other five were incubated in thepresence of 1027 M OHT before nucleus isolation. Cells were then carefullystripped, suspended in 40 ml of ice-cold PBS, which had been supplementedeither with 1029 M estradiol or with 1027 M OHT (buffer H, buffer C, andbuffer W were similarly supplemented), and centrifuged for 5 min at 1000 rpm.The supernatant was discarded, and the pellet was resuspended in 7 ml ofice-cold buffer H (15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mMspermidine, 1 mM EDTA, 0.1 mM EGTA, 0.2% NP40, 5% saccharose, and 10mM Tris-HCl, pH 7.4). The cell suspension was homogenized with 15 strokesof a cold Dounce homogenizer. The state of the resulting nuclei was checkedunder a microscope. The resulting slurry was centrifuged over 4 ml of ice-coldbuffer C (buffer H containing 10% saccharose) for 20 min at 3000 rpm. Thesupernatant was discarded, and the pellet was washed twice with 13 ml ofice-cold buffer W (15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mMspermidine, and 10 mM Tris-HCl, pH 7.4). After a last centrifugation (3 min at3000 rpm) the pellet was finally resuspended in 2 ml of buffer W.

DNase I Digestion.Aliquots containing 500ml of nuclei suspension (cor-responding to 250–300mg of DNA) were digested for 10 min at 37°C withvarious amounts of DNase I (ranging from 1.6 to 8 units/500ml) in thepresence of 0.5 mM CaCl2 and 1 mM MgCl2. DNase I digestion was stoppedby overnight incubation with 200ml of proteinase K buffer (25 mM EDTA, 2%SDS, and 200mg/ml proteinase K). Proteinase K was removed by phenol-chloroform extraction and ethanol precipitation. The DNA was resuspended in10 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA.

Southern Blot Experiments. Approximately 40–60mg of DNase I-digested DNA were cleaved byStuI in 500ml of the corresponding digestionbuffer (usually 3 units/mg DNA for 2 h; complete digestion was checked byadding 0.5mg of a known cleavable plasmid to a 20-ml aliquot of the reactionsolution). Digested DNA was then ethanol precipitated in the presence ofglycogen and 0.3 M NaOAc and resuspended in the sample buffer. Fortymgof DNA were separated electrophoretically on a 0.8% agarose gel and trans-ferred to a nylon membrane. The membrane was hybridized overnight with theappropriate32P-labeled probes. Positions of the molecular weight markerbands were measured before transfer onto the nylon membrane.

Chemical Modification of DNA with Sodium Bisulfite

The sodium bisulfite reaction was performed as described by Frommeret al.(19) with slight modifications partially reported by Raiziset al. (20). Briefly,

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sodium bisulfite (1.9 g) was mixed with 2.5 ml of water. Then 0.7 ml of 2 MNaOH and 0.5 ml of 1 M hydroquinone were added. Template DNA (5mg) in30–50ml of water was denatured by a 10-min incubation at room temperatureafter the addition of 0.1 volume of 2M NaOH. DNA was resuspended in 1 mlof bisulfite solution and then successively incubated for 4 h at 50°C, heated at95°C for 5 min, cooled to 50°C for 1 h, and then cooled to 4°C. DNA waspurified with the Wizard DNA Clean-Up System and dissolved in 100ml ofwater. After neutralization (43ml of 1 N NaOH) and precipitation, the pelletwas resuspended in 50ml of water.

PCRs, Cloning, and Sequencing of the Products

Direct PCR Products. The three pairs of oligonucleotides, D3 and D2, D8and D6, and D4 and D5 (see Fig. 4B), generated 994-, 612-, and 617-bp-longfragments, respectively. These fragments covered the entire promoter A andpart of promoter B as defined by Kastneret al. (21). The PCR fragments werepurified on an agarose gel, cloned in the T-vector, and sequenced.

