Estrogen and antiestrogen-dependent regulation of breast cancer cell proliferation in multicellular spheroids: Influence of cell microenvironment.

Multicellular tumor spheroids, an in vitro 3-D model that simulates malignant-cell contacts within a tumor, can be used to evaluate tumor response to therapeutic agents. We found that MELN (derived from MCF-7 cells) cells grown in 3-D as spheroids, remain highly sensitive to estradiol in terms of growth, down-regulation of ERalpha expression and ERalpha-induced transcriptional activity. Estradiol induces cyclin D1 and CDK1 proteins in Ki-67 positive proliferating cells, whereas survivin is up-regulated in both Ki-67 positive proliferative outer layer of cells and around the necrotic zone in non-proliferating cells. OH-Tam inhibits both estradiol-induced transcriptional activity and estradiol-dependent growth of MELN spheroids. Consistent with its antiproliferative effect, we observed that OH-Tam induces an important decrease in the proportion of proliferating cells, positive for Ki-67, cyclin D1 and CDK1. But, in contrast to what was expected, OH-Tam treatment resulted in a decrease in the proportion of p21 positive cells. Furthermore, despite its ability to down-regulate survivin in MELN spheroids, OH-Tam did not trigger apoptosis. Taken together, these results indicate that this model, is more relevant to an in vivo situation than monolayer cultures. It could be useful to identify new markers of the response to endocrine treatment and to investigate the effects of drugs combination.

Role of histone N-terminal tails and their acetylation in nucleosome dynamics.

Histone N-terminal tails are central to the processes that modulate nucleosome structure and function. We have studied the contribution of core histone tails to the structure of a single nucleosome and to a histone (H3-H4)(2) tetrameric particle assembled on a topologically constrained DNA minicircle. The effect of histone tail cleavage and histone tail acetylation on the structure of the nucleoprotein particle was investigated by analyzing the DNA topoisomer equilibrium after relaxation of DNA torsional stress by topoisomerase I. Removal of the H3 and H4 N-terminal tails, as well as their acetylation, provoked a dramatic change in the linking-number difference of the (H3-H4)(2) tetrameric particle, with a release of up to 70% of the negative supercoiling previously constrained by this structure. The (H3-H4)(2) tetramers containing tailless or hyperacetylated histones showed a striking preference for relaxed DNA over negatively supercoiled DNA. This argues in favor of a change in tetramer structure that constrains less DNA and adopts a relaxed flat conformation instead of its left-handed conformation within the nucleosome. In contrast neither removal or hyperacetylation of H3 and H4 tails nor removal or hyperacetylation of H2A and H2B N-terminal tails affected the nucleosome structure. This indicates that the globular domain of H2A and H2B is sufficient to stabilize the tailless or the hyperacetylated (H3-H4)(2) tetramer in a left-handed superhelix conformation. These results suggest that the effect of histone tail acetylation that facilitates transcription may be mediated via transient formation of an (H3-H4)(2) tetrameric particle that could adopt an open structure only when H3 and/or H4 tails are hyperacetylated.

Independent glucocorticoid induction and repression of two contiguous responsive genes.

Specific DNA sequence elements which contain binding sites for the glucocorticoid receptor mediate the action of glucocorticoid hormones on gene transcription. In glucocorticoid-inducible genes, these glucocorticoid-responsive elements behave as hormone-inducible enhancers of transcription. We have taken advantage of the bovine papillomavirus (BPV) system to test the stringency of glucocorticoid regulation of transcription. BPV episomes were constructed to contain two hormone-regulated transcription units in close proximity; one transcription unit is under control of a glucocorticoid-inducible promoter (mouse mammary tumor virus) while the other is under control of a glucocorticoid-inhibited promoter (pro-opiomelanocortin). Glucocorticoids independently regulated transcription of the two physically linked transcription units, irrespective of their relative orientation and of their proximity on the BPV episomes. This result contrasts with the so-called position-independent activity of enhancers and suggests that the multicomponent organization of eucaryotic promoters restricts the action of hormone-responsive regulatory elements to a specific transcription unit, thus accounting for the stringency of hormonal regulation observed in vivo.

