Cancer Research Cancer Epigenetics Genetics and Biology of Brain Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Demirpence, E.
Right arrow Articles by Pons, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Demirpence, E.
Right arrow Articles by Pons, M.
[Cancer Research 62, 6519-6528, November 15, 2002]
© 2002 American Association for Cancer Research

Endocrinology

An Estrogen-responsive Element-targeted Histone Deacetylase Enzyme Has an Antiestrogen Activity That Differs from That of Hydroxytamoxifen1

Ediz Demirpence, Abdelhabib Semlali, Joan Oliva, Patrick Balaguer, Eric Badia, Marie-Josèphe Duchesne, Jean-Claude Nicolas and Michel Pons2

INSERM U439, 34090 Montpellier, France


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We showed previously that prolonged treatment of a MCF-7-derived cell line with hydroxytamoxifen (OHT) induces the irreversible silencing of some estrogen-responsive genes, whereas OHT-resistant cell growth appears simultaneously (E. Badia et al., Cancer Res., 60: 4130–4138, 2000). Based on the hypothesis that particular gene silencings could be involved in triggering the resistance phenomenon, we focused our study on the mechanism of OHT-induced silencing. More precisely, we wished to determine to what extent the recruited histone deacetylase (HDAC) activity, which is known to be involved in the repressive effect induced by antagonist ligands of nuclear receptors, could participate in various aspects of OHT effects, particularly in gene silencing. A fusion protein (HDAC-EG) of human HDAC1 fused with the estrogen receptor DNA-binding domain and the glucocorticoid receptor ligand-binding domain allowed targeting of chimeric HDAC1 activity on estrogen-responsive elements (EREs) in the presence of glucocorticoid ligands. When HDAC-EG was transiently expressed in HeLa cells together with estrogen receptor, an antiestrogen-like effect was obtained on an ERE-controlled luciferase reporter gene in the presence of agonist or antagonist glucocorticoids. In MCF-7-derived cells stably expressing HDAC-EG and an estrogen-regulated luciferase, liganded HDAC-EG again produced an antiestrogenic effect on expression of natural estrogen-regulated genes such as pS2, progesterone receptor, and cathepsin D and cell growth together with chimeric luciferase gene expression. However, a prolonged HDAC-EG-mediated antiestrogen effect did not lead to irreversible luciferase gene silencing, as OHT does. It nevertheless accelerated the OHT-driven phenomenon. The antiestrogen effect of OHT thus differs from that of an ERE-targeted HDAC1 activity that might participate in irreversible silencing but is not sufficient to trigger it.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antiestrogens are widely used to treat estrogen-dependent breast cancers, but their use is limited by the associated critical side effects, such as the opposite pattern of action in tissues or the almost unavoidable acquisition of resistance. In previous studies (1, 2, 3) , we showed that prolonged OHT3 treatment of MVLN cells, i.e., a MCF-7-derived cell line, induces irreversible silencing of estrogen-responsive genes. The expression of a chimeric E2-regulated luciferase gene was thus rapidly silenced, with a half-inactivation time of 7 days, which was incompatible with a selection process but revealed a new facet of antiestrogen action. Similarly, the estrogen-dependent expression of the natural PR was half-silenced after 3 months of OHT treatment, whereas that of the pS2 gene remained fully inducible after a 9 month-treatment. Moreover, OHT-resistant cell growth appeared simultaneously. Assuming that silencing of particular (but undetermined) gene(s) may be involved in the resistance-triggering phenomenon, we decided to study the mechanism of such silencing.

Steroid hormone receptors are transcription factors that regulate gene expression in a ligand-dependent manner (4) . The binding of a hormone agonist to its specific receptor induces transcriptional activation of the receptor, whereas the binding of a hormone antagonist inhibits this process (5) . Depending on the cell and promoter context, receptors can either activate or repress the transcription of various target genes and regulate cellular responses such as proliferation, differentiation, development, and apoptosis. In the past few years, proteins that regulate the transcriptional efficiency of nuclear receptors have been identified (6 , 7) and reviewed (8 , 9) . They are coactivators and corepressors that activate or repress transcription, respectively, and are mainly recruited as large complexes on the LBD of receptors (10 , 11) .

Several coactivators were found to be associated with histone acetyltransferase activity (12, 13, 14, 15) . Histone acetylation is an important mechanism in gene activation (16, 17, 18) , and its absence has been correlated with transcriptional silencing (19 , 20) . The receptor-mediated transactivation inhibition may involve active repression through the recruitment of corepressors (21) . The major corepressors associated with unliganded and/or antagonist-bound receptors are N-CoR and SMRT, which contain repression domains (8 , 9) . Corepressors are associated with deacetylase activity through the recruitment of HDACs (22, 23, 24) , which possess different functional domains responsible for deacetylase activity and interaction with other proteins (25) . The amount of histone acetylation is thus determined by an equilibrium between acetyltransferases and deacetylases, and it has been suggested that the ratio of corepressors to coactivators is the modulator of transcription in a given context (26, 27, 28) . ER bound to an antiestrogen is thus unable to activate transcription, and it results mainly from the recruitment of a repressor complex with HDAC activity. Moreover, HDAC activity has already been associated with gene silencing in multicellular eukaryotes (29) .

We thus wished to determine to what extent the recruited HDAC activity could participate in various facets of OHT effects, particularly in gene silencing. A new type of in vitro antiestrogenic-like activity, associated with the targeting of transcriptional repressors to specific gene sites (30, 31, 32) , was recently described as a tool for antiestrogenic repression study and also as a new potential therapeutic approach. In a strategy analogous to that described for inhibiting oncogene-activated genes through a protein fused with the KRAB repressor domain (33) and in relation to the already observed HDAC1 repression of reporter genes (24 , 34) , our aim was to determine whether the direct targeting of HDAC1 activity to EREs could mimic or modulate OHT antiestrogen reversible or irreversible effects. To this end, we constructed a fusion protein (HDAC-EG) of human HDAC1 fused with the ER DBD and the GR LBD. This GR domain was preferred over its homologue in ER for two reasons. First, on the basis of previous results on targeted repressors (30, 31, 32) that showed that these fusion proteins had a dominant repressive effect in the presence of agonist, we considered that in an estrogen-dependent cell line, a fusion protein containing the LBD of ER would have such a repressive effect during cloning of a stable transfectant and therefore certainly prevent cloning. We were thus able to avoid this problem by using the GR LBD. Secondly, HDAC-EG allowed the targeting of chimeric HDAC1 activity on EREs in presence of (anti)glucocorticoids. Hence, in transfected cells, the expected antiestrogen effect obtained despite the absence of antiestrogens could be assigned to the fusion protein. The behaviors of this HDAC-EG fusion protein and of some other related fusion proteins were evaluated by transient transfection in HeLa cells to characterize their repressive effect. Then the HDAC-EG fusion protein-mediated effects on natural estrogen-responsive genes, as well as its long-term effect, were evaluated in MVLN-HEG cells, a stable transfectant issued from MVLN cells, a MCF-7-derived cell line.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Materials for cell culture came from Life Technologies, Inc. E2, Dex, MTT, and DAPI were purchased from Sigma. OHT was from Zeneca. RU486 and bimedrazol were from Roussel-Uclaf. Stock solutions were prepared in ethanol at 10-3 M. Further dilutions were made in culture medium. Luciferin was synthesized by G. Auzou (INSERM, Montpellier, France) according to Bowie (35) . Random labeling kits, nylon Hybond-N+ membrane, and the enhanced chemiluminescence system were from Amersham. Ultrahyb hybridization buffer for Northern blots was from Ambion. Biomax-MR films were from Kodak. The monoclonal (mouse) PR antibody (sc-811) and the polyclonal HDAC1 antibody (sc-7872) were from Santa Cruz Biotechnology, and the ß-actin antibody (A2066) was from Sigma. A single photon counting camera (ARGUS-100) from Hamamatsu Photonics was used to detect luciferase activity in whole cells and to analyze Western blots. A phosphorimager FUJIX BAS 1000 was used to analyze Northern blots, and a Luminoskan Ascent from Labsystem was used for luciferase assay of cell lysates.