PCR Products from Bisulfite-treated DNA. PCR was performed on 2.5ml of the reacted DNA with oligonucleotides that matched the new se-quence. The upper (coding) strand of thePR gene promoter was amplifiedwith the two pairs of oligonucleotides, F1 and F2, and F3 and F4 describedin Fig. 4B. The sequences of oligonucleotides F1 and F3 were deducedfrom the natural upper strand by changing C to T, and those of F2 and F4were deduced from the natural lower strand by changing G to A. Thenatural sequences corresponding to these oligonucleotides are devoid ofmethylatable CpGs; therefore, they fit perfectly with the gene sequencemodified after the bisulfite reaction. The PCR products were purified onagarose gel, cloned in the T-vector, and sequenced.

RESULTS

OHT-treated MVLN Cells Acquired Growth Independent ofThis AE

Growth Rate Evaluated by Passage Frequency.The MVLN cellline, a stably transfected MCF-7 cell line, is clonal. As for the MCF-7cell line, MVLN cell proliferation is stimulated by estradiol andinhibited by AEs (22). At the beginning of continuous 1027

M OHTtreatment, the MVLN cell doubling time was as long as 120 h (Fig. 1).However, after 6 weeks of culture, the MVLN doubling time short-ened, reaching a steady state of 40 h after 10 weeks. The experimentalresults quite closely fit a theoretical model whereby an MVLN cellpopulation grown in the presence of OHT (doubling time, 120 h)acquires AE resistance due to the modification of three cells permillion per day, the doubling time of which drops to 40 h. Althoughspeculative, this model suggests that as much as 95% of the cellsgrown for 3 and 6 months in the presence of OHT could derive from10 to 20 resistant cells modified in the first week of treatment.Selection then became the driving force of cell proliferation.

Growth Rate Evaluated by Measuring DNA Content. At theend of 3-, 6-, and 9-month OHT treatment times, 35 clones wereisolated from the culture. All of these clones grew in the presence ofOHT, which notably stimulated proliferation. Table 1 shows the

proliferation of MVLN cells and eight clones isolated from 6-monthtreated cells (CL 6.1, CL 6.5, CL 6.7, CL 6.8, CL 6.20, CL 6.27, CL6.28, and CL 6.32) after 8 days incubation of cells with variouseffectors. In DCC medium, it was 2–3-fold higher than at seeding(data not shown). The DNA content of 1027

M OHT- and 1029M

estradiol-treated resistant clones was;2- and 4-fold higher, respec-tively, than that of cells grown in DCC, whereas it was 2-fold lowerin 1027

M ICI 164384-treated cells. Conversely, in parental MVLNcells, OHT as well as ICI 164384 inhibited cell proliferation by half.These results were confirmed by experiments in which cell growthwas evaluated by the [3H]thymidine incorporation method (results notshown).

Estrogen-stimulated PR Expression of MVLN Cells IsIrreversibly Decreased as a Function of OHT Treatment Time

In a previous paper (9), it was shown that the MVLN cell expres-sion of firefly luciferase was irreversibly inactivated by a short OHTtreatment (,3 weeks), and we showed that this inactivation resultedfrom a 50-fold inactivation of individual cells. We describe here theirreversible inactivation of PR expression, a natural estrogenic re-sponse. The estrogen-stimulated PR content of MVLN cell culturesdecreased with OHT treatment time during the first 6 months oftreatment (Fig. 2A). Because this PR content was only a mean value,we also measured the PR content of individual clones issued from 3-and 6-month OHT-treated MVLN cells, respectively, (Fig. 2,B andC)and observed that PR content (mean6 SD) was 606 26 and25 6 23%, respectively (100% is the MVLN cell PR content,i.e.,

Fig. 1. Growth of MVLN cells during long-term OHT treatment. MVLN cells wereseeded in a T25 flask and grown in DCC medium with 1027 M OHT for 25 weeks. As soonas cells reached confluence, one-tenth of them were plated in a new T25 flask. Cellnumber was determined on the basis of the dilution at each passage. The logarithm of thiscell number was plotted against the number of treatment weeks (filled symbols). Celldoubling time was then graphically determined. A theoretical model was constructed,which simulates variations in growth rates of a population of cells in which threecells/106/day raise their growth rate instantaneously from 120- to 40-h doubling time(open symbols).