Glucocorticoid regulation of transcription at an amplified, episomal promoter.

The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been introduced into cultured murine cells, using the 69% transforming fragment of bovine papilloma virus type 1 (BPV). Transformed cells contain up to 200 copies of the chimeric molecules per diploid genome. The restriction endonuclease map of the acquired recombinants, as well as the physical structure of the DNA, indicates that the LTR-BPV molecules present in these cells occur exclusively as unintegrated, extrachromosomal episome. When a 72-base pair direct repeat "enhancer" element (derived from the Harvey sarcoma retrovirus) was included in the MMTV LTR-BPV chimeric plasmids, DNA acquired through transfection, with a single exception, was integrated or rearranged or both. The transcriptional potential of the episomal MMTV promoter present in these cells was tested in two ways. First, steady-state levels of MMTV-initiated RNA were measured by quantitative S1 mapping. Second, the relative number of transcription complexes initiated in vivo was determined by using a subnuclear fraction highly enriched for MMTV-BPV minichromosomes in an in vitro transcription extension assay. Both approaches showed that the MMTV LTR present in the episomal state was capable of supporting glucocorticoid hormone-regulated transcription. We have therefore demonstrated the hormone response for the first time in a totally defined primary sequence environment. Significant differences both in the basal level of MMTV-initiated transcription and in the extent of glucocorticoid induction were observed in individual cell lines with similar episomal copy numbers. These phenotypic variations suggest that epigenetic structure is an important component of the mechanism of regulation.

Expression level-dependent contribution of glucocorticoid receptor domains for functional interaction with STAT5.

The action of the glucocorticoid receptor (GR) on beta-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the beta-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent beta-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for beta-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.

Long-term hydroxytamoxifen treatment of an MCF-7-derived breast cancer cell line irreversibly inhibits the expression of estrogenic genes through chromatin remodeling.

Antiestrogen resistance is frequently observed in patients after longterm treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitro studies 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 1-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.

Chromatin structure of the regulatory regions of pS2 and cathepsin D genes in hormone-dependent and -independent breast cancer cell lines.

We have compared the DNase I hypersensitivity of the regulatory region of two estrogen-regulated genes, pS2 and cathepsin D in hormone-dependent and -independent breast carcinoma cell lines. This strategy allowed the identification of two important control regions, one in pS2 and the other in cathepsin D genes. In the hormone-dependent MCF7 cell line, within the pS2 gene 5'-flanking region, we detected two major DNase I hypersensitive sites, induced by estrogens and/or IGFI: pS2-HS1, located in the proximal promoter and pS2-HS4, located -10.5 Kb from the CAP site, within a region that has not been cloned. The presence of these two DNase I hypersensitive sites correlates with pS2 expression. Interestingly in MCF7 cells, estrogens and IGFI induced indistinguishable chromatin structural changes over the pS2 regulatory region, suggesting that the two transduction-pathways converge to a unique chromatin target. In two cell lines that do not express pS2, MDA MB 231, a hormone-independent cell line that lacks the estrogen receptor alpha, and HE5, a cell line derived from MDA MB 231 by transfection that expresses estrogen receptor alpha, there was only one hormone-independent DNase I hypersensitive site. This site, pS2-HS2, was located immediately upstream of pS2-HS1. In MCF7 cells, two major DNase I hypersensitive sites were present in the 5'-flanking sequences of the cathepsin D gene, which is regulated by estrogens in these cells. These sites, catD-HS2 and catD-HS3, located at positions -2.3 Kb and -3.45 Kb, respectively, were both hormone-independent. A much weaker site, catD-HS1, covered the proximal promoter. In MDA MB 231 cells, that express cathepsin D constitutively, we detected an additional strong hormone-independent DNase I hypersensitive site, catD-HS4, located at position -4.3 Kb. This region might control the constitutive over-expression of cathepsin D in hormone-independent breast cancer cells. All together, these data demonstrate that a local reorganization of the chromatin structure over pS2 and cathepsin D promoters accompanies the establishment of the hormone-independent phenotype of the cells.

