Antiestrogen resistance is frequently observed in patients after long-term treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitrostudies in resistant cells showed that the expression of natural estrogen-responsive genes is frequently altered. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrated that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estrogen-regulated chimeric luciferase response by a 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, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irreversibly decreased in some of these clones, whereas the PRA:PRB ratio of residual PR remained unchanged. The irreversible inactivation of both chimeric luciferase gene and PR gene expression was associated with the disappearance of DNase I-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Genomic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promoter region. In all of the resistant clones tested and independently of their PR content,estrogen receptor expression was only lowered by half and remained functional, whereas pS2 expression was not modified. We also observed that the residual luciferase activity level (1–2%) of the MVLN clones, the luciferase expression of which had been irreversibly inactivated, was raised 4-fold by trichostatin A treatment. We conclude that long-term OHT treatment may modify the chromatin structure and thus could contribute to differentially silencing natural target genes.

Long-term tamoxifen therapy is widely used in the management of hormone-responsive breast cancer and is also being evaluated as a preventive treatment for breast cancer in healthy women at risk for breast cancer (1). Although curative tamoxifen treatment is efficient in many patients, most of them experience further tumor progression after several years of treatment, i.e.,so-called acquired tamoxifen resistance. The exact molecular mechanisms that account for acquired tamoxifen resistance remain unclear, but there is an emerging body of evidence that multiple mechanisms could be involved, concerning, for most of them, modifications in trans-elements at different points along the ER3pathway: (a) alterations in the pharmacology and metabolism of the AE (2); (b) ER mutations(3); and (c) alterations in cross-talk between signal transduction pathways (4, 5) or in the balance of nuclear receptor coeffectors (6, 7). Although each of these mechanisms has been invoked to explain the tamoxifen resistance process, none of them could account for the triggering of resistance acquisition.

Acquired resistance to tamoxifen may be due either to a simple selection process involving cells from an already heterogeneous population or to a two-stage process first implying cell alteration by the drug (here termed “direct effect”) followed by the selection of the new phenotype, provided that cell growth is no longer inhibited by tamoxifen. 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 an ERE (10), we showed that OHT was able to induce rapid and irreversible silencing of the luciferase gene. The subsequent appearance of cell heterogeneity (cells expressing or not expressing luciferase) was clearly incompatible with a selection process, because the time of half inactivation was as short as 7 days. We hypothesize that irreversibly affecting natural estrogen-responsive gene(s) by an AE direct effect could be a primordial event that triggers the resistance process; it then would lead to altered expression of other genes involved in cell proliferation. In vitro studies on tamoxifen- or OHT-resistant variant cell lines showed that expressions of some natural estrogen-responsive genes are frequently altered in these cells. The process leading to such alterations has not yet been elucidated (11, 12, 13).

The aim of this paper is to show that long-term OHT treatment can induce irreversible inhibition of the expression of estrogen-responsive genes along with having a direct effect on chromatin structure. By analyzing several OHT-resistant clones issued from OHT-treated MVLN cells, we show that the AE irreversibly inactivated the expression of the natural estrogen-controlled PR gene. This inactivation required a longer treatment period than for the luciferase gene. Conversely, the pS2 gene, another estrogen-regulated gene, was not inactivated by such treatment. Inactivations of luciferase and PR genes were associated with chromatin remodeling as revealed by the disappearance of DHSSs, one of which was localized close to the ERE in the chimeric gene.

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

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). Estradiol,sodium bisulfite, hydroquinone, spermine, spermidine, proteinase K, and 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, Montigny le Bretonneux, France). The T-vector ligation kit,Wizard DNA Clean-Up System, and competent JM 109 cells used for cloning PCR products were from Promega (Charbonnières, France). The sequencing kit, random labeling kits, nylon Hybond-N+ membrane, and the ECL system were from Amersham Pharmacia Biotech (Les Ulis, France). Detection of luciferase activity on cell-free extracts was performed with an LKB-Wallac (Sundyberg, Sweden) 1251 luminometer. A photon-counting camera (Argus 100) from Hamamatsu Photonics (Hamamatsu,Japan) was used to detect luciferase activity in intact cells and to analyze Western blots. The monoclonal anti-PR antibody (MA1–410) was from Affinity-BioReagents (Interchim, Montluçon, France). A Fujix BAS1000 PhosphorImager was used to analyze Northern blots.

Cell Line, Culture Conditions, and Luciferase Assay

The stable MVLN transfectant expressing the luciferase reporter gene under estrogen control has been described elsewhere(10). These MCF-7-derived cells express the firefly luciferase gene Luc under control of the 5′ flanking region of the Xenopus vitellogenin A2 gene Vit(15), inserted in front of the herpes simplex virus promoter for tk. The stability of this expression was checked over 70 passages in DMEM with phenol red, supplemented with 5%FCS (FCS medium). For long-term treatments, cells were cultured in DMEM without phenol red supplemented with 3% of a steroid-free,dextran-coated charcoal-treated FCS (DCC medium). Medium was replaced every other day. Luciferase activity was determined as described by Badia et 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 clones in tissue culture flasks. They were grown for ∼1 month in FCS medium until the clones were visible without magnification. Their NL or luminous phenotype was determined using a photon-counting camera (after supplementing the medium with 0.3 mm sterile luciferin). Clones were individually harvested and grown for 1 extra month in FCS medium. Clones NL 6 and NL 10 are NL clones obtained after a 12-day OHT treatment, whereas clones CL 3.1–CL 3.35 and clones CL 6.1–CL 6.35 represent two NL clone series obtained after 3 and 6 months of treatment, respectively. Clone CL 32inf was obtained by culturing clone CL 6.32 in the presence of 10−7m OHT for 12 extra months.