Table 1 Growth rate and PR and ER content of MVLN cells and clones isolated from 6-month OHT-treated cells

Among the clones issued from 6-month OHT-treated MVLN cells, eight were selected for their different PR contents,i.e., very low for CL 6.7 and CL 6.32, low for CL 6.1 andCL 6.20, average for CL 6.5 and CL 6.8, and high (similar to PR content in untreated cells) for CL 6.27 and CL 6.28. Results (mean6 SD) are given as femtomoles per milligramof mg proteins. The numbers of experiments (each performed in duplicate) are given in parentheses for PR content; the number was three for ER content. The proliferation was studiedin various culture media (DCC or in the presence of estradiol (E2), OHT, and ICI 164384). Growth rates are presented as a percentage of DNA content (mean6 SD for threedeterminations) of cells grown in the presence of estradiol.

Cell clones MVLN CL 6.1 CL 6.5 CL 6.7 CL 6.8 CL 6.20 CL 6.27 CL 6.28 CL 6.32

DNA contentDCC 21.66 1.7 17.16 0.8 29.46 1.5 24.76 1.3 37.46 1.5 42.96 1.7 246 2.1 41.36 1.7 30.36 1.4E2 100 100 100 100 100 100 100 100 100OHT 13.46 2.1 65.86 8.4 636 0.7 54.86 3.3 57.66 8.6 79.96 3.1 55.56 0.4 70.46 2 74.86 4.1ICI 6.6 6 1.2 6.96 0.1 8.86 1.2 13.46 1.3 8.26 0.5 11.76 0.5 7.86 1 15.16 0.4 7.56 3.8

PR content 6486 110 (5) 1456 55 (4) 2946 20 (3) 176 3 (3) 2626 43 (2) 1116 30 (3) 7646 161 (3) 5746 104 (3) 236 6 (2)ER content 2926 15 1066 12 1426 8 1056 25 1686 4 1756 32 1416 26 1996 3 1186 31

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6486 110 fmol/mg of proteins). This indicated that (a) the mean PRcontent decreased as the treatment time increased; and (b) the PRcontents of individual clones varied markedly as suggested in eachcase by the high SD value (see above: 26 and 23%), reflecting theprogressive appearance of heterogeneity and the randomness of thisevent. After 3 or 6 months of OHT treatment, clones were grown for2 months in FCS medium (an estrogenic culture condition) beforeharvesting; hence the fact that the PR content was still low indicatedthat the loss of PR expression was irreversible. Northern blot exper-iments suggested that this inactivation occurred at the transcriptionlevel (results not shown). As a control, the estrogen-stimulated PRcontent of 12 clones issued from the parental MVLN cells was108 6 20%.

Approximately 100 clones issued from MVLN cells treated for 3, 6,or 9 months with OHT were analyzed by Western blotting for theirrespective PRA and PRB contents. On seven clones obtained after 3months of treatment and expressing various PR levels, Fig. 2D showsthat both PR isoforms disappeared proportionally regardless of the PRcontent (wells were loaded with an equal cytosol protein amount).PRA:PRB ratios remained stable around the 3–4 value obtained withMVLN cells. This suggests that both promoters were equally modifiedby the OHT treatment. Although the band intensities in many clonesissued from cells treated for 6 and 9 months (data not shown) werefaint, no differences in the PRA:PRB ratios were noted in the clonesanalyzed. We also observed (Fig. 2D) that MVLN cells expressedsignificantly less PR than T47D cells, used as a control in theseexperiments with a smaller PRA:PRB ratio.

Estradiol Receptor Expression Is Decreased by Half in OHT-treated MVLN Cells

The eight clones (CL 6.1, CL 6.5, CL 6.7, CL 6.8, CL 6.20, CL6.27, CL 6.28, and CL 6.32) expressing various amounts of PR wereanalyzed for their ER content (Table 1). They exhibited nearly 50% ofthe MVLN cell ER content (292 fmol/mg of protein). Very low PRlevels in CL 6.7 (2.6%) and CL 6.32 (3.5%) or high PR levels in CL6.27 (117%) and CL 6.28 (88.5%) were thus associated with compa-rable ER levels (36, 40, 48, and 68%, respectively), suggesting thatthe PR expression level is not directly related to the ER level.