Interaction with the nuclear matrix of a chimeric construct containing a replication origin and a transcription unit.

We have studied the interaction of a chimeric construct containing an origin of replication (from bovine papilloma virus) and a hormonally regulated transcription unit (long terminal repeat from the mouse mammary tumor virus, driving the v-Ha-ras gene) with the nuclear scaffold and matrix from mouse fibroblasts. We used two experimental approaches because the nuclear matrix protein composition depends largely on the isolation conditions, making its definition mostly operational. In situ studies and in vitro experiments performed in 1361.5 cells, a cell line in which multiple copies of the construct have been established, indicate that two interesting regions of the construct interact with the nuclear matrix. The first region is located in the v-Ha-ras gene 5'-flanking sequences. These sequences come from the Harvey virus and contain a piece of the virus like 30S (VL30) sequences in which the v-Ha-ras gene is embedded. This DNA fragment was coupled to the thymidine kinase (TK) promoter driving the reporter luciferase gene and assayed in transient transfection experiments. Its insertion, in the sense orientation, upstream of the TK promoter resulted in a moderate enhancement (2-3-fold) of the luciferase activity. The second region is the most interesting from a physiological point of view. It contains the plasmid maintenance sequence 1 (PMS-1) and the core origin of replication of the bovine papilloma virus. Differences in the results from in situ (nuclear scaffold) and in vitro (nuclear matrix) experiments suggest that the components involved in the interaction with PMS-1 and the viral origin of replication are different. This may be of importance in the context of the recently proposed view that PMS-1 could be part of a composite origin of replication and provide information at a distance.

The establishment of the long terminal repeat of the mouse mammary tumor virus into CV-1 cells allows a functional analysis of steroid receptors.

To analyze in situ the effects of mineralocorticoid receptor (MR) on the nucleo-protein organization of the target MMTV promoter, we have established a new cell line by integrating in CV-1 cells a construct containing the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR). The MMTV-LTR contains glucocorticoid response elements (GREs), known to interact with MR. CV-1 cells were selected because they lack glucocorticoid receptor (GR). The absence of GR in the host cell line allows the selective analysis of transcription activation by aldosterone in cells expressing MR transiently. The CV-1 cells were transfected with the construct pMAMneoCAT, a plasmid containing the MMTV promoter driving the chloramphenicol acetyl transferase (CAT) gene and a gene for neomycin selection. A neomycin-resistant clone (M8), which contains two copies of the unrearranged construct was characterized. The integrated MMTV promoter is functional, as demonstrated by the induction of the CAT activity upon addition of aldosterone, dexamethasone, and R5020 to M8 cells transiently transfected with MR, GR, and progesterone receptor (PR) expression vectors, respectively. Induction of the CAT activity by dexamethasone or progesterone was 2 to 3-fold higher than by aldosterone. These differences in CAT activities were not related to differences in the levels of receptor expression. In the transiently transfected M8 cells, MR and PR contents were similar (50-70 fmol/mg protein) while GR content was higher (250 fmol/mg protein). Thus, this new cell line M8, provides a useful tool for selectively studying the effect of MR on a target promoter organized into chromatin.

Mechanism of dexamethasone 21-mesylate antiglucocorticoid action: II. Receptor-antiglucocorticoid complexes do not interact productively with mouse mammary tumor virus long terminal repeat chromatin.