Plasmids.
The HDAC1 gene (GenBank U50079) was a gift from Dr. S. Emiliani (Institut de Génétique Humaine, Montpellier, France). The gene was PCR-amplified using the primers 5'-ATCGATCGCAATTGATGGCGCAGACGCAGGGCACC-3' and 5'-TGACTGACGAATTCCGGGGTACCGGCCAACTTGACCTCCTCCTT-3' that introduced a MunI site into the 5' end and KpnI and EcoRI sites into the 3' end. The MunI-EcoRI restriction fragment of the PCR product was cloned in the EcoRI site of pSG5 vector to obtain pHDAC. The short KpnI-BglII fragment of pHDAC was exchanged with the KpnI-BglII fragment of pGR-ERcass (36) bearing the DBD of ER and the LBD of GR to obtain pHDAC-EG (see the structure of the corresponding protein in Fig. 1Citation ). The GR LBD has the I747T mutation, which alters its ligand specificity for transactivation (37) ; the mutated receptor is not activated by natural glucocorticoids but is only activated by high-affinity synthetic steroids such as Dex and bimedrazol and is efficiently inhibited by the antiglucocorticoid RU486. A gene that confers resistance to puromycin was later inserted into pHDAC-EG to obtain pHDAC-EG-puro. To construct p{Delta}HDAC-EG-puro, in which the HDAC sequence was absent, the sense 5'-AATTCACCATGGACTCCAAAGAATCAGGTACCA-3' and antisense 5'-GATCTGGTACCTGATTCTTTGGAGTCCATGGTG-3' oligonucleotides containing an internal ATG in a Kozac consensus sequence (38) and an appropriately in-frame KpnI site were first subcloned in the EcoRI-BglII site of pSG5, and the Xmn I-Kpn I fragment of this plasmid was introduced instead of the corresponding fragment of pHDAC-EG-puro. Two mutated plasmids, pHDACm141-EG and pHDACm176-EG, carrying either the H141A or D176N mutation in the HDAC1 sequence, respectively (34) , were obtained from pHDAC-EG using the QuikChange site-directed mutagenesis kit (Stratagene). All constructions were verified by sequencing, and structures of the corresponding proteins are shown in Fig. 1Citation . The reporter plasmids pERE-ßGlob-Luc+ (pEBL) responding to estrogens (39) and pFC31-Luc responding to glucocorticoids through the GRE of mouse mammary tumor virus (40) , the plasmids coding for ER{alpha} (pSG5-ER{alpha}; Ref. 41 ), the natural GR (pHG0), and pGR-ERcass (36) were defined elsewhere. For homogeneity and better comprehension, pGR-ERcass is referred to as pGAB-EG in the remainder of the text and codes for a GR in which the DBD of GR is replaced by the DBD of ER. The ß-galactosidase expression vector pCMV-ßGal was kindly provided by T. Lerouge. Standard recombinant DNA technology protocols (42) were used.



View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Schematic representation of GR and ER steroid receptors and of five chimeric receptors coded by the plasmids pHDAC-EG, p{Delta}HDAC-EG, pHDACm141-EG, and pHDACm176-EG and by pGAB-EG [the name by which we refer to pGR-ERcass (36) in this article]. The abbreviation -EG corresponds to the sequence of ER(C), the DBD of ER, followed by that of GR(DE), the hinge region, and the LBD of GR. Various point mutations are numbered as in the native GR and HDAC1 proteins. The GR LBD has a I747T mutation that alters its ligand specificity for transactivation (37) ; the mutated LBD is only transactivated by high-affinity synthetic glucocorticoids. The two H141A and D176N mutations were described in the HDAC1 sequence by Hassig et al. (34) . The D176N mutant was described to have almost no deacetylase activity and not to interact with corepressors, whereas the H141A mutant only had a reduced deacetylase activity and was still able to interact with corepressors. Black bars under HDAC1 regions represent the epitope corresponding to amino acids 432–482 mapped at the COOH terminus of HDAC1 against which the antibody was grown.

 
Cell Lines and Cell Culture Conditions.
The MVLN cell line, a MCF-7-derived cell line that stably expresses the luciferase gene under control of E2, was described previously (43 , 44) . The MVLN-HEG cell line was obtained by stably transfecting MVLN cells with HDAC-EG. MVLN, HeLa, and COS cells were routinely cultured in DMEM with phenol red supplemented with 5% FCS. For experiments, cells were first incubated in DMEM without phenol red and supplemented with 3% dextran-coated charcoal-stripped serum (DMEM-DCC) for 4 days, and steroid effectors were then added for an additional incubation time, as indicated.

Transient and Stable Transfection Experiments.
Transfections were performed in medium supplemented with 10% FCS. HeLa cells were seeded at 30% confluence in 24-well plates, and plasmid DNA was added the next day using the calcium phosphate precipitation method. Twenty-four h after transfection, cells were washed once with PBS, and the culture medium was replaced with DMEM-DCC. Effectors were added to this medium 24 h before analysis.

For stable transfections, MVLN cells were seeded in 10-cm cell culture dishes and transiently transfected with 10 µg of plasmid carrying the puromycin resistance gene. Twenty-four h after transfection, the culture medium was replaced with a medium containing 0.5 µg/ml puromycin, and cells were grown for 3 days. Cells were washed once a day to eliminate dead cells, and fresh medium was added. After 1 week, puromycin resistant cell clones were allowed to grow in the presence of puromycin. Clones were selected according to their (anti)hormone response using single photon detecting camera.

Luciferase Assay in Transiently and Stably Transfected Cells.
Cell lysates were prepared as recommended by Promega. Briefly, cells were washed twice with 1 ml of PBS and lysed with 0.4 ml of lysis buffer [25 mM Tris-phosphate (pH 7.8), 2 mM EDTA, 10% glycerol, and 1% Triton X-100] for 10 min. Cell lysate (100 µl) was transferred to wells of a 96-well plate, and luminescence was detected after injection of 100 µl of luciferase detection buffer [20 mM Tricine (pH 7.8), 1.07 mM (MgCO3)4 Mg(OH)2, 2.67 mM MgSO4, 0.2 mM EDTA, 0.53 mM ATP, 0.27 mM CoA, and 0.48 mM luciferin]. Arbitrary units in transient transfection experiments represent a ratio of luminescence to ß-galactosidase activity used as transfection control. In stable transfections, arbitrary units represent the ratio of luminescence to protein content in the assay.

Cell Growth Assay.
MVLN and MVLN-HEG cells (5000 cells/well) were plated in 24-well plates in DMEM-DCC. One day later, effectors were added at various concentrations. Cells were grown for 1 week, with replenishment of ligands in fresh medium every 2 days. Cell growth was determined by MTT assay or by fluorescence enhancement of DAPI (45) . By assessing mitochondrial activity through the selective ability of living cells to reduce the yellow soluble salt MTT to a purple-blue insoluble formazan precipitate, the MTT assay was validated as a cell proliferation assay (46) . Briefly, the culture medium was removed and replaced with 250 µl of fresh medium containing 10% reagent (4 mg MTT/ml PBS). After 4 h at 37°C, the reaction was stopped by removing medium, and formazan salt was allowed to dry. It was solubilized by adding 200 µl of DMSO and measured at 490 nm.

Northern Blot Analysis of pS2 and Cathepsin D Expression.
MVLN and MVLN-HEG cells were seeded in 75-cm2 flasks and grown for 4 days in DMEM-DCC. The medium was then supplemented with various effectors for 24 h. Ten µg of the total RNA isolated using a Qiagen kit were electrophoretically separated on a 1% agarose denaturing gel and transferred to a nylon membrane. The membrane was hybridized overnight in Ultrahyb buffer with 32P-labeled probes corresponding to pS2 (47) , cathepsin D (48 , 49) , and 18S RNA cDNAs (50) at 42°C, in 50% formamide, as already described. After stringency washes, filters were first exposed to the phosphorimager screen to evaluate pS2, cathepsin D, and 18S RNA expression.