pS2Expression Is Not Modified in OHT-treated MVLN Cells

Fig. 3Ashows the results of the analysis of thepS2transcriptionlevel of MVLN cell cultures treated for various times with OHT.Estradiol-stimulated and basal (in the presence of OHT)pS2mRNA levels, compared with those of the36B4mRNA standard,were not significantly modified. The same results were obtainedwith clones issued from these treated MVLN cells (Fig. 3B). It isnoteworthy thatpS2expression was constant regardless of the PRexpression levels (Fig. 2). For example, CL 3.23 and CL 6.27 onone hand and CL 3.24 and CL 6.7 on the other expressed a normalPR level and a very low PR level, respectively. As discussedbelow, this latter result shows that the different natural estrogen-controlled responses may have different degrees of sensitivity tolong-term OHT treatment. In addition, when administered withestradiol, OHT was still able to counteractpS2 induction in cellspretreated for 6 months with OHT, indicating that its antiestrogeniceffect was still efficient on this estrogen-controlled response ofresistant cells (results not shown). These results also show that theER and the basal transcription machinery functions were not af-fected by prolonged exposure to OHT and were still able to elicitsome of the estrogenic responses.

Irreversible Inhibition of Vit-tk-Luc Gene Expression in MVLNCells Is Associated with Complete Disappearance of DHSSs

In a previous study (9), Southern blotting experiments suggestedthat three copies of the Vit-tk-Luc plasmid placed in a head-to-tailconfiguration were integrated in the MVLN cells (Fig. 4A). It wasshown that theXhoI-XhoI restriction fragment, which contains thecomplete gene unit composed of the luciferase gene, the regulatoryand promoter elements, and the polyadenylation site, was entire ineach copy, and the copies were not rearranged by OHT treatment. Inthe present study, digestion withStuI, which does not cleave pVit-tk-Luc, gave only one band with a length (21 kbp; Fig. 4A) compatiblewith that of an insert containing the three plasmid copies flanked by

Fig. 2. PR content of MVLN and OHT-treated resistant cells and PRA:PRB ratioanalysis. PR content of MVLN cells and individual clones was determined after 4-daystimulation with 1 nM estradiol. PR content of untreated MVLN cells was taken as 100%.A, MVLN cells were grown in DCC medium containing 1027 M OHT for 1, 3, 6, and 9months.B andC, clones1–35were isolated as described in “Materials and Methods” fromMVLN cells grown in DCC medium in the presence of 1027 M OHT for 3 months (B) and6 months (C).D, each clone was grown for 4 days with 1 nM estradiol, after which 20mgof cell extracts were submitted to 12% SDS-PAGE, transblotted, and processed forWestern analysis.

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short genomic sequences. In thisStuI-StuI fragment, DHSSs wereanalyzed in cells with luciferase that was either expressed or irrevers-ibly inactivated. In MVLN cells, at least four DHSSs were visible, asindicated by thearrows in Fig. 5, all of them being inducible by

estradiol stimulation. In clones NL 6, CL 6.32, and CL 32inf, theluciferase expression of which was irreversibly inactivated, DHSSs2–4 completely disappeared, whereas site 1 was dramatically de-creased.

Localization of DHSSs in Integrated pVit-tk-Luc Copies

To localize DHSSs in theStuI-StuI fragment of the pVit-tk-Lucintegrated copies, DNA from estradiol-stimulated MVLN cells wasrestricted byApaLI andBanII (Fig. 4A) and gave a fragment encom-passing the ERE near its 59end. The pattern obtained after Southernblotting and hybridization with probe P1, a 665-bpBanI-BanII frag-ment strictly included in theApaLI-BanII fragment, gave, as expected,a single 1950-bp-long band for DNA undigested by DNase I (Fig. 6,line A). After digestion with DNase I, theApaLI-BanII DNA fragmentwas partially cleaved, and a single extra band of;1400 bp wasgenerated (Fig. 6,line B). Because the ERE is located 1450 bpupstream of theBanII restriction site, the cartographic analysis re-vealed that at least one of the four above-mentioned DHSSs waslocated close the ERE of one pVit-tk-Luc copy.