Cell Growth Assay

Each MVLN population or clone assayed was cultured for 5 days in DCC medium. Cells were harvested, and 2 × 104 cells per well were seeded in 24-well tissue culture cluster plates in the same medium. One day later, the medium 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 media were renewed every 2 days. After an 8-day growth period with the different effectors, triplicate wells were assayed for DNA content using 4′,6-diamidino-2-phenylindole(16).

PR Binding Assay and PR Isoform Analysis

Each MVLN population or clone was cultured in DCC medium for 5 days and then in DCC medium containing either 1 nmestradiol or 100 nm OHT in 0.1% ethanol. After a 4-day incubation, cells were harvested in PBS-EDTA, washed twice with PBS,pelleted, and frozen at −20°C until assay. The pellets were 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 180,000 × g for 40 min. Cytosol aliquots (200 μl) were incubated at 0°C for 4 h in the presence of either 10 nm[3H]promegestone or both 10 nm [3H]promegestone and 2μ m [1H]promegestone. Binding activity was measured by liquid scintillation counting after dextran-coated charcoal treatment. Specific stimulated (with estradiol)or unstimulated (with OHT) binding was expressed as the number of femtomoles of [3H]promegestone bound per milligram of cytosol proteins, measured by the Bradford technique. Relative expression of PRA and PRB isoforms was examined by Western blotting on a 15–25-μl cytosol aliquot (i.e., 20 μg of protein), using the ECL system after incubation with monoclonal mouse anti-PR. The chemiluminescence level was quantified using a photon-counting camera (Argus 100; Hamamatsu).

ER Binding Assay

Each tested clone was cultured in DCC medium for 5 days. Cells were then harvested in PBS-EDTA, washed twice with PBS, pelleted, and frozen at −20°C until assay. The pellets were treated as described above for PR binding; cytosol aliquots (200 μl) were incubated at 0°C for 2 h with either 5 nm[3H]estradiol or both 5 nm[3H]estradiol and 1 μm[1H]estradiol.

Northern Blot Analysis of pS2 Expression

MVLN cells that had grown for 3, 6, and 9 months in DCC medium alone or in DCC medium containing 10−7m OHT and clones isolated from MVLN cells treated with OHT for 1, 3, and 6 months were cultured in 25-cm2dishes and grown for 5 days in DCC medium and then in DCC medium supplemented with 10−9m estradiol or 10−7m OHT for 4 days. Total RNA was isolated according to the method of Chomczynski and Sacchi(17) using RNAzol B reagent. Ten μg of total RNA were electrophoretically separated on a 1% agarose denaturing gel and transferred to a nylon membrane. The membrane was hybridized overnight with 32P-labeled pS2 and 36B4 probes at 42°C in 50% formamide, as described previously (8). After stringency washes, filters were first exposed to the PhosphorImager screen to evaluate pS2versus36B4expression and autoradiographed.

DHSS Analysis

The methodology was adapted from that of Richard-Foy et al. (18) as follows.

Nucleus Isolation.

Nuclei were isolated either from untreated MVLN cells or from clones CL 6.32, CL 6.32inf, and NL 6. Cells contained in 10 T150 flasks were brought near confluence; five flasks were then incubated for 2 h in the presence of 10−9m estradiol, and the other five were incubated in the presence of 10−7m OHT before nucleus isolation. Cells were then carefully stripped, suspended in 40 ml of ice-cold PBS, which had been supplemented either with 10−9m estradiol or with 10−7m OHT (buffer H, buffer C, and buffer W were similarly supplemented), and centrifuged for 5 min at 1000 rpm. The supernatant was discarded, and the pellet was resuspended in 7 ml of ice-cold buffer H (15 mm NaCl, 60 mmKCl, 0.15 mm spermine, 0.5 mm spermidine, 1 mm EDTA, 0.1 mm EGTA, 0.2% NP40, 5%saccharose, and 10 mm Tris-HCl, pH 7.4). The cell suspension was homogenized with 15 strokes of a cold Dounce homogenizer. The state of the resulting nuclei was checked under a microscope. The resulting slurry was centrifuged over 4 ml of ice-cold buffer C (buffer H containing 10% saccharose) for 20 min at 3000 rpm. The supernatant was discarded, and the pellet was washed twice with 13 ml of ice-cold buffer W (15 mm NaCl, 60 mm KCl,0.15 mm spermine, 0.5 mm spermidine, and 10 mm Tris-HCl, pH 7.4). After a last centrifugation (3 min at 3000 rpm) the pellet was finally resuspended in 2 ml of buffer W.

DNase I Digestion.

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

Southern Blot Experiments.

Approximately 40–60 μg of DNase Idigested DNA were cleaved by StuI in 500 μl of the corresponding digestion buffer(usually 3 units/μg DNA for 2 h; complete digestion was checked by adding 0.5 μg of a known cleavable plasmid to a 20-μl aliquot of the reaction solution). Digested DNA was then ethanol precipitated in the presence of glycogen and 0.3 M NaOAc and resuspended in the sample buffer. Forty μg of DNA were separated electrophoretically on a 0.8%agarose gel and transferred to a nylon membrane. The membrane was hybridized overnight with the appropriate 32P-labeled probes. Positions of the molecular weight marker bands were measured before transfer onto the nylon membrane.