Western Blot Analysis of HDAC1 and PR.
COS cells were seeded at 20% confluence in 10-cm cell culture dishes and transiently transfected with 10 µg of the constructed plasmids. Seventy-two h after transfection, 20 µg of whole cell lysate were electrophoresed on a 12% SDS-acrylamide gel. The blots were hybridized with HDAC1 and ß-actin antibodies and detected by peroxidase-coupled antirabbit IgG in the enhanced chemiluminescence system. Luminescent bands were visualized by autoradiography. PR expression under various conditions was examined in MVLN and MVLN-HEG cells by Western blotting of 40 µg of proteins.

Tritiated Dex Binding Site Assay in MVLN or MVLN-HEG Cells.
Two T150 flasks of each of the MVLN and MVLN-HEG cell lines were cultured in DMEM-DCC medium for 5 days. Cells were harvested in PBS-EDTA, pooled, washed twice with PBS, and then pelleted. The pellets were resuspended in 2 ml of buffer [10 mM Tris-HCl (pH 7.4), 1.5 mM EDTA, and 1 mM DTT], sonicated for 5 s at 0°C, and then centrifuged at 180,000 x g for 40 min. Cytosol aliquots (200 µl) were incubated at 0°C for 4 h in the presence of 5, 15, 50, or 150 nM [3H]Dex (total binding) and of 150 nM [3H]Dex plus 10 µM [1H]Dex (nonspecific binding). Binding activity was measured by liquid scintillation counting after dextran-coated charcoal treatment. Specific binding activity determined by Scatchard analysis was expressed as the number of femtomoles of [3H]Dex bound per milligram of cytosol proteins, measured by the Lowry method.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HDAC-EG Is Expressed as a 95-kDa Protein in COS and MVLN-HEG Cells.
Western blot analysis was performed to visualize HDAC-EG expression after transient and stable transfection. COS cells were transiently transfected with pHDAC-EG, pHDAC-EG-puro, p{Delta}HDAC-EG, or pGAB-EG (Fig. 2A)Citation . Cells were allowed to express proteins for 72 h, and whole cell extracts were prepared. The fusion proteins were visualized using HDAC1 antibody. The natural HDAC1 protein was detected in transfected and nontransfected cells as a 60-kDa protein, whereas HDAC-EG was detected as a 95-kDa protein in cells transfected with pHDAC-EG (Fig. 2ACitation , Lane 2) and pHDAC-EG-puro (Fig. 2ACitation , Lane 3). Because the HDAC sequence was missing in {Delta}HDAC-EG (Fig. 2ACitation , Lane 4) and GAB-EG (Fig. 2ACitation , Lane 5), only the natural 60-kDa HDAC band was visible in cells transfected with these plasmids. HDAC-EG was also detected as a 95-kDa protein in the stably transfected MVLN-HEG cells (Fig. 2BCitation , Lane 1), whereas the natural 60-kDa protein was observed in both MVLN and MVLN-HEG cells. The glucocorticoid binding activity in MVLN-HEG and control MVLN cells was evaluated (Fig. 2C)Citation by the Scatchard plot method (51) to quantify HDAC-EG expression level. The fact that a natural Dex binding activity of 100 fmol/mg protein was observed in MVLN cells and the total binding activity was 1060 fmol/mg protein in MVLN-HEG indicates that the binding activity brought by HDAC-EG was about 1000 fmol/mg protein. The difference observed in the Kd of Dex for the natural and chimeric Dex binding sites reflects the slight decrease in Dex affinity for the chimeric HDAC-EG, due to the I747T mutation in the GR LBD used. This mutation, which is present in the HG1 construction (36 , 37) and in the derived GAB-EG, allows the high-affinity synthetic glucocorticoids Dex and bimedrazol to specifically stimulate GR transactivation and RU486 to inhibit this transactivation, whereas natural glucocorticoids cortisol and corticosterone are inactive.



View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Cellular expression of HDAC-EG in transient and stable transfectants. A, whole cell extracts were prepared from nontransfected (Lane 1) and transfected COS cells by a plasmidic DNA without (HDAC-EG, Lane 2) or with (HDAC-EG*, Lane 3) the resistance gene to puromycin or plasmidic DNAs that were not coding for HDAC1, i.e., {Delta}HDAC-EG (Lane 4) and GAB-EG (Lane 5). B, whole cell extracts were prepared from MVLN-HEG (Lane 1) and MVLN (Lane 2) cells. Twenty µg of each extract were electrophoresed, blotted, and probed with HDAC1 and ß-actin antibodies as described in "Materials and Methods." The scales on both sides indicate the position and the molecular mass of marker proteins. C, Scatchard representation of tritiated Dex binding in MVLN and MVLN-HEG cells. Dissociation constant (Kd) and total specific binding sites are given for each cell line.

 
HDAC-EG Does Not Activate ERE-mediated Transcription in Transiently Transfected HeLa Cells.
The plasmid pHDAC-EG was cotransfected in HeLa cells with the reporter plasmid pEBL expressing luciferase under ERE control. Plasmids pGAB-EG and p{Delta}HDAC-EG were also transfected with the same reporter as controls. Because all these constructions contain the GR LBD, their transcriptional activity could be measured in the presence of agonist (Dex) or antagonist (RU486) glucocorticoids. As shown in Fig. 3ACitation , both GAB-EG and {Delta}HDAC-EG activated ERE-controlled transcription in the presence of the agonist, whereas there was no activation in the presence of the antagonist. Dex-induced activation was inhibited by adding RU486 [the high RU486 efficiency in inhibiting the Dex-induced response is due to the I747T mutation (37) in the GR LBD]. Conversely, HDAC-EG did not activate ERE-controlled transcription even in the presence of the agonist; instead, it slightly and reproducibly inhibited basal transcription by about 50%. In addition, when they were unliganded, the three fusion proteins GAB-EG, {Delta}HDAC-EG, and HDAC-EG had only a limited effect on the basal (Fig. 3BCitation , compare Lanes 3–5 with Lane 1) or ER-stimulated (Fig. 3BCitation , compare Lanes 6–8 with Lane 2) transcription level. In agreement with Berthois et al. (52) , the ER-mediated luciferase expression level (Fig. 3BCitation , Lanes 2 and 6–8) was stimulated by the estrogen activity of the phenol red-containing and FCS-supplemented culture medium. This expression level was not significantly altered by the presence of unliganded HDAC-EG, even though it was transfected five times in excess against ER (Fig. 3BCitation , Lane 8).



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Lack of HDAC-EG-mediated transcriptional activation. A, differential transcriptional activity mediated by chimeric receptors GAB-EG, {Delta}HDAC-EG, and HDAC-EG. HeLa cells were transfected with 0.4 µg/well pEBL, 0.08 µg/well chimeric receptors, and 0.08 µg/well pCMV-ßGal. B, unliganded HDAC-EG had no effect on ERE-mediated transactivation. HeLa cells were transfected with 0.4 µg/well pEBL, 0.08 µg/well pCMV-ßGal, and as indicated in the absence or presence of plasmids coding for the indicated receptors (0.08 or 04 µg/well). After transfection, cells were treated and assayed with luciferase as described in "Materials and Methods." The results are expressed as the mean ± SD (in arbitrary units) of at least three independent transfections performed in triplicate.

 
Liganded HDAC-EG Inhibits ERE-mediated Transcription and Involves ERE-targeted HDAC1 Activity in Transiently Transfected HeLa Cells.
When pSG5-ER{alpha} coding for ER was cotransfected with pHDAC-EG (Fig. 4A)Citation , the E2-induced expression of luciferase was inhibited by adding Dex or RU486. The higher the cotransfected pHDAC-EG concentration (versus pSG5-ER{alpha} concentration), the higher the observed inhibition.