We have studied the interaction of covalent dexamethasone 21-mesylate (DM) labeled, activated glucocorticoid receptor with mouse mammary tumor virus (MMTV) chromatin. Studies were performed on a murine cell line (904.13) which contains 200 copies per cell of a MMTV long terminal repeat (LTR) v-rasH casette mobilized on bovine papilloma virus based episomes. DM binds covalently to glucocorticoid receptors and displays almost full antagonist activity in this cell line. In situ transcription extension assays indicate that activated receptor-DM complex cannot stimulate LTR-initiated transcription. The receptor-DM complex also fails to induce DNase I hypersensitivity (HSR) and transcription factor loading at the MMTV promoter. Transcription activation, HSR formation, and factor binding induced with the agonist dexamethasone are blocked by covalent occupation of the receptor by DM. Although DM-activated receptor binds specifically to receptor sites on purified LTR DNA, the antagonist receptor complex does not interact productively with MMTV LTR chromatin in vivo.

Spironolactone, an aldosterone antagonist, acts as an antiglucocorticosteroid on the mouse mammary tumor virus promoter.

The ability of the glucocorticosteroid receptor to bind mineralocorticosteroids suggests that spironolactone, a potent aldosterone antagonist, may also interact with the glucocorticosteroid receptor, resulting in an agonist or antagonist glucocorticosteroid activity. We have investigated the effect of this drug on the activity of the glucocorticosteroid-regulated mouse mammary tumor virus (MMTV) promoter. For these studies we used the mouse fibroblast cell line 1471.1. It contains about 200 copies of a permanently established chimeric DNA construct comprising a transcription unit [MMTV long terminal repeat (LTR)] driving the reporter gene chloramphenicol acetyltransferase linked to the 69% transforming fragment of the bovine papilloma virus genome. This cell line has a high level of glucocorticosteroid receptor (1200 fmol/mg protein) and no detectable mineralocorticosteroid receptor. Competition experiments showed a binding of spironolactone to glucocorticosteroid receptor, with an affinity 50-fold lower than that of dexamethasone. In these cells, spironolactone behaves as an antiglucocorticosteroid, inhibiting in a dose-dependent fashion dexamethasone-induced chloramphenicol acetyltransferase activity, with an ED50 of 8 microM. The absence of agonist activity, even at a high concentration of this compound (10 microM), demonstrates that spironolactone is a pure antiglucocorticosteroid in this cell line. MMTV LTR DNase-I hypersensitivity studies demonstrated that spironolactone, when administered in combination with dexamethasone, inhibits formation of the hormone-induced hypersensitive site located about 160 basepairs up-stream of the MMTV cap site. Furthermore, spironolactone alone failed to induce this DNase-I-hypersensitive site, suggesting that the antagonist-receptor complex does not interact productively with MMTV LTR chromatin.

Chromatin structure and dynamics: functional implications.

In eucaryotes, DNA packaging into nucleosomes and its organization in a chromatin fiber generate constraints for all processes involving DNA, such as DNA-replication, -repair, -recombination, and -transcription. Transient changes in chromatin structure allow overcoming these constraints with different requirements in regions where processes described above are initiated. Mechanisms involved in chromatin dynamics are complex. Multiprotein complexes which can contain histone-acetyltransferase, -deacetylase, -methyltransferase or -kinase activities are targeted by regulatory factors to precise regions of the genome. These enzymes have been shown to modify histone-tails within specific nucleosomes. Post-translational modifications of histone-tails constitute a code that is thought to contribute to the nucleosome or to the chromatin fiber remodeling, either directly, or through the recruitment of other proteins. Other multiprotein complexes, such as ATP-dependent remodeling complexes, play an essential role in chromatin fiber dynamics allowing nucleosome sliding and redistribution on the DNA. We will focus here on the chromatin structure and its consequences for DNA damaging, replication, repair, and transcription and we will discuss the mechanisms of chromatin remodeling.

Affinity chromatography of the uterine estradiol receptor on estradiol-PAB-cellulose: an artefact.