The sodium bisulfite reaction was performed as described by Frommer et al. (19) with slight modifications partially reported by Raizis et al. (20). Briefly, sodium bisulfite (1.9 g) was mixed with 2.5 ml of water. Then 0.7 ml of 2 M NaOH and 0.5 ml of 1 M hydroquinone were added. Template DNA (5 μg) in 30–50 μl of water was denatured by a 10-min incubation at room temperature after the addition of 0.1 volume of 2 m NaOH. DNA was resuspended in 1 ml of bisulfite solution and then successively incubated for 4 h at 50°C, heated at 95°C for 5 min, cooled to 50°C for 1 h, and then cooled to 4°C. DNA was purified with the Wizard DNA Clean-Up System and dissolved in 100 μl of water. After neutralization (43 μl of 1 n NaOH) and precipitation, the pellet was resuspended in 50 μl of water.

Direct PCR Products.

The three pairs of oligonucleotides, D3 and D2, D8 and D6, and D4 and D5 (see Fig. 1.,Fig. 4 B), generated 994-, 612-, and 617-bp-long fragments, respectively. These fragments covered the entire promoter A and part of promoter B as defined by Kastner et al.(21). The PCR fragments were purified on an agarose gel,cloned in the T-vector, and sequenced.

PCR Products from Bisulfite-treated DNA.

PCR was performed on 2.5 μl of the reacted DNA with oligonucleotides that matched the new sequence. The upper (coding)strand of the PR gene promoter was amplified with the two pairs of oligonucleotides, F1 and F2, and F3 and F4 described in Fig. 4 B. The sequences of oligonucleotides F1 and F3 were deduced from the natural upper strand by changing C to T, and those of F2 and F4 were deduced from the natural lower strand by changing G to A. The natural sequences corresponding to these oligonucleotides are devoid of methylatable CpGs; therefore, they fit perfectly with the gene sequence modified after the bisulfite reaction. The PCR products were purified on agarose gel, cloned in the T-vector, and sequenced.

OHT-treated MVLN Cells Acquired Growth Independent of This AE

Growth Rate Evaluated by Passage Frequency.

The MVLN cell line, a stably transfected MCF-7 cell line, is clonal. As for the MCF-7 cell line, MVLN cell proliferation is stimulated by estradiol and inhibited by AEs (22). At the beginning of continuous 10−7m OHT treatment, the MVLN cell doubling time was as long as 120 h (Fig. 1). However, after 6 weeks of culture, the MVLN doubling time shortened,reaching a steady state of 40 h after 10 weeks. The experimental results quite closely fit a theoretical model whereby an MVLN cell population grown in the presence of OHT (doubling time, 120 h)acquires AE resistance due to the modification of three cells per million per day, the doubling time of which drops to 40 h. Although speculative, this model suggests that as much as 95% of the cells grown for 3 and 6 months in the presence of OHT could derive from 10 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 the end of 3-, 6-, and 9-month OHT treatment times, 35 clones were isolated from the culture. All of these clones grew in the presence of OHT, which notably stimulated proliferation. Table 1 shows the proliferation of MVLN cells and eight clones isolated from 6-month treated cells (CL 6.1, CL 6.5, CL 6.7, CL 6.8, CL 6.20, CL 6.27, CL 6.28, and CL 6.32) after 8 days incubation of cells with various effectors. In DCC medium, it was 2–3-fold higher than at seeding (data not shown). The DNA content of 10−7m OHT- and 10−9m estradiol-treated resistant clones was ∼2- and 4-fold higher, respectively, than that of cells grown in DCC, whereas it was 2-fold lower in 10−7m ICI 164384-treated cells. Conversely, in parental MVLN cells, OHT as well as ICI 164384 inhibited cell proliferation by half. These results were confirmed by experiments in which cell growth was evaluated by the[3H]thymidine incorporation method (results not shown).

In a previous paper (9), it was shown that the MVLN cell expression of firefly luciferase was irreversibly inactivated by a short OHT treatment (<3 weeks), and we showed that this inactivation resulted from a 50-fold inactivation of individual cells. We describe here the irreversible inactivation of PR expression, a natural estrogenic response. The estrogen-stimulated PR content of MVLN cell cultures decreased with OHT treatment time during the first 6 months of treatment (Fig. 2,A). 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 and C) and observed that PR content (mean ± SD)was 60 ± 26 and 25 ± 23%, respectively(100% is the MVLN cell PR content, i.e., 648 ± 110 fmol/mg of proteins). This indicated that (a)the mean PR content decreased as the treatment time increased; and(b) the PR contents of individual clones varied markedly as suggested in each case by the high SD value (see above: 26 and 23%),reflecting the progressive appearance of heterogeneity and the randomness of this event. After 3 or 6 months of OHT treatment, clones were grown for 2 months in FCS medium (an estrogenic culture condition)before harvesting; hence the fact that the PR content was still low indicated that the loss of PR expression was irreversible. Northern blot experiments suggested that this inactivation occurred at the transcription level (results not shown). As a control, the estrogen-stimulated PR content of 12 clones issued from the parental MVLN cells was 108 ± 20%.

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

The eight clones (CL 6.1, CL 6.5, CL 6.7, CL 6.8, CL 6.20, CL 6.27, CL 6.28, and CL 6.32) expressing various amounts of PR were analyzed for their ER content (Table 1). They exhibited nearly 50% of the MVLN cell ER content (292 fmol/mg of protein). Very low PR levels in CL 6.7 (2.6%) and CL 6.32 (3.5%) or high PR levels in CL 6.27(117%) and CL 6.28 (88.5%) were thus associated with comparable ER levels (36, 40, 48, and 68%, respectively), suggesting that the PR expression level is not directly related to the ER level.