Irreversible Inhibition of PR Gene Expression in MVLN CellsIs Associated with Partial Disappearance of a DHSS

Hypersensitive sites were studied in the 59region of thePRgene inuntreated MVLN cells and in CL 6.32, a 6-month OHT-treated clonewith very low PR expression. At least two DHSSs could be distin-guished on the autoradiograph: site 1 located around position2570 bpand site 2 (faint) around position1530 (Fig. 7). Site 3 around position11130 of thePR gene promoter also present in undigested DNAcould not be considered a DHSS. Although these sites were notobviously inducible by 2-h estradiol stimulation (results not shown),

Fig. 3. Induction ofpS2 in MVLN cells and clones isolated from cells treated withOHT. A, MVLN cells that had grown for 3, 6, and 9 months in DCC medium alone or inDCC medium containing 1027 M OHT were treated as described in “Materials andMethods” with either 1029 M estradiol (E2) or 1027 M OHT. Total RNA was then isolatedand processed for Northern blot analysis using a pS2 probe as described in “Materials andMethods.”B, various clones were isolated from MVLN cells treated with 1027 M OHT for1 month (CL 1.4, CL 1.15, andCL 1.7), 3 months (CL 3.23andCL 3.24), and 6 months(CL 6.7andCL 6.27). Each of these clones was analyzed as inA.

Fig. 4. Scheme ofPRpromoter and cartography of Vit-tk-Luc copiesintegrated in MVLN cells.A, schematic representation of the insertcontaining the pVit-tk-Luc copies integrated in the MVLN cell line. Eachof the three copies described contained the entire gene unit (encompassedin the XhoI-XhoI restriction fragment, here denotedX) including thevitellogenin gene ERE, the tk promoter, and the luciferase gene, as wellas the polyadenylation site, although the junction between two consec-utive copies is not identical.P1 represents a 665-bp probe edged byBanI-BanII restriction sites, andP2 is a 1654-bp probe edged byXbaI-XbaI restriction sites.B, schematic representation of the promoter part ofPR gene. Eachvertical dash represents a CpG dinucleotide.Filledtrianglesanddiamondsrepresent sites of transcription initiation for PRAand PRB, respectively. The sequences and locations of the variousoligonucleotides used in the experiments are depicted.

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the intensity of bands corresponding to sites 1 and 2 was much lowerin clone CL 6.32 than in MVLN cells.

DHSS Disappearance in thePR Gene Is Not Associated withMethylation or with Mutations in Its Promoter Part

Fig. 4Bshows the location of the CpG dinucleotides belonging tothe promoter part of the humanPR gene (21). They are denselyassociated in promoter A and in the 39 end of promoter B. Using thebisulfite reaction, an exhaustive analysis of the methylation of thisregion was performed on genomic DNA from clone CL 6.7, whichexhibits a very low PR expression level. The PCR products obtainedwith primer pairs F1 and F2, and F3 and F4 were cloned, andindividual molecules belonging to 10 different “PCR clones” weresequenced (to ensure that both alleles would be analyzed). On a totalof 53 CpGs present between positions2164 and11044, no meth-ylation was observed in any of the 10 tested PCR clones. Examples ofsuch sequences are shown in Fig. 8,C andD.

The PR gene promoter part (unreacted with bisulfite) was alsoamplified using three pairs of primers, D3 and D2, D8 and D6, and D4and D5 (Fig. 4B). PCR products were cloned, and individual mole-cules belonging to five different PCR clones were sequenced. No

mutation was observed in this part of the promoter, which mainlycontrols PRA expression (21).

Trichostatin A Experiments

A histone deacetylase inhibitor such as trichostatin A increases theacetylated histone level in many cell types and thus was expected toenhance the expression of some repressed genes. The effect of tricho-statin A was investigated on cells from the parental MVLN cell lineand from NL 6 and CL 32inf clones (Fig. 9). These two clonesdisplayed a residual luciferase activity that could be estradiol inducedto 2% at most of the maximal value reached by the parental MVLNcell line. We observed a small but reproducible increase (by 4-fold) ofthe estradiol-induced luciferase expression on cell lines in whichluciferase was irreversibly inactivated but not in MVLN cells. Nosignificant effect of TSA (used at 200 nM for 48 h) was observedeither in cell lines cultured in DCC (control, results not shown) or inthe presence of OHT. Conversely, this treatment had no effect on PRinduction in CL 6.32 (results not shown).