View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. A, inhibition of ER-mediated transactivation by liganded HDAC-EG. HeLa cells were transfected with 0.4 µg/well pEBL, 0.08 µg/well pCMV-ßGal, or 0.08 µg/well pSG5-ER{alpha} in the absence or presence of pHDAC-EG (0.08 or 0.4 µg/well). B, HDAC activity was involved in the ligand-dependent inhibitory effect of HDAC-EG. HeLa cells were transfected with 0.4 µg/well pEBL, 0.08 µg/well pCMV-ßGal, or 0.08 µg/well pSG5-ER{alpha} with or without 0.4 µg/well pHDAC-EG, p{Delta}HDAC-EG, pHDACm141-EG, or pHDACm176-EG. In the inset, Western blots show the expression of HDAC-EG (WT), HDACm141-EG (H141A), and HDACm176-EG (D176N) fusion proteins (closed arrow) and natural HDAC1 enzyme (open arrow) in transiently transfected COS cells in a parallel experiment. C and D, inhibition of ERE-mediated transactivation by liganded HDAC-EG. HeLa cells were transfected with 0.4 µg/well pEBL, 0.08 µg/well pGAB-EG (C) or p{Delta}HDAC-EG (D), and 0.08 or 0.4 µg/well pHDAC-EG. After transfection, cells were treated and assayed as described in "Materials and Methods." Luciferase activity was calculated as a percentage of agonist (E2 or Dex)-induced transactivation. The results are expressed as the mean ± SD of at least three independent transfections performed in triplicate. The average luciferase activities taken as 100% were 21.72 arbitrary units for ER in the presence of E2 and 14.28 and 19.71 arbitrary units for GAB-EG and {Delta}HDAC-EG, respectively, in the presence of Dex.

 
To assess whether the targeted HDAC1 activity was involved in this inhibition, pSG5-ER{alpha} coding for ER{alpha} was cotransfected with each of the plasmids coding for HDAC-EG, {Delta}HDAC-EG, and mutated HDACm141-EG or HDACm176-EG to compare their effects on ERE-driven transactivation. HDAC-EG inhibited E2-induced luciferase expression (Fig. 4BCitation , Lane 3), whereas {Delta}HDAC-EG had no inhibitory effect (Fig. 4BCitation , Lane 4). Like {Delta}HDAC-EG, GAB-EG had no inhibitory effect in the presence of Dex (data not shown). It is clear that liganded HDACm176-EG (Fig. 4BCitation , Lane 6), corresponding to the D176N mutant, which has almost no deacetylase activity and does not interact with corepressors (34) , had no inhibitory effect on the E2-induced luciferase expression. Moreover, liganded HDACm141-EG (Fig. 4BCitation , Lane 5), corresponding to the H141A mutant, whose deacetylase activity is reduced by >80% (34) but whose protein is still able to interact with corepressors, inhibited E2-induced luciferase expression but to a lesser extent than HDAC-EG. This latter result may reflect the residual HDAC1 activity of this mutated fusion protein. The relative expression of each fusion protein was controlled in a parallel experiment by Western blotting. The polyclonal HDAC antibody, grown against the COOH-terminal part of HDAC1 (see Fig. 1Citation ), was used. The inset of Fig. 4BCitation shows that the three plasmids pHDAC-EG, pHDACm141-EG, and pHDACm176-EG, transiently transfected in COS cells, led to a comparable protein expression level, for identical expression of the natural HDAC1 protein enzyme taken as a standard. Similar results (data not shown) were obtained in HeLa cells, although fusion protein expressions were lower. The lack of inhibition with the mutants compared with that observed with HDAC-EG was therefore probably not due to a difference in protein expression but strongly suggests that this inhibition was correlated with the expression of functional HDAC enzyme activity.

It is shown (Fig. 3ACitation ; Fig. 4CCitation and Fig. 4DCitation , compare Lanes 5 and 8 with Lane 2) that GAB-EG and {Delta}HDAC-EG-mediated Dex-induced expression of an ERE-driven luciferase was efficiently inhibited by the antiglucocorticoid RU486. The concomitant expression of HDAC-EG led to strong inhibition, easily observed in the presence of Dex alone (Fig. 4, C and DCitation , compare Lanes 3 and 4 versus Lane 2), and to substantial additional inhibition in the presence of RU486 (Fig. 4, C and DCitation , see Lanes 6 and 7 versus Lane 5 and Lanes 9 and 10 versus Lane 8).

Because HDAC-EG, which has a repressive effect in the presence of Dex, and chimeric {Delta}HDAC-EG and GAB-EG, which mediate the agonistic activity of Dex, share a common EG protein part (Fig. 1)Citation , the fact that their behaviors differ indicated that the inhibitory action of HDAC-EG must involve the active HDAC part. As in the case of other constructions with the repressors KRAB or N-CoR, HDAC-EG led to a dominant negative receptor. On the whole, these results suggest that Dex-induced inhibition is correlated with ERE-targeted HDAC1 activity. As observed in Fig. 4ACitation , this targeting was significantly more efficient (P < 0.01) with the agonist Dex than with the antagonist RU486, i.e., inhibitions in the presence of Dex were greater than inhibitions in the presence of RU486. This might reflect a differential affinity for the ERE of complexes recruited by HDAC-EG liganded to the two compounds.

HDAC-EG Does Not Inhibit GRE-mediated Transcription in Transiently Transfected HeLa Cells.
To show that the inhibitory effect of HDAC-EG was specific to ERE-mediated transactivation and actually mimicked an antiestrogenic effect, pHDAC-EG was cotransfected with pHG0, the wild-type GR-expressing plasmid, and pFC31-Luc into HeLa cells. In these cells, Dex activates GR-mediated luciferase transcription under the control of the GRE of mouse mammary tumor virus. The concomitant expression of HDAC-EG did not inhibit the luciferase expression mediated by GR (Fig. 5A)Citation , whereas when transfected at the same concentration, it inhibited GAB-EG-mediated transcription (Fig. 5B)Citation . This shows that the fusion protein was specifically targeted to ERE and that its inhibitory effect was not a nonspecific squelching or a global nontargeted deacetylating effect.



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. HDAC-EG does not inhibit GRE-mediated transactivation. A, HeLa cells were transfected with 0.4 µg/well pFC31-Luc (GRE-Luc reporter plasmid) and 0.08 µg/well pHG0 coding for GR with or without 0.4 µg/well pHDAC-EG. B, HeLa cells were transfected with 0.4 µg/well pEBL (ERE-Luc reporter plasmid) and 0.08 µg/well pGAB-EG with or without 0.4 µg/well pHDAC-EG. After transfection, cells were treated and assayed as described in "Materials and Methods." Luciferase activity was calculated as a percentage of agonist (Dex)-induced transactivation. The results are representative of at least two independent experiments and expressed as the mean ± SD of triplicate transfections. The average luciferase activities taken as 100% were 16.3 arbitrary units for GAB-EG in the presence of Dex, and 14.3 arbitrary units for GR in the presence of Dex.

 
Stably Transfected HDAC-EG Inhibits Estrogen-induced Luciferase Expression in the MVLN-HEG Cell Line.
Because HDAC-EG showed a specific antiestrogen behavior in transiently transfected ER- cells, we wondered whether it would also have an antiestrogenic effect on estrogenic responses in ER+ breast cancer cells. We addressed this question by developing the MVLN-HEG cell line, i.e., a double stable transfectant that derives, as does its parental MVLN cell line, from MCF-7 cells and allows stable expression of the estrogen-dependent luciferase reporter gene. MVLN-HEG cells also stably express HDAC-EG. As described above for transient tranfections, HDAC-EG was expressed in MVLN-HEG (Fig. 2)Citation as an expected 95-kDa fusion protein to a high level of about 1000 fmol/mg protein, i.e., 3.3-fold more than the natural ER present in these cells, a necessary requirement to observe a marked effect.