Estradiol-PAB-cellulose, an easily prepared adsorbent, has been proposed to purify the uterine estradiol receptor according to the principle of biospecific affinity chromatography. It apparently removes all hormone binding sites when cytosol preparations are incubated with it. A systematic study of this adsorbent was undertaken, including the synthesis and testing of the radioactive material. Two main results were obtained: 1) Estradiol-PAB-cellulose is heavily contaminated with free ligand and releases it during the normal chromatographic conditions. 2) Estradiol spacer derivatives (hydroxyethylphenyl-diazo (2 or 4)-estradiol) have a very low affinity for the receptor (Ki = 10 muM). The conclusion is that estradiol-PAB-cellulose is unsuitable for affinity chromatography of estradiol receptor.

Chromatin structure of hormono-dependent promoters.

Transient transfections of mutated MMTV LTRs, driving the luciferase reporter gene, have shown the presence of at least one cis-acting element cooperating with the GREs. Studies of the chromatin structure of two glucocorticoid-regulated promoters, the mouse mammary tumor virus (MMTV) long terminal repeat (LTR), a retroviral promoter, and the rat tyrosine aminotransferase (TAT) promoter, demonstrate that both DNAs are organized into precisely positioned nucleosomes. Hormonal activation of transcription is accompanied by structural changes of one (MMTV LTR) or two (TAT promoter) nucleosomes associated with the hormone-response elements (HREs). These changes can be visualized by the appearance of DNasel hypersensitive sites. Association of the hormone-receptor complex with the nucleus is necessary to induce the DNasel hypersensitive site and to maintain transcription, but is not necessary to maintain DNasel hypersensitivity. Anti-hormones, even when able to promote a strong binding of the receptor to the nucleus, are unable to induce the chromatin structural change. Using cell lines containing approx. 200 copies of a MMTV LTR/Hv-ras chimeric construct, we have demonstrated a strong, hormono-independent nuclear matrix interaction of sequences located just upstream and downstream of the ras coding sequences.

A method for efficient extraction of bovine papilloma virus-based minichromosomes that preserves native chromatin structure.

Aiming to create an adequate model for investigation of the molecular mechanisms involved in transcriptional regulation by steroid hormones, a number of cell lines carrying bovine papilloma virus (BPV) based constructs containing the mouse mammary tumor virus long terminal repeat (LTR) were established (Ostrowski et al., Mol. Cell. Biol. 3, 2945-2957, 1983). However, all our attempts to extract from the cells such minichromosomes as nucleoprotein complexes using a method previously described (Ostrowski, Nucleic Acids Res. 15, 6957-6971, 1987) failed. Here, we show that this failure was attributable to DNA rearrangements in most of the cell lines, resulting in the integration of the BPV-based constructs into the host cell genome. We have identified two cell lines where the constructs are episomal. Micrococcal nuclease digestion of the nuclei demonstrated the presence of nucleosomes positioned over the episomal MMTV LTR. We managed to optimize conditions for preparation of nuclei and minichromosomes, which allowed extraction of approximately 40% of the minichromosomes, most of them being in circular superhelical form. Our data show clearly that the main factor preventing the release of minichromosomes from the nuclei is the presence of polyamines in the cell lysis buffer. The organization of MMTV promoter chromatin was unaffected by the extraction procedure, suggesting that these minichromosomes could be valuable templates for in vitro transcription studies and to identify proteins involved in chromatin remodeling during transcription.

Chromatin remodeling in hormone-dependent and -independent breast cancer cell lines.