Fig. 5. DNase I hypersensitivity sites of the insert containingpVit-tk-Luc copies in MVLN, NL 6, CL 6.32, and CL 32inf

cells. MVLN cells and clones NL 6, CL 6.32, and CL 32inf werecultured in FCS medium. Two h before nucleus isolation, theywere incubated with 1029 M estradiol (E2) or 1027 M OHT, asindicated below the autoradiograph. Nucleus isolation andDNase I digestion were performed as described in “Materialsand Methods.” DNase I amounts (units/500ml) are indicatedaboveeach lane on the autoradiograph. The Southern blottedDNA was hybridized with P2 containing the luciferase part ofthe Vit-tk-Luc plasmid.Arrows,DNase I hypersensitivity sites.

Fig. 6. Localization of the DNase I hypersensitivity sites of the insert containingpVit-tk-Luc copies in MVLN cells. Fortymg of DNA obtained from MVLN cell nucleistimulated by estradiol were digested or not with 8 units/500ml of DNase I, as indicatedabove the autoradiograph, and then extracted by phenol-chloroform and cleaved byApaLI 1 BanII restriction endonucleases (3 units/mg of DNA). The digestion productswere analyzed by Southern blotting on a 0.8% agarose gel and probed with P1.

Fig. 7. DNase I hypersensitivity sites contained in the promoter part of thePRgene inMVLN cells and clone CL 6.32. Treatment and DNase I digestion of MVLN cell nucleiwere performed as described in Fig. 5. A 1029 M estradiol treatment was performed 2 hbefore nucleus isolation. The amount of DNase I is indicated in thetop part(units/500ml).Southern blotted DNA was hybridized with a 625-bp PCR probe amplified betweenoligonucleotides D4 and D5 (Fig. 4A).Arrows,DNase I hypersensitivity sites.

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DISCUSSION

Apart from the well-known beneficial antiproliferative or antitumoraleffect of the drug, studies on the direct side effects of tamoxifen havemainly been restricted to investigating the possible formation of drugadducts with DNA, prompting mutation events (23–25). However, it hasnot been clearly proven that these mutations could lead to gene inacti-vation. The present study, performed on MVLN cells derived fromMCF-7 cells, was designed to show that tamoxifen could also alter theexpression of natural and chimeric genes in breast cancer cells (PRandVit-tk-Luc genes) through epigenetic modifications of the chromatintemplate. In the case of the chimericVit-tk-Luc gene, the high geneinactivation rate (t1/2, 7 days) is clearly incompatible with a selectionprocess (8). This does nota priori seem to be as clear for natural genesand, in particular, the PR gene, because a few months of OHT treatmentare required to irreversibly inhibit its expression in most cells. A homo-geneous (tagged with the chimeric luciferase gene) MVLN cell line wasisolated and used for the long-term OHT treatment experiments. Duringthe first 3 months of treatment, the antiproliferative effect of OHTpromoted the selection of resistant clones, and many but not all resistantclones irreversibly lost PR expression. In this regard, Grahamet al. (26)already observed that tamoxifen treatment could alter PR content inT47-D cells, leading to mixed subpopulations of cells. Once resistantcells had emerged and the growth rate was stabilized (after 3 months of

OHT treatment), the mean level of PR expression continued to decrease(see Fig. 2 after 6 months of treatment). Therefore, no synchronismbetween OHT resistance acquisition and PR inactivation was observed,showing that PR gene inactivation is not a prerequisite for the emergenceof resistant cells.