In two parallel experiments, MVLN and MVLN-HEG cells were treated with hormones and/or antihormones. In both cell lines, E2 induced luciferase expression (Fig. 6Citation , Lanes 2, 5, 10, and 16), whereas OHT (Lane 3) inhibited this E2-induced luciferase. As already observed in transient transfection experiments, E2-induced luciferase expression was inhibited in MVLN-HEG cells in a dose-dependent manner by the two glucocorticoid agonists Dex (Lanes 6b-8b) and bimedrazol (Lanes 17b-20b) and also by the antiglucocorticoid RU486 (Lanes 11b-14b). As a control, no significant alteration of luciferase expression was observed in MVLN cells undergoing identical treatments (Lanes a). Bimedrazol (EC50 = ~1 nM) is efficient at a 30–100-fold lower concentration than Dex (EC50 = ~100 nM) for inhibiting E2-controlled luciferase induction. For this reason, bimedrazol was used in most of the following experiments with MVLN-HEG cells. A stronger inhibition was obtained with the agonist compounds than with RU486, as already observed with transient transfectants and discussed. However, here it could be partially due to the fact that, contrary to Dex and bimedrazol (Fig. 6Citation , Lanes 9, 21, and 22), RU486 exerted partial ER-mediated agonistic activity (Lanes 15 compared with Lanes 1) in the ER+ cells as observed previously (53) .



View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Liganded HDAC-EG stably expressed in MVLN-HEG cells inhibits E2-induced luciferase expression. Like MVLN cells, MVLN-HEG cells stably express an estrogen-dependent luciferase gene. The two cell lines were treated in parallel. E2 was used at 1 nM, whereas OHT, Dex, RU486, and bimedrazol ligands were used at the indicated concentrations. The results are expressed as a percentage of the mean ± SD of triplicate values (in arbitrary units/mg protein). The average luciferase activities taken as 100% were 33,500 and 36,000 arbitrary units/mg protein for MVLN and MVLN-HEG, respectively.

 
Stably Transfected HDAC-EG Inhibits the Estrogen-induced Expression of Natural Estrogen Responses in MVLN-HEG Cells.
The effects of HDAC-EG on the expression of three natural estrogen-responsive genes (pS2, cathepsin D, and PR) were studied. Bimedrazol inhibited both basal and E2-induced pS2 expression, whereas the inhibition produced by RU486 was again weaker (Fig. 7A)Citation . Similar bimedrazol inhibitory effects were obtained in MVLN-HEG cells when the expression of two other E2-responsive genes, cathepsin D (Fig. 7B)Citation and PR (Fig. 7C)Citation , were studied by Northern blot and Western blot, respectively, whereas little or no effect was observed in MVLN cells.



View larger version (52K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Bimedrazol liganded HDAC-EG stably expressed in MVLN-HEG cells inhibits E2-induced natural gene expressions. Typical experiments were obtained on the two cell lines treated in parallel for 2 (A and B) or 4 (C) days. A, pS2 Northern blot; B, cathepsin D Northern blot; C, RP Western blot. In these experiments, E2 (E), OHT (T), bimedrazol (B), and RU486 (R) were used at 1, 100, 100, and 300 nM, respectively.

 
The effect of HDAC-EG expression on the proliferation of MVLN-HEG cells was also studied. Cell growth is a classical estrogenic response in ER+ breast cancer cells, and its inhibition in hormone-dependent cancer cells remains the main target in the quest of new types of antiestrogen activity. It was evaluated here using the MTT assay. E2 induced and OHT inhibited cell growth in both cell lines as they did with luciferase gene expression and natural estrogen responses (Fig. 8A)Citation . An inhibitory effect was observed with Dex (about 40%) and RU486 (about 20%) when MVLN-HEG cell growth was compared with that of MVLN cells. The inhibitory effect of the glucocoticoid effector was confirmed in an experiment in which cell growth was evaluated by measuring DNA by fluorescence enhancement of DAPI. In the presence of bimedrazol (Fig. 8B)Citation , a significant effect (P < 0.01) was indeed observed, but a significant effect was not observed in the presence of RU486.



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. Bimedrazol liganded HDAC-EG stably expressed in MVLN-HEG cells inhibits their E2-induced growth. The two cell lines were treated in parallel. A, results are expressed as the mean ± SD of triplicate experiments. The mean MTT activities taken as 100% were 0.728 and 0.948 DO490 units for MVLN and MVLN-HEG cells, respectively. B, the results are expressed as the mean ± SD of six individual values of an experiment performed in a 24-well plate (four conditions). The mean DNA contents taken as 100% were 6.5 and 6.3 µg for MVLN and MVLN-HEG cells, respectively.

 
Together, these results show that classical antiestrogen effects could be efficiently mimicked by a HDAC1 activity targeted directly to an ERE in an estrogen-dependent cell line. We then wondered whether HDAC-EG expression may also lead to the irreversible silencing of some genes, as OHT does.

Prolonged Targeting of HDAC-EG on ERE Binding Sites Does Not Irreversibly Inactivate Expression of the Estrogen-responsive Luciferase Reporter Gene but Increases the Rate of Its Irreversible Inactivation by OHT.
Prolonged incubation of MVLN cells with OHT leads to quick (t1/2 = ~7 days) and irreversible silencing of the luciferase reporter gene through chromatin remodeling (3) . Because bimedrazol inhibited luciferase gene expression in MVLN-HEG cells as efficiently as OHT did in MVLN cells (Fig. 6)Citation , we tested whether an irreversible silencing of the luciferase gene would also occur by treating MVLN-HEG cells with bimedrazol for a prolonged incubation time. Both MVLN-HEG and MVLN cell lines were therefore treated or not treated (day 0) with OHT or bimedrazol for up to 21 days and then stimulated by 48 h of E2 treatment to recover luciferase activity (Fig. 9)Citation . As expected, the luciferase activity was gradually and irreversibly inactivated when MVLN and MVLN-HEG cells were treated with OHT [the OHT-induced irreversibility was demonstrated elsewhere and observable after E2 stimulation for 2–20 days (1) ]. When cells were treated with bimedrazol for several days, we noted (Fig. 9)Citation partial decrease in luciferase expression recovery, but it was HDAC-EG independent because it occurred in both cell lines and was probably due to a general nonspecific effect of glucocorticoids. Bimedrazol has no antiestrogen activity on E2-induced luciferase expression (data not shown) that could explain this inhibition, which instead would result from nonclassical antiestrogenic actions of glucocorticoids on more global responses, such as that described on cell proliferation in breast cancer cell lines (54, 55, 56, 57) . Therefore, the irreversible silencing obtained with OHT could not be specifically mimicked by a HDAC1 activity targeted directly to an ERE.



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 9. Bimedrazol liganded HDAC-EG stably expressed in MVLN-HEG cells does not irreversibly silence luciferase gene expression. MVLN and MVLN-HEG cells were treated with or without the effectors OHT or bimedrazol at 200 and 100 nM, respectively, for 0, 6, or 21 days. The recovered luciferase activity was measured after treatment with 1 nM E2 for 48 h. The results are the mean ± SD of triplicate values (arbitrary units/mg protein) and are expressed as the percentage of luciferase recovery obtained under control conditions at day 0 (100% = 35,300 and 38,500 arbitrary units/mg protein for MVLN and MVLN-HEG cells, respectively).

 
Because the prolonged incubation of MVLN-HEG cells with bimedrazol did not lead to specific irreversible silencing, we wondered whether this treatment would nevertheless modulate this phenomenon. We thus found that the rate of this irreversible OHT-induced silencing was increased by the presence of bimedrazol (Fig. 10Citation , compare OHT and OHT+B). A 60–80% lower recovered luciferase activity was indeed observed when MVLN-HEG cells were treated with bimedrazol and OHT rather than with OHT alone. The decrease was much smaller (20–25%) when treating MVLN cells the same way. This suggests that the activity of the targeted HDAC1 could be involved in this phenomenon. The results were confirmed in experiments under various other conditions, such as lower bimedrazol concentrations and longer incubation times (data not shown).