Fig. 3,A shows the results of the analysis of the pS2transcription level of MVLN cell cultures treated for various times with OHT. Estradiol-stimulated and basal (in the presence of OHT) pS2 mRNA levels, compared with those of the 36B4mRNA standard, were not significantly modified. The same results were obtained with clones issued from these treated MVLN cells (Fig. 3,B). It is noteworthy that pS2 expression was constant regardless of the PR expression levels (Fig. 2). For example,CL 3.23 and CL 6.27 on one hand and CL 3.24 and CL 6.7 on the other expressed a normal PR level and a very low PR level, respectively. As discussed below, this latter result shows that the different natural estrogen-controlled responses may have different degrees of sensitivity to long-term OHT treatment. In addition, when administered with estradiol, OHT was still able to counteract pS2 induction in cells pretreated for 6 months with OHT, indicating that its antiestrogenic effect was still efficient on this estrogen-controlled response of resistant cells (results not shown). These results also show that the ER and the basal transcription machinery functions were not affected by prolonged exposure to OHT and were still able to elicit some of the estrogenic responses.

In a previous study (9), Southern blotting experiments suggested that three copies of the Vit-tk-Luc plasmid placed in a head-to-tail configuration were integrated in the MVLN cells (Fig. 4,A). It was shown that the XhoI-XhoI restriction fragment, which contains the complete gene unit composed of the luciferase gene, the regulatory and promoter elements, and the polyadenylation site, was entire in each copy, and the copies were not rearranged by OHT treatment. In the present study, digestion with StuI, which does not cleave pVit-tk-Luc, gave only one band with a length (21 kbp; Fig. 4,A) compatible with that of an insert containing the three plasmid copies flanked by short genomic sequences. In this StuI-StuI fragment, DHSSs were analyzed in cells with luciferase that was either expressed or irreversibly inactivated. In MVLN cells, at least four DHSSs were visible, as indicated by the arrows in Fig. 5, all of them being inducible by estradiol stimulation. In clones NL 6,CL 6.32, and CL 32inf, the luciferase expression of which was irreversibly inactivated, DHSSs 2–4 completely disappeared, whereas site 1 was dramatically decreased.

To localize DHSSs in the StuI-StuI fragment of the pVit-tk-Luc integrated copies, DNA from estradiol-stimulated MVLN cells was restricted by ApaLI and BanII(Fig. 4,A) and gave a fragment encompassing the ERE near its 5′ end. The pattern obtained after Southern blotting and hybridization with probe P1, a 665-bp BanI-BanII fragment strictly included in the ApaLI-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, the ApaLI-BanII DNA fragment was partially cleaved,and a single extra band of ∼1400 bp was generated (Fig. 6, line B). Because the ERE is located 1450 bp upstream of the BanII restriction site, the cartographic analysis revealed that at least one of the four above-mentioned DHSSs was located close the ERE of one pVit-tk-Luc copy.

Hypersensitive sites were studied in the 5′ region of the PR gene in untreated MVLN cells and in CL 6.32, a 6-month OHT-treated clone with very low PR expression. At least two DHSSs could be distinguished on the autoradiograph: site 1 located around position−570 bp and site 2 (faint) around position +530 (Fig. 7). Site 3 around position +1130 of the PR gene promoter also present in undigested DNA could not be considered a DHSS. Although these sites were not obviously inducible by 2-h estradiol stimulation(results not shown), the intensity of bands corresponding to sites 1 and 2 was much lower in clone CL 6.32 than in MVLN cells.

Fig. 4,B shows the location of the CpG dinucleotides belonging to the promoter part of the human PR gene(21). They are densely associated in promoter A and in the 3′ end of promoter B. Using the bisulfite reaction, an exhaustive analysis of the methylation of this region was performed on genomic DNA from clone CL 6.7, which exhibits a very low PR expression level. The PCR products obtained with primer pairs F1 and F2, and F3 and F4 were cloned, and individual molecules belonging to 10 different “PCR clones” were sequenced (to ensure that both alleles would be analyzed). On a total of 53 CpGs present between positions −164 and+1044, no methylation was observed in any of the 10 tested PCR clones. Examples of such sequences are shown in Fig. 8 and D.

The PR gene promoter part (unreacted with bisulfite) was also amplified using three pairs of primers, D3 and D2, D8 and D6, and D4 and D5 (Fig. 4 B). PCR products were cloned, and individual molecules belonging to five different PCR clones were sequenced. No mutation was observed in this part of the promoter, which mainly controls PRA expression (21).

A histone deacetylase inhibitor such as trichostatin A increases the acetylated histone level in many cell types and thus was expected to enhance the expression of some repressed genes. The effect of trichostatin A was investigated on cells from the parental MVLN cell line and from NL 6 and CL 32inf clones (Fig. 9). These two clones displayed a residual luciferase activity that could be estradiol induced to 2% at most of the maximal value reached by the parental MVLN cell line. We observed a small but reproducible increase(by 4-fold) of the estradiol-induced luciferase expression on cell lines in which luciferase was irreversibly inactivated but not in MVLN cells. No significant effect of TSA (used at 200 nm for 48 h) was observed either in cell lines cultured in DCC (control,results not shown) or in the presence of OHT. Conversely, this treatment had no effect on PR induction in CL 6.32 (results not shown).