Although it cannot be definitively excluded that drug-induced muta-tions could be responsible for the inactivation of PR or luciferase genes,no mutation was observed in their promoter parts, suggesting that theseinactivation processes might rather involve a change in chromatin struc-ture. The humanPR gene promoter has been extensively studied byKastneret al. (21, 27), who found that PR expression is controlled by twopromoters, one located between2711 and131 (promoter B) and onelocated between1464 and11105 (promoter A), that direct the synthesisof mRNA transcripts originating from two clusters of transcription startsites and coding for PRB and PRA proteins, respectively. The expressionof PRA and PRB differs in a cell type-, promoter-, or ligand-specificmanner: for example, Grahamet al. (28) showed that estradiol inducedpreferential stimulation of PRB expression in human T47-D breast cancercells. This suggests that if both promoters can be differentially activated,their inactivation might also occur independently. We investigated thispoint by Western blotting experiments. Clearly, PR expression wasirreversibly decreased, whereas the PRA:PRB ratio of residual PR re-mained unchanged,suggesting that the inhibition process did not affect a

Fig. 8. Genomic sequencing of CL 6.7 bisulfite-treatedDNA. A and B, control reaction. DNA from the pVit-tk-Lucplasmid (30 pg mixed with 10mg of DNA extracted fromMCF-7 cells, which do not contain pVit-tk-Luc) was methylated(A) or not (B) withSssI CpG methylase. Each DNA was thensubjected to the bisulfite reaction. Products were PCR amplifiedusing two pairs of oligonucleotides surrounding the tk promoter,which amplify the coding strand only, cloned in the T-vector,and sequenced with the primer located on the 39end sequence.The sequence is thus read as the original DNA in which all ofthe guanine residues (facing a reacted cytosine) have beenconverted to adenines except those facing a methylated cyto-sine. In the native nonmethylated plasmid (B), all guanines wereread as adenines, and no bands appeared in theG track. In theplasmid methylated withSssI CpG methylase (A), guanineswere read as adenines except for those facing a CpG motifcytosine and, therefore, appeared as bands in theG track.C andD, PR promoter methylation status of a OHT-treated clone.DNA from clone CL6.7 was reacted with bisulfite, and theupper (coding) strand of the reaction product was amplifiedusing F1 and F2 primers (Fig. 4B). PCR products were se-quenced with the primer located on the 39end (C) and 59end(D); the sequences are thus read as the original DNA, in whichall of the guanine residues have been converted to adeninesexcept those facing a methylated cytosine (C) and the cytosineresidues have been converted to thymines except those thatwere methylated (D).

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limited part of the gene but involved a large portion of the chromatintemplate.

Because gene expression is usually associated with the induction orat least the presence of DHSSs (29), we wished to determine whetherthe DHSS pattern of the two inactivated genes would be coherent withthat observation. Such was the case, because (a) the four estrogen-inducible DHSSs in the luciferase gene were absent in two clones withluciferase expression that was irreversibly inactivated; and (b) theintensities of DHSS 1 and DHSS 2 bands belonging to thePRpromoter were much weaker in the PR-negative clone CL 6.32 than inuntreated MVLN cells, although the sites were not estradiol inducible.This could be compared with a study on the mouse uterusPR gene(30) in which three DHSSs were found in the 59 region, and no clearinducibility of any of these sites was observed. These results highlysuggested that both luciferase and PR genes were irreversibly inhib-ited along with a parallel closure of the promoter chromatin template.

To date, the epigenetic long-term silencing of genes has been shown tobe involved in situations as diverse as position-effect variegation inDrosophila, telomeric position effect in yeast, X chromosome inactiva-tion, control of homeotic gene clusters during the development andimprinting in mammals (31–34), as well as some silencing of reportertransgenes (35). DNA methylation could be involved in permanent si-lencing (36) through either direct interference of methylation with thebinding of transcription factors or the binding of specific repressors suchas MeCP1 or MeCP2 to methyl-CpG (37). Our results suggest that DNAmethylation was not involved in PR gene silencing, because no meth-ylated cytosine was found in the part ofPRgene promoter that containsdensely associated CpGs, and because the strongest DHSS1 was locatedin a part of promoter B that contains very few CpGs. With regard toclones in which luciferase expression is inhibited, one CpG methylationwas previously observed (9) and strictly correlated with gene expressiondisappearance; albeit this methylation site belongs to one of the twoNotIrestriction sites of the reporter gene polylinker and, therefore, outside ofthe gene promoter part. The luciferase expression inhibition was further-more not reversed by a 5-azacytidine treatment (9). CpG methylationtherefore does not seem to be the main mechanism leading to theinactivation of PR and luciferase expressions in OHT-treated MVLNcells. In a recent work, Fergusonet al. (38) showed that a few CpGslocated downstream of promoter A ofPR were methylated in ER-andPR-negative MDA-MB-231 cells but that these methylationsper secannot prevent PR gene induction by transfected ER. This result again