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 10. Bimedrazol liganded HDAC-EG stably expressed in MVLN-HEG cells potentializes the OHT-induced irreversible silencing of luciferase gene expression. MVLN and MVLN-HEG cells were treated with 100 nM bimedrazol (B) or 200 nM OHT separately or together (B+OHT) for up to 6 days. The recovered luciferase expression was induced in the presence of 1 nM E2 for 48 h. The results are expressed as the mean ± SD of triplicate values (RLU/mg protein) and as the percentage of luciferase recovery obtained under control conditions at day 0 (100% = 35.3 x 104 and 38.5 x 104 RLU for MVLN and MVLN-HEG cells, respectively).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this work, to investigate whether the antiestrogenic effects of OHT involve the targeting of HDAC activities to specific sites of action (i.e., EREs) through the recruitment of large complexes with corepressors, we assessed the extent to which the direct targeting of the HDAC1 enzyme on EREs via a fusion protein would mimic or modulate all of the antiestrogenic effects of OHT, including the irreversible silencing process.

Criteria for an Antiestrogenic Action of ERE-targeted HDAC1 Activity and Evidence on the Involvement of the HDAC Enzyme Activity.
In a first group of experiments using transient transfections in HeLa cells, the nature and specificity of effects due to the activity of targeted HDAC-EG were addressed. As observed by others (30, 31, 32) with several fusion proteins that associate the DBD of ER with other known repressive domains (KRAB, N-CoR), the HDAC-EG fusion protein did not activate ERE-mediated transcription and inhibited ER-mediated transactivation, regardless of whether it was liganded to Dex or RU486, its specific ligands. We attempted to estimate the HDAC activity of the HDAC-EG fusion protein because it has been suggested that the inhibitory effect of HDACs could result from deacetylase activity as well as from the recruitment of other repressor proteins. It was, however, difficult to unequivocally evaluate the enzymatic activity of the in vitro-translated HDAC-EG protein because of the high basal activity levels of the translation systems (data not shown). We then used trichostatin A, a HDAC inhibitor classically used to inhibit deacetylase activity, and only observed a slight reduction in HDAC-EG-induced inhibition, and these results were overshadowed by the effect of trichostatin A on the overall gene expression leading to a marked reduction of luciferase expression (data not shown). A different approach involving modulation of HDAC activity was nevertheless used to clarify the role of the enzymatic HDAC activity. The effects of four homologous fusion proteins targeted on EREs through the EG protein part were investigated. Firstly, when the HDAC sequence was deleted ({Delta}HDAC-EG) or replaced with the A/B domain of GR (GAB-EG), the modified fusion proteins liganded to Dex could activate an estrogen response similar to that of the wild-type ER, and they did not inhibit ER-mediated transactivation. Because the EG protein part bears all of the elements capable of recruiting coactivators that lead to transcription activation, the HDAC part in HDAC-EG could therefore be responsible for the inhibition mediated by the fusion protein and its lack of activating transcription. To confirm the role of enzymatic HDAC activity, two previously defined point mutations in the HDAC sequence were introduced, according to the work of Hassig et al. (34) , to modulate the deacetylase activity and/or corepressor binding capacity. The H141A mutation, which reduced the deacetylase activity by >80% without affecting corepressor-associated protein (Sin3A or RbAp48) binding, partially decreased the inhibitory effect of the fusion protein, whereas the D176N mutation, which disrupted both corepressor binding and deacetylase activity, completely abrogated the inhibitory effect. These results strongly suggest that the functional HDAC sequence enforced its inhibitory effect and that the fusion protein exerted a dominant negative character due to the expression of this constitutive HDAC activity. Finally, it is noteworthy that the effect of targeted HDAC-EG was specific for estrogen responses because the fusion protein did not affect GRE-controlled gene transactivation. Together, these results show that the reversible inhibitory activity of this fusion protein meets the criteria of antiestrogen activity.

In a MCF-7-derived Cell Line, the Observed Antiestrogenic Effect of the ERE-targeted HDAC1 Activity Differs from That of OHT.
In a second group of experiments in which stable transfectants were used, we assessed whether such antiestrogen activity would occur on natural estrogen responses and studied the effect of prolonged HDAC-EG targeting. The MVLN-HEG cell line is a MCF-7-derived cell line that stably expresses HDAC-EG. Like its parental MVLN cell line, MVLN-HEG cells express the estrogen-responsive luciferase gene and are estrogen dependent; cell proliferation is increased by estrogens, and cells express natural estrogen-regulated genes including pS2, cathepsin D, and PR. Because HDAC-EG has a high expression level in these cells (about 3-fold higher than that of natural ER), substantial antiestrogen activity was expected and actually observed on responses that were efficiently inhibited in the presence of bimedrazol.

Although HDAC1 targeted to ERE seems to faithfully mimic short-term antiestrogen effects of OHT, it did not lead to the irreversible inactivation that was observed after prolonged OHT treatment (3) , suggesting that complexes targeted on the response elements are different. Various hypotheses could be put forward to account for the incapability of HDAC1 to lead to luciferase silencing. Among them, there could be a situation in which the ERE is blocked by the presence of a complex containing HDAC-EG that would be unable to evolve further because of inappropriate or limited properties of the targeted HDAC1. Indeed, taking into account that the recently described epigenetic "histone code" (58, 59, 60) strongly suggests that transient or long-term changes in gene expression depend on a specific sequence or combination of histone modifications by various histone modifiers, the permanent presence of the sole HDAC activity could not be sufficient to engage the silencing process. However, the ERE blockade could be released by the binding of an OHT liganded ER (or the complex that it recruits) that is able to compete with HDAC-EG on ERE, as suggested by the observed increased OHT-induced silencing rate (see below). If the HDAC nature is involved in the lack of irreversible silencing, it should be noted that it was shown that N-CoR and SMRT, two corepressors that may be recruited by the OHT-ER complex (26 , 28 , 61 , 62) , also interact with HDAC3 (63 , 64) . Moreover, Underhill et al. (63) showed that a complex containing N-CoR also contains, in addition to HDAC3, KAP-1 (or TIF1ß), which is known to be involved in heterochromatin remodeling, which may lead to gene silencing through recruitment of the HP1 (65, 66, 67) . However, it was also very recently described that SU(VAR)3-9, the Drosophila homologue of human Suv39H1, another heterochomatin-associated protein (see below), is physically and functionally associated with HDAC1 and not HDAC3 in Drosophila (68) .

Because treatment with glucocorticoid bimedrazol did not enable HDAC-EG to irreversibly silence luciferase expression, we wondered whether the bimedrazol liganded HDAC-EG would protect the luciferase gene, by competition on the ERE, against ER-dependent OHT-induced irreversible silencing. On the contrary, the OHT-induced silencing rate was substantially increased by bimedrazol. Hence, the activity of the directly targeted HDAC (and/or that of the complex in which it is engaged) was thus not sufficient to irreversibly silence this gene but nevertheless may participate in and potentialize this gene silencing. This increased rate of chromatin remodeling might be associated with alternate exchanges of complexes targeted by HDAC-EG with complexes recruited by ER-OHT, which may either, in turn, favor different steps of chromatin remodeling or increase HDAC-HEG concentration in the close ERE environment, whose HDAC activity would be required by the OHT-ER complex to trigger silencing. HDAC activities have indeed been described to be recruited at an early step to remodel chromatin in Sin3A complexes that mediate the repressive effect of N-CoR or SMRT and have also been described to be recruited at a later step by HP1 to silence gene expression in a hypoacetylated heterochromatin environment (65 , 69) . Along with HP1 proteins, gene silencing may involve the recently described Suv39h1, a histone methyl transferase that creates a strong binding site for HP1 proteins after methylation of lysine 9 in histone H3 (70 , 71) . Interestingly, this methylation was also recently proposed to be involved in the transcriptional repression of euchromatic genes by retinoblastoma protein (72) and during retinoblastoma protein-mediated differentiation (69 , 73) . Studies on protein determination are thus currently under way to elucidate the nature of chromatin-bound complexes in promoters of cells expressing or not expressing the luciferase reporter gene and to explain this ER-dependent OHT-induced silencing.