Apart from the well-known beneficial antiproliferative or antitumoral effect of the drug, studies on the direct side effects of tamoxifen have mainly been restricted to investigating the possible formation of drug adducts with DNA, prompting mutation events(23, 24, 25). However, it has not been clearly proven that these mutations could lead to gene inactivation. The present study,performed on MVLN cells derived from MCF-7 cells, was designed to show that tamoxifen could also alter the expression of natural and chimeric genes in breast cancer cells (PR and Vit-tk-Lucgenes) through epigenetic modifications of the chromatin template. In the case of the chimeric Vit-tk-Luc gene, the high gene inactivation rate (t1/2, 7 days) is clearly incompatible with a selection process (8). This does not a priori seem to be as clear for natural genes and, in particular, the PR gene, because a few months of OHT treatment are required to irreversibly inhibit its expression in most cells. A homogeneous (tagged with the chimeric luciferase gene) MVLN cell line was isolated and used for the long-term OHT treatment experiments. During the first 3 months of treatment, the antiproliferative effect of OHT promoted the selection of resistant clones, and many but not all resistant clones irreversibly lost PR expression. In this regard,Graham et al. (26) already observed that tamoxifen treatment could alter PR content in T47-D cells, leading to mixed subpopulations of cells. Once resistant cells 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 synchronism between OHT resistance acquisition and PR inactivation was observed, showing that PR gene inactivation is not a prerequisite for the emergence of resistant cells.

Although it cannot be definitively excluded that drug-induced mutations could be responsible for the inactivation of PR or luciferase genes, no mutation was observed in their promoter parts, suggesting that these inactivation processes might rather involve a change in chromatin structure. The human PR gene promoter has been extensively studied by Kastner et al. (21, 27),who found that PR expression is controlled by two promoters, one located between −711 and +31 (promoter B) and one located between +464 and +1105 (promoter A), that direct the synthesis of mRNA transcripts originating from two clusters of transcription start sites and coding for PRB and PRA proteins, respectively. The expression of PRA and PRB differs in a cell type-, promoter-, or ligand-specific manner: for example, Graham et al. (28) showed that estradiol induced preferential stimulation of PRB expression in human T47-D breast cancer cells. This suggests that if both promoters can be differentially activated, their inactivation might also occur independently. We investigated this point by Western blotting experiments. Clearly, PR expression was irreversibly decreased, whereas the PRA:PRB ratio of residual PR remained unchanged,suggesting that the inhibition process did not affect a limited part of the gene but involved a large portion of the chromatin template.

Because gene expression is usually associated with the induction or at least the presence of DHSSs (29), we wished to determine whether the DHSS pattern of the two inactivated genes would be coherent with that observation. Such was the case, because (a) the four estrogen-inducible DHSSs in the luciferase gene were absent in two clones with luciferase expression that was irreversibly inactivated;and (b) the intensities of DHSS 1 and DHSS 2 bands belonging to the PR promoter were much weaker in the PR-negative clone CL 6.32 than in untreated MVLN cells, although the sites were not estradiol inducible. This could be compared with a study on the mouse uterus PR gene (30) in which three DHSSs were found in the 5′ region, and no clear inducibility of any of these sites was observed. These results highly suggested that both luciferase and PR genes were irreversibly inhibited along with a parallel closure of the promoter chromatin template.

To date, the epigenetic long-term silencing of genes has been shown to be involved in situations as diverse as position-effect variegation in Drosophila, telomeric position effect in yeast, X chromosome inactivation, control of homeotic gene clusters during the development and imprinting in mammals (31, 32, 33, 34),as well as some silencing of reporter transgenes (35). DNA methylation could be involved in permanent silencing (36)through either direct interference of methylation with the binding of transcription factors or the binding of specific repressors such as MeCP1 or MeCP2 to methyl-CpG (37). Our results suggest that DNA methylation was not involved in PR gene silencing, because no methylated cytosine was found in the part of PR gene promoter that contains densely associated CpGs, and because the strongest DHSS1 was located in a part of promoter B that contains very few CpGs. With regard to clones in which luciferase expression is inhibited, one CpG methylation was previously observed (9)and strictly correlated with gene expression disappearance; albeit this methylation site belongs to one of the two NotI restriction sites of the reporter gene polylinker and, therefore, outside of the gene promoter part. The luciferase expression inhibition was furthermore not reversed by a 5-azacytidine treatment (9). CpG methylation therefore does not seem to be the main mechanism leading to the inactivation of PR and luciferase expressions in OHT-treated MVLN cells. In a recent work, Ferguson et al.(38) showed that a few CpGs located downstream of promoter A of PR were methylated in ER-and PR-negative MDA-MB-231 cells but that these methylations per se cannot 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 involved in gene silencing (39, 40, 41), whereas histone acetylation was found to be involved in transcriptional activation mediated by nuclear receptors (42, 43, 44). However, the influence of histone acetylation on steroid hormone gene expression is not clear, because both inhibitory (45) and stimulatory(46) effects have been reported. Treatment of OHT-inactivated clones with trichostatin A, a deacetylase inhibitor,increased luciferase expression 4-fold at most but was ineffective on PR gene expression. No synergy between trichostatin A and azacytidine was observed (results not shown) such as that reported in a recent review (47).

Differences observed concerning DNA methylation and histone deacetylation of PR and luciferase genes may reflect a difference in mechanisms between the rapid Vit-tk-Lucinactivation and the much slower PR inactivation.

Another gene-silencing mechanism could involve the formation of“inactive heterochromatin-like” condensed structures initiated by appropriate factors (48, 49, 50), suggesting that mechanisms other than methylation or acetylation should be investigated. Recently,a functional link between these mechanisms and those involving nuclear receptors began to emerge (51, 52, 53, 54). These findings substantiated a mechanism that would involve a direct effect of nuclear receptors in chromatin structure remodeling. It is not yet known whether such a mechanism is involved in OHT-induced silencing.