Chromatin restricts the accessibility of DNA to regulatory factors; its remodelling over the regulatory regions contributes to the control of gene expression. An increasing number of evidence links defects in chromatin remodelling machinery and cancer. Our aim is to elucidate the role of chromatin structure in the control of the expression of hormone-induced genes in breast cell lines oestrogen-dependent or -independent for growth. Mammary tumour growth is controlled by steroid hormones via their nuclear receptor and by growth factors via tyrosine kinase receptors. 50% of these tumours elude to hormonal control. This limits the anti-oestrogen therapy. As a model, we have analysed in several cell lines the chromatin organisation of the regulatory regions of two genes, pS2 that is associated with a good prognostic, and cathepsin D (catD) that is a bad prognostic marker. The expression of the two genes is oestrogen-regulated in oestrogen-dependent cell line MCF7. In contrast in the hormone-independent cell line MDA MB 231, pS2 is not expressed and catD is constitutively expressed. Within the regulatory regions of pS2 gene, we have localised two regions that undergo a hormone-dependent change in chromatin structure in MCF7 cells but not in MDA MB 231 and that can be correlated with gene expression. In contrast catD regulatory regions did not display hormone-dependent changes in chromatin structure, suggesting that hormone regulation takes place within regions with a constitutively open chromatin structure.

[Chromatin remodeling in estrogen-dependent and independent human breast cancer cell lines]. / Remodelage de la structure chromatinienne des régions régulatrices de deux gènes oestrogéno-régulés dans des lignées de cellules cancéreuses mammaires humaines.

Chromatin restricts the accessibility of DNA to regulatory factors; its remodeling over the regulatory regions contributes to the control of gene expression. An increasing number of evidence links defects in chromatin remodeling machinery and cancer. Our aim is to elucidate the role of chromatin structure in the control of the expression of hormone-induced genes in breast cell lines estrogen-dependent or -independent for growth. Mammary tumor growth is controlled by steroid hormones via their nuclear receptor and by growth factors via tyrosine kinase receptors. 50 % of these tumors elude to hormonal control. This limits the anti-estrogen therapy. As a model, we have analyzed in several cell lines the chromatin organization of the regulatory regions of two genes, pS2 that is associated with a good prognostic, and cathepsin D (catD) that is a bad prognostic marker. The expression of the two genes is estrogen-regulated in estrogen-dependent cell line MCF7. In contrast in the hormone-independent cell line MDA MB 231, pS2 is not expressed and catD is constitutively expressed. Within the regulatory regions of pS2 gene, we have localized two regions that undergo a hormone-dependent change in chromatin structure in MCF7 cells but not in MDA MB 231. The lack of chromatin remodeling in MDA MB 231 cells is not due to the absence of expression of the estrogen receptor in the cell line. The expression of pS2 gene can be correlated with chromatin remodeling over the regulatory regions of pS2 gene. In contrast catD regulatory regions did not display hormone-dependent changes in chromatin structure, suggesting that hormone regulation takes place within regions with a constitutively open chromatin structure.

Eliminating epigenetic barriers induces transient hormone-regulated gene expression in estrogen receptor negative breast cancer cells.

In breast cancer, approximately one-third of tumors express neither the estrogen receptor (ERalpha) nor estrogen-regulated genes such as the progesterone receptor gene (PR). Our study provides new insights into the mechanism allowing hormone-activated expression of ERalpha target genes silenced in ERalpha-negative mammary tumor cells. In cell lines derived from ERalpha-negative MDA-MB231 cells, stable expression of different levels of ERalpha from a transgene did not result in transcription of PR. A quantitative comparative analysis demonstrates that inhibiting DNA methyltransferases using 5-aza-2'-deoxycytidine or specific disruption of DNMT1 by small interfering RNAs and treatment with the histone-deacetylase inhibitor trichostatin A enabled ERalpha-mediated hormone-dependent expression of endogenous PR. We show that demethylation of a CpG island located in the first exon of PR was a prerequisite for ERalpha binding to these regulatory sequences. Although not a general requirement, DNA demethylation is also necessary for derepression of a subset of ERalpha target genes involved in tumorigenesis. PR transcription did not subsist 4 days after removal of the DNA methyltransferase blocking agents, suggesting that hormone-induced expression of ERalpha target genes in ERalpha-negative tumor cells is transient. Our observations support a model where an epigenetic mark confers stable silencing by precluding ERalpha access to promoters.