   ACKNOWLEDGMENTS
 
We thank Carine Charron and Vincent Delauzun for skillful technical help, Dr. J-L. Borgna for critical reading of the manuscript, and David Manley for correcting the English. We are grateful to Drs. Stephane Emiliani, Thierry Lerouge, and Pierre Chambon for providing us with plasmids (coding for HDAC1, ß-galactosidase, ER/GR derived receptors, respectively).


   FOOTNOTES
 
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 Supported by the Institut National de la Santé et de la Recherche Médicale, the Association pour la Recherche sur le Cancer (Grant 5002), and the Groupement des Entreprises Françaises dans la Lutte contre le Cancer. Back

2 To whom requests for reprints should be addressed, at INSERM U439, 70 rue de Navacelles, 34090 Montpellier France. Phone: 33-4-67-04-37-13; Fax: 33-4-67-04-37-15; E-mail: pons{at}montp.inserm.fr Back

3 The abbreviations used are: OHT, 4-hydroxytamoxifen; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen-responsive element; HDAC, histone deacetylase, GR, glucocorticoid receptor; GRE, glucocorticoid-responsive element; Dex, dexamethasone; PR, progesterone receptor; LBD, ligand-binding domain; DBD, DNA-binding domain; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI, 4',6-diamidino-2-phenylindole; HP1, heterochromatin-associated protein 1. Back