In conclusion, the present study demonstrated that, besides its reported genotoxic effects, tamoxifen could also directly and irreversibly alter estrogen-dependent gene expression in cultured breast cancer cells, through epigenetic modifications of the chromatin template. This is the first documented example of long-term gene silencing induced by an antihormone. In our resistant clones, this phenomenon differentially affects hormone-responsive genes, because we observed that it modified the expression of two estrogen-induced genes at different rates and that the expression of a third one, i.e., the pS2 gene, was not modified. It should still be determined whether such irreversible inhibition of gene expression could be involved in OHT resistance acquisition by shutting off the expression of putative growth suppressors. 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 deserves special attention, because it may be the consequence of a direct and primordial effect of the AE. Using the OHT-resistant cells described in this paper, we are presently addressing this question by an exhaustive search for irreversibly inactivated genes.

Fig. 1.

Growth of MVLN cells during long-term OHT treatment. MVLN cells were seeded in a T25 flask and grown in DCC medium with 10−7m OHT for 25 weeks. As soon as cells reached confluence, one-tenth of them were plated in a new T25 flask. Cell number was determined on the basis of the dilution at each passage. The logarithm of this cell number was plotted against the number of treatment weeks (filled symbols). Cell doubling time was then graphically determined. A theoretical model was constructed, which simulates variations in growth rates of a population of cells in which three cells/106/day raise their growth rate instantaneously from 120- to 40-h doubling time (open symbols).

Fig. 1.

Growth of MVLN cells during long-term OHT treatment. MVLN cells were seeded in a T25 flask and grown in DCC medium with 10−7m OHT for 25 weeks. As soon as cells reached confluence, one-tenth of them were plated in a new T25 flask. Cell number was determined on the basis of the dilution at each passage. The logarithm of this cell number was plotted against the number of treatment weeks (filled symbols). Cell doubling time was then graphically determined. A theoretical model was constructed, which simulates variations in growth rates of a population of cells in which three cells/106/day raise their growth rate instantaneously from 120- to 40-h doubling time (open symbols).

Close modal
Fig. 2.

PR content of MVLN and OHT-treated resistant cells and PRA:PRB ratio analysis. PR content of MVLN cells and individual clones was determined after 4-day stimulation with 1 nm estradiol. PR content of untreated MVLN cells was taken as 100%. A, MVLN cells were grown in DCC medium containing 10−7m OHT for 1, 3, 6, and 9 months. B and C, clones 1–35 were isolated as described in “Materials and Methods” from MVLN cells grown in DCC medium in the presence of 10−7mOHT for 3 months (B) and 6 months (C). D, each clone was grown for 4 days with 1 nmestradiol, after which 20 μg of cell extracts were submitted to 12%SDS-PAGE, transblotted, and processed for Western analysis.

Fig. 2.

PR content of MVLN and OHT-treated resistant cells and PRA:PRB ratio analysis. PR content of MVLN cells and individual clones was determined after 4-day stimulation with 1 nm estradiol. PR content of untreated MVLN cells was taken as 100%. A, MVLN cells were grown in DCC medium containing 10−7m OHT for 1, 3, 6, and 9 months. B and C, clones 1–35 were isolated as described in “Materials and Methods” from MVLN cells grown in DCC medium in the presence of 10−7mOHT for 3 months (B) and 6 months (C). D, each clone was grown for 4 days with 1 nmestradiol, after which 20 μg of cell extracts were submitted to 12%SDS-PAGE, transblotted, and processed for Western analysis.

Close modal
Fig. 3.

Induction of pS2 in MVLN cells and clones isolated from cells treated with OHT. A, MVLN cells that had grown for 3, 6, and 9 months in DCC medium alone or in DCC medium containing 10−7m OHT were treated as described in “Materials and Methods” with either 10−9m estradiol (E2) or 10−7m OHT. Total RNA was then isolated and processed for Northern blot analysis using a pS2 probe as described in “Materials and Methods.” B, various clones were isolated from MVLN cells treated with 10−7m OHT for 1 month(CL 1.4, CL 1.15, and CL 1.7), 3 months(CL 3.23 and CL 3.24), and 6 months(CL 6.7 and CL 6.27). Each of these clones was analyzed as in A.

Fig. 3.

Induction of pS2 in MVLN cells and clones isolated from cells treated with OHT. A, MVLN cells that had grown for 3, 6, and 9 months in DCC medium alone or in DCC medium containing 10−7m OHT were treated as described in “Materials and Methods” with either 10−9m estradiol (E2) or 10−7m OHT. Total RNA was then isolated and processed for Northern blot analysis using a pS2 probe as described in “Materials and Methods.” B, various clones were isolated from MVLN cells treated with 10−7m OHT for 1 month(CL 1.4, CL 1.15, and CL 1.7), 3 months(CL 3.23 and CL 3.24), and 6 months(CL 6.7 and CL 6.27). Each of these clones was analyzed as in A.

Close modal
Fig. 4.

Scheme of PR promoter and cartography of Vit-tk-Luc copies integrated in MVLN cells. A, schematic representation of the insert containing the pVit-tk-Luc copies integrated in the MVLN cell line. Each of the three copies described contained the entire gene unit (encompassed in the XhoI-XhoI restriction fragment, here denoted X) including the vitellogenin gene ERE, the tk promoter, and the luciferase gene, as well as the polyadenylation site,although the junction between two consecutive copies is not identical. P1 represents a 665-bp probe edged by BanI-BanII restriction sites, and P2 is a 1654-bp probe edged by XbaI-XbaI restriction sites. B, schematic representation of the promoter part of PR gene. Each vertical dash represents a CpG dinucleotide. Filled triangles and diamonds represent sites of transcription initiation for PRA and PRB, respectively. The sequences and locations of the various oligonucleotides used in the experiments are depicted.