suggests that the methylation process is not responsible for the PR-negative phenotype.

It was recently shown that histone deacetylation could also be involvedin gene silencing (39–41), whereas histone acetylation was found to beinvolved in transcriptional activation mediated by nuclear receptors (42–44). However, the influence of histone acetylation on steroid hormonegene expression is not clear, because both inhibitory (45) and stimulatory(46) effects have been reported. Treatment of OHT-inactivated cloneswith trichostatin A, a deacetylase inhibitor, increased luciferase expres-sion 4-fold at most but was ineffective on PR gene expression. Nosynergy between trichostatin A and azacytidine was observed (results notshown) such as that reported in a recent review (47).

Differences observed concerning DNA methylation and histonedeacetylation ofPR and luciferase genes may reflect a difference inmechanisms between the rapidVit-tk-Luc inactivation and the muchslower PR inactivation.

Another gene-silencing mechanism could involve the formation of“inactive heterochromatin-like” condensed structures initiated by ap-propriate factors (48–50), suggesting that mechanisms other thanmethylation or acetylation should be investigated. Recently, a func-tional link between these mechanisms and those involving nuclearreceptors began to emerge (51–54). These findings substantiated amechanism that would involve a direct effect of nuclear receptors inchromatin structure remodeling. It is not yet known whether such amechanism is involved in OHT-induced silencing.

In conclusion, the present study demonstrated that, besides itsreported genotoxic effects, tamoxifen could also directly and irrevers-ibly alter estrogen-dependent gene expression in cultured breast can-cer cells, through epigenetic modifications of the chromatin template.This is the first documented example of long-term gene silencinginduced by an antihormone. In our resistant clones, this phenomenondifferentially affects hormone-responsive genes, because we observedthat it modified the expression of two estrogen-induced genes atdifferent rates and that the expression of a third one,i.e., thepS2gene,was not modified. It should still be determined whether such irrevers-ible inhibition of gene expression could be involved in OHT resis-tance acquisition by shutting off the expression of putative growthsuppressors. Besides the classical ways mentioned in “Introduction”that could explain the resistance process, we think that estrogen-controlled gene alteration at the chromatin structure level deservesspecial attention, because it may be the consequence of a direct and

Fig. 9. Effect of trichostatin A on the luciferase expression ofOHT-treated clones. MVLN cells, clone NL 6, and clone CL 32inf

were cultured either in FCS medium or in DCC medium for 24 h.FCS and DCC media were then changed and supplemented with 1nM estradiol (E2) with or without 200 nM trichostatin (TSA) or 1027

M OHT with or without 200 nM trichostatin (TSA), respectively,for 48 h.

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primordial effect of the AE. Using the OHT-resistant cells describedin this paper, we are presently addressing this question by an exhaus-tive search for irreversibly inactivated genes.

ACKNOWLEDGMENTS

We thank David Manley for correcting the English in the manuscript.

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2000;60:4130-4138. Cancer Res Eric Badia, Marie-Josèphe Duchesne, Abdelhabib Semlali, et al. Estrogenic Genes through Chromatin RemodelingBreast Cancer Cell Line Irreversibly Inhibits the Expression of Long-Term Hydroxytamoxifen Treatment of an MCF-7-derived

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Long-Term Hydroxytamoxifen Treatment of an MCF-7-derived ...cancerres.aacrjournals.org/content/canres/60/15/4130.full.pdf · Eric Badia,2 Marie-Jose`phe Duchesne, Abdelhabib Semlali,