Fig. 4.

Scheme of PR promoter and cartography of Vit-tk-Luc copies integrated in MVLN cells. A, schematic representation of the insert containing the pVit-tk-Luc copies integrated in the MVLN cell line. Each of the three copies described contained the entire gene unit (encompassed in the XhoI-XhoI restriction fragment, here denoted X) including the vitellogenin gene ERE, the tk promoter, and the luciferase gene, as well as the polyadenylation site,although the junction between two consecutive copies is not identical. P1 represents a 665-bp probe edged by BanI-BanII restriction sites, and P2 is a 1654-bp probe edged by XbaI-XbaI restriction sites. B, schematic representation of the promoter part of PR gene. Each vertical dash represents a CpG dinucleotide. Filled triangles and diamonds represent sites of transcription initiation for PRA and PRB, respectively. The sequences and locations of the various oligonucleotides used in the experiments are depicted.

Close modal
Fig. 5.

DNase I hypersensitivity sites of the insert containing pVit-tk-Luc copies in MVLN, NL 6, CL 6.32, and CL 32infcells. MVLN cells and clones NL 6, CL 6.32, and CL 32inf were cultured in FCS medium. Two h before nucleus isolation, they were incubated with 10−9mestradiol (E2) or 10−7m OHT,as indicated below the autoradiograph. Nucleus isolation and DNase I digestion were performed as described in“Materials and Methods.” DNase I amounts (units/500 μl) are indicated above each lane on the autoradiograph. The Southern blotted DNA was hybridized with P2 containing the luciferase part of the Vit-tk-Luc plasmid. Arrows, DNase I hypersensitivity sites.

Fig. 5.

DNase I hypersensitivity sites of the insert containing pVit-tk-Luc copies in MVLN, NL 6, CL 6.32, and CL 32infcells. MVLN cells and clones NL 6, CL 6.32, and CL 32inf were cultured in FCS medium. Two h before nucleus isolation, they were incubated with 10−9mestradiol (E2) or 10−7m OHT,as indicated below the autoradiograph. Nucleus isolation and DNase I digestion were performed as described in“Materials and Methods.” DNase I amounts (units/500 μl) are indicated above each lane on the autoradiograph. The Southern blotted DNA was hybridized with P2 containing the luciferase part of the Vit-tk-Luc plasmid. Arrows, DNase I hypersensitivity sites.

Close modal
Fig. 6.

Localization of the DNase I hypersensitivity sites of the insert containing pVit-tk-Luc copies in MVLN cells. Forty μg of DNA obtained from MVLN cell nuclei stimulated by estradiol were digested or not with 8 units/500 μl of DNase I, as indicated abovethe autoradiograph, and then extracted by phenol-chloroform and cleaved by ApaLI + BanII restriction endonucleases (3 units/μg of DNA). The digestion products were analyzed by Southern blotting on a 0.8%agarose gel and probed with P1.

Fig. 6.

Localization of the DNase I hypersensitivity sites of the insert containing pVit-tk-Luc copies in MVLN cells. Forty μg of DNA obtained from MVLN cell nuclei stimulated by estradiol were digested or not with 8 units/500 μl of DNase I, as indicated abovethe autoradiograph, and then extracted by phenol-chloroform and cleaved by ApaLI + BanII restriction endonucleases (3 units/μg of DNA). The digestion products were analyzed by Southern blotting on a 0.8%agarose gel and probed with P1.

Close modal
Fig. 7.

DNase I hypersensitivity sites contained in the promoter part of the PR gene in MVLN cells and clone CL 6.32. Treatment and DNase I digestion of MVLN cell nuclei were performed as described in Fig. 5. A 10−9m estradiol treatment was performed 2 h before nucleus isolation. The amount of DNase I is indicated in the top part (units/500μl). Southern blotted DNA was hybridized with a 625-bp PCR probe amplified between oligonucleotides D4 and D5 (Fig. 4 A). Arrows, DNase I hypersensitivity sites.

Fig. 7.

DNase I hypersensitivity sites contained in the promoter part of the PR gene in MVLN cells and clone CL 6.32. Treatment and DNase I digestion of MVLN cell nuclei were performed as described in Fig. 5. A 10−9m estradiol treatment was performed 2 h before nucleus isolation. The amount of DNase I is indicated in the top part (units/500μl). Southern blotted DNA was hybridized with a 625-bp PCR probe amplified between oligonucleotides D4 and D5 (Fig. 4 A). Arrows, DNase I hypersensitivity sites.

Close modal
Fig. 8.

Genomic sequencing of CL 6.7 bisulfite-treated DNA. A and B, control reaction. DNA from the pVit-tk-Luc plasmid (30 pg mixed with 10 μg of DNA extracted from MCF-7 cells, which do not contain pVit-tk-Luc) was methylated(A) or not (B) with SssI CpG methylase. Each DNA was then subjected to the bisulfite reaction. Products were PCR amplified using 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 3′end sequence. The sequence is thus read as the original DNA in which all of the guanine residues (facing a reacted cytosine) have been converted to adenines except those facing a methylated cytosine. In the native nonmethylated plasmid (B), all guanines were read as adenines, and no bands appeared in the G track. In the plasmid methylated with SssI CpG methylase(A), guanines were read as adenines except for those facing a CpG motif cytosine and, therefore, appeared as bands in the G track. C and D, PR promoter methylation status of a OHT-treated clone. DNA from clone CL6.7 was reacted with bisulfite, and the upper (coding)strand of the reaction product was amplified using F1 and F2 primers(Fig. 4 B). PCR products were sequenced with the primer located on the 3′ end (C) and 5′ end (D);the sequences are thus read as the original DNA, in which all of the guanine residues have been converted to adenines except those facing a methylated cytosine (C) and the cytosine residues have been converted to thymines except those that were methylated(D).

Fig. 8.

Genomic sequencing of CL 6.7 bisulfite-treated DNA. A and B, control reaction. DNA from the pVit-tk-Luc plasmid (30 pg mixed with 10 μg of DNA extracted from MCF-7 cells, which do not contain pVit-tk-Luc) was methylated(A) or not (B) with SssI CpG methylase. Each DNA was then subjected to the bisulfite reaction. Products were PCR amplified using 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 3′end sequence. The sequence is thus read as the original DNA in which all of the guanine residues (facing a reacted cytosine) have been converted to adenines except those facing a methylated cytosine. In the native nonmethylated plasmid (B), all guanines were read as adenines, and no bands appeared in the G track. In the plasmid methylated with SssI CpG methylase(A), guanines were read as adenines except for those facing a CpG motif cytosine and, therefore, appeared as bands in the G track. C and D, PR promoter methylation status of a OHT-treated clone. DNA from clone CL6.7 was reacted with bisulfite, and the upper (coding)strand of the reaction product was amplified using F1 and F2 primers(Fig. 4 B). PCR products were sequenced with the primer located on the 3′ end (C) and 5′ end (D);the sequences are thus read as the original DNA, in which all of the guanine residues have been converted to adenines except those facing a methylated cytosine (C) and the cytosine residues have been converted to thymines except those that were methylated(D).

Close modal
Fig. 9.

Effect of trichostatin A on the luciferase expression of OHT-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 1 nm estradiol (E2) with or without 200 nm trichostatin (TSA) or 10−7m OHT with or without 200 nm trichostatin(TSA), respectively, for 48 h.

Fig. 9.

Effect of trichostatin A on the luciferase expression of OHT-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 1 nm estradiol (E2) with or without 200 nm trichostatin (TSA) or 10−7m OHT with or without 200 nm trichostatin(TSA), respectively, for 48 h.

Close modal

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1

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

3

The abbreviations used are: ER, estrogen receptor; AE, antiestrogen; ERE, estrogen response 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.

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 and CL 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 (mean ± SD) are given as femtomoles per milligram of 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 studied in 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 (mean ± SD for three determinations) of cells grown in the presence of estradiol.

Cell clonesMVLNCL 6.1CL 6.5CL 6.7CL 6.8CL 6.20CL 6.27CL 6.28CL 6.32
DNA content          
DCC 21.6 ± 1.7 17.1 ± 0.8 29.4 ± 1.5 24.7 ± 1.3 37.4 ± 1.5 42.9 ± 1.7 24 ± 2.1 41.3 ± 1.7 30.3 ± 1.4 
E2 100 100 100 100 100 100 100 100 100 
OHT 13.4 ± 2.1 65.8 ± 8.4 63 ± 0.7 54.8 ± 3.3 57.6 ± 8.6 79.9 ± 3.1 55.5 ± 0.4 70.4 ± 2 74.8 ± 4.1 
ICI 6.6 ± 1.2 6.9 ± 0.1 8.8 ± 1.2 13.4 ± 1.3 8.2 ± 0.5 11.7 ± 0.5 7.8 ± 1 15.1 ± 0.4 7.5 ± 3.8 
PR content 648 ± 110 (5) 145 ± 55 (4) 294 ± 20 (3) 17 ± 3 (3) 262 ± 43 (2) 111 ± 30 (3) 764 ± 161 (3) 574 ± 104 (3) 23 ± 6 (2) 
ER content 292 ± 15 106 ± 12 142 ± 8 105 ± 25 168 ± 4 175 ± 32 141 ± 26 199 ± 3 118 ± 31 
Cell clonesMVLNCL 6.1CL 6.5CL 6.7CL 6.8CL 6.20CL 6.27CL 6.28CL 6.32
DNA content          
DCC 21.6 ± 1.7 17.1 ± 0.8 29.4 ± 1.5 24.7 ± 1.3 37.4 ± 1.5 42.9 ± 1.7 24 ± 2.1 41.3 ± 1.7 30.3 ± 1.4 
E2 100 100 100 100 100 100 100 100 100 
OHT 13.4 ± 2.1 65.8 ± 8.4 63 ± 0.7 54.8 ± 3.3 57.6 ± 8.6 79.9 ± 3.1 55.5 ± 0.4 70.4 ± 2 74.8 ± 4.1 
ICI 6.6 ± 1.2 6.9 ± 0.1 8.8 ± 1.2 13.4 ± 1.3 8.2 ± 0.5 11.7 ± 0.5 7.8 ± 1 15.1 ± 0.4 7.5 ± 3.8 
PR content 648 ± 110 (5) 145 ± 55 (4) 294 ± 20 (3) 17 ± 3 (3) 262 ± 43 (2) 111 ± 30 (3) 764 ± 161 (3) 574 ± 104 (3) 23 ± 6 (2) 
ER content 292 ± 15 106 ± 12 142 ± 8 105 ± 25 168 ± 4 175 ± 32 141 ± 26 199 ± 3 118 ± 31 

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

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