Received 12/26/01. Accepted 9/26/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Badia E., Duchesne M. J., Fournier-Bidoz S., Simar-Blanchet A. E., Terouanne B., Nicolas J. C., Pons M. Hydroxytamoxifen induces a rapid and irreversible inactivation of an estrogenic response in an MCF-7-derived cell line. Cancer Res., 54: 5860-5866, 1994.[Abstract/Free Full Text]
  2. Badia E., Duchesne M. J., Nicolas J. C., Pons M. Rapid tamoxifen-induced inactivation of an estrogenic response is accompanied by a localized epigenetic modification but not by mutations. Breast Cancer Res. Treat., 47: 71-81, 1998.[Medline]
  3. Badia E., Duchesne M. J., Semlali A., Fuentes M., Giamarchi C., Richard-Foy H., Nicolas J. C., Pons M. Long-term hydroxytamoxifen treatment of an MCF-7-derived breast cancer cell line irreversibly inhibits the expression of estrogenic genes through chromatin remodeling. Cancer Res., 60: 4130-4138, 2000.[Abstract/Free Full Text]
  4. Moras D., Gronemeyer H. The nuclear receptor ligand-binding domain: structure and function. Curr. Opin. Cell Biol., 10: 384-391, 1998.[Medline]
  5. Webster N. J., Green S., Jin J. R., Chambon P. The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell, 54: 199-207, 1988.[Medline]
  6. Chakravarti D., LaMorte V. J., Nelson M. C., Nakajima T., Schulman I. G., Juguilon H., Montminy M., Evans R. M. Role of CBP/P300 in nuclear receptor signalling. Nature (Lond.), 383: 99-103, 1996.[Medline]
  7. Chen J. D., Evans R. M. A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature (Lond.), 377: 454-457, 1995.[Medline]
  8. Torchia J., Glass C., Rosenfeld M. G. Co-activators and co-repressors in the integration of transcriptional responses. Curr. Opin. Cell Biol., 10: 373-383, 1998.[Medline]
  9. Xu L., Glass C. K., Rosenfeld M. G. Coactivator and corepressor complexes in nuclear receptor function. Curr. Opin. Genet. Dev., 9: 140-147, 1999.[Medline]
  10. McInerney E. M., Rose D. W., Flynn S. E., Westin S., Mullen T. M., Krones A., Inostroza J., Torchia J., Nolte R. T., Assa-Munt N., Milburn M. V., Glass C. K., Rosenfeld M. G. Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. Genes Dev., 12: 3357-3368, 1998.[Abstract/Free Full Text]
  11. Darimont B. D., Wagner R. L., Apriletti J. W., Stallcup M. R., Kushner P. J., Baxter J. D., Fletterick R. J., Yamamoto K. R. Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev., 12: 3343-3356, 1998.[Abstract/Free Full Text]
  12. Chen H., Lin R. J., Schiltz R. L., Chakravarti D., Nash A., Nagy L., Privalsky M. L., Nakatani Y., Evans R. M. Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell, 90: 569-580, 1997.[Medline]
  13. Bannister A. J., Kouzarides T. The CBP co-activator is a histone acetyltransferase. Nature (Lond.), 384: 641-643, 1996.[Medline]
  14. Spencer T. E., Jenster G., Burcin M. M., Allis C. D., Zhou J., Mizzen C. A., McKenna N. J., Onate S. A., Tsai S. Y., Tsai M. J., O’Malley B. W. Steroid receptor coactivator-1 is a histone acetyltransferase. Nature (Lond.), 389: 194-198, 1997.[Medline]
  15. Brown C. E., Lechner T., Howe L., Workman J. L. The many HATs of transcription coactivators. Trends Biochem. Sci., 25: 15-19, 2000.[Medline]
  16. Grunstein M. Histone acetylation in chromatin structure and transcription. Nature (Lond.), 389: 349-352, 1997.[Medline]
  17. Workman J. L., Kingston R. E. Alteration of nucleosome structure as a mechanism of transcriptional regulation. Annu. Rev. Biochem., 67: 545-579, 1998.[Medline]
  18. Struhl K. Histone acetylation and transcriptional regulatory mechanisms. Genes Dev., 12: 599-606, 1998.[Free Full Text]
  19. Sasaki S., Lesoon-Wood L. A., Dey A., Kuwata T., Weintraub B. D., Humphrey G., Yang W. M., Seto E., Yen P. M., Howard B. H., Ozato K. Ligand-induced recruitment of a histone deacetylase in the negative-feedback regulation of the thyrotropin ß gene. EMBO J., 18: 5389-5398, 1999.[Medline]
  20. Chen W. Y., Townes T. M. Molecular mechanism for silencing virally transduced genes involves histone deacetylation and chromatin condensation. Proc. Natl. Acad. Sci. USA, 97: 377-382, 2000.[Abstract/Free Full Text]
  21. Zhang X., Jeyakumar M., Petukhov S., Bagchi M. K. A nuclear receptor corepressor modulates transcriptional activity of antagonist-occupied steroid hormone receptor. Mol. Endocrinol., 12: 513-524, 1998.[Abstract/Free Full Text]
  22. Li H., Leo C., Schroen D. J., Chen J. D. Characterization of receptor interaction and transcriptional repression by the corepressor SMRT. Mol. Endocrinol., 11: 2025-2037, 1997.[Abstract/Free Full Text]
  23. Alland L., Muhle R., Hou H., Jr., Potes J., Chin L., Schreiber-Agus N., DePinho R. A. Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature (Lond.), 387: 49-55, 1997.[Medline]
  24. Nagy L., Kao H. Y., Chakravarti D., Lin R. J., Hassig C. A., Ayer D. E., Schreiber S. L., Evans R. M. Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell, 89: 373-380, 1997.[Medline]
  25. Grozinger C. M., Hassig C. A., Schreiber S. L. Three proteins define a class of human histone deacetylases related to yeast Hda1p. Proc. Natl. Acad. Sci. USA, 96: 4868-4873, 1999.[Abstract/Free Full Text]
  26. Smith C. L., Nawaz Z., O’Malley B. W. Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol. Endocrinol., 11: 657-666, 1997.[Abstract/Free Full Text]
  27. Takimoto G. S., Graham J. D., Jackson T. A., Tung L., Powell R. L., Horwitz L. D., Horwitz K. B. Tamoxifen resistant breast cancer: coregulators determine the direction of transcription by antagonist-occupied steroid receptors. J. Steroid Biochem. Mol. Biol., 69: 45-50, 1999.[Medline]
  28. Lavinsky R. M., Jepsen K., Heinzel T., Torchia J., Mullen T. M., Schiff R., Del-Rio A. L., Ricote M., Ngo S., Gemsch J., Hilsenbeck S. G., Osborne C. K., Glass C. K., Rosenfeld M. G., Rose D. W. Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc. Natl. Acad. Sci. USA, 95: 2920-2925, 1998.[Abstract/Free Full Text]
  29. Mottus R., Sobel R. E., Grigliatti T. A. Mutational analysis of a histone deacetylase in Drosophila melanogaster: missense mutations suppress gene silencing associated with position effect variegation. Genetics, 154: 657-668, 2000.[Abstract/Free Full Text]
  30. Ma Z. Q., Tsai M. J., Tsai S. Y. Suppression of gene expression by tethering KRAB domain to promoter of ER target genes. J. Steroid Biochem. Mol. Biol., 69: 155-163, 1999.[Medline]
  31. Chien P. Y., Ito M., Park Y., Tagami T., Gehm B. D., Jameson J. L. A fusion protein of the estrogen receptor (ER) and nuclear receptor corepressor (NCoR) strongly inhibits estrogen-dependent responses in breast cancer cells. Mol. Endocrinol., 13: 2122-2136, 1999.[Abstract/Free Full Text]
  32. de Haan G., Chusacultanachai S., Mao C., Katzenellenbogen B. S., Shapiro D. J. Estrogen receptor-KRAB chimeras are potent ligand-dependent repressors of estrogen-regulated gene expression. J. Biol. Chem., 275: 13493-13501, 2000.[Abstract/Free Full Text]
  33. Ayyanathan K., Fredericks W. J., Berking C., Herlyn M., Balakrishnan C., Gunther E., Rauscher F. J. Hormone-dependent tumor regression in vivo by an inducible transcriptional repressor directed at the PAX3-FKHR oncogene. Cancer Res., 60: 5803-5814, 2000.[Abstract/Free Full Text]
  34. Hassig C. A., Tong J. K., Fleischer T. C., Owa T., Grable P. G., Ayer D. E., Schreiber S. L. A role for histone deacetylase activity in HDAC1-mediated transcriptional repression. Proc. Natl. Acad. Sci. USA, 95: 3519-3524, 1998.[Abstract/Free Full Text]
  35. Bowie L. J. Synthesis of firefly luciferin and structural analogs DeLuca M. A. eds. . Methods in Enzymology, Vol. LVII: 15-28, Academic Press New York 1978.
  36. Kumar V., Green S., Stack G., Berry M., Jin J. R., Chambon P. Functional domains of the human estrogen receptor. Cell, 51: 941-951, 1987.[Medline]
  37. Roux S., Terouanne B., Balaguer P., Jausons-Loffreda N., Pons M., Chambon P., Gronemeyer H., Nicolas J. C. Mutation of isoleucine 747 by a threonine alters the ligand responsiveness of the human glucocorticoid receptor. Mol. Endocrinol., 10: 1214-1226, 1996.[Abstract/Free Full Text]
  38. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res., 15: 8125-8148, 1987.[Abstract/Free Full Text]
  39. Metzger D., Berry M., Ali S., Chambon P. Effect of antagonists on DNA binding properties of the human estrogen receptor in vitro and in vivo. Mol. Endocrinol., 9: 579-591, 1995.[Abstract/Free Full Text]
  40. Gouilleux F., Sola B., Couette B., Richard-Foy H. Cooperation between structural elements in hormono-regulated transcription from the mouse mammary tumor virus promoter. Nucleic Acids Res., 19: 1563-1569, 1991.[Abstract/Free Full Text]
  41. Green S., Issemann I., Sheer E. A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res., 16: 369 1988.[Free Full Text]
  42. Sambrook J., Fritsch E. F., Maniatis T. . Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, NY 1989.
  43. Pons M., Gagne D., Nicolas J. C., Mehtali M. A new cellular model of response to estrogens: a bioluminescent test to characterize (anti) estrogen molecules. Biotechniques, 9: 450-459, 1990.[Medline]
  44. Demirpence E., Duchesne M. J., Badia E., Gagne D., Pons M. MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity. J. Steroid Biochem. Mol. Biol., 46: 355-364, 1993.[Medline]
  45. Brunk C. F., Jones K. C., James T. W. Assay for nanogram quantities of DNA in cellular homogenates. Anal. Biochem., 92: 497-500, 1979.[Medline]
  46. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65: 55-63, 1983.[Medline]
  47. Jakowlew S. B., Breathnach R., Jeltsch J. M., Masiakowski P., Chambon P. Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF-7. Nucleic Acids Res., 12: 2861-2878, 1984.[Abstract/Free Full Text]
  48. Augereau P., Garcia M., Mattei M. G., Cavailles V., Depadova F., Derocq D., Capony F., Ferrara P., Rochefort H. Cloning and sequencing of the 52K cathepsin D complementary deoxyribonucleic acid of MCF7 breast cancer cells and mapping on chromosome 11. Mol. Endocrinol., 2: 186-192, 1988.[Abstract/Free Full Text]
  49. Cavailles V., Garcia M., Rochefort H. Regulation of cathepsin-D and pS2 gene expression by growth factors in MCF7 human breast cancer cells. Mol. Endocrinol., 3: 552-558, 1989.[Abstract/Free Full Text]
  50. Torczynski R. M., Fuke M., Bollon A. P. Cloning and sequencing of a human 18S ribosomal RNA gene. DNA, 4: 283-291, 1985.[Medline]
  51. Scatchard G. The attraction of proteins for small molecules and ions. Ann. N. Y. Acad. Sci., 51: 660-672, 1949.
  52. Berthois Y., Katzenellenbogen J. A., Katzenellenbogen B. S. Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc. Natl. Acad. Sci. USA, 83: 2496-2500, 1986.[Abstract/Free Full Text]
  53. Jeng M. H., Langan-Fahey S. M., Jordan V. C. Estrogenic actions of RU486 in hormone-responsive MCF-7 human breast cancer cells. Endocrinology, 132: 2622-2630, 1993.[Abstract/Free Full Text]
  54. Lippman M., Bolan G., Huff K. The effects of glucocorticoids and progesterone on hormone-responsive human breast cancer in long-term tissue culture. Cancer Res., 36: 4602-4609, 1976.[Abstract/Free Full Text]
  55. Osborne C. K., Monaco M. E., Kahn C. R., Huff K., Bronzert D., Lippman M. E. Direct inhibition of growth and antagonism of insulin action by glucocorticoids in human breast cancer cells in culture. Cancer Res., 39: 2422-2428, 1979.[Abstract/Free Full Text]
  56. Zhou F., Bouillard B., Pharaboz-Joly M. O., Andre J., Lippman M., Bolan G., Huff K. Non-classical antiestrogenic actions of dexamethasone in variant MCF-7 human breast cancer cells in culture. Mol. Cell. Endocrinol., 66: 189-197, 1989.[Medline]
  57. Hundertmark S., Buhler H., Rudolf M., Weitzel H. K., Ragosch V., Lippman M., Bolan G., Huff K. Inhibition of 11ß-hydroxysteroid dehydrogenase activity enhances the antiproliferative effect of glucocorticosteroids on MCF-7 and ZR-75-1 breast cancer cells. J. Endocrinol., 155: 171-180, 1997.[Abstract/Free Full Text]
  58. Grunstein M. Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell, 93: 325-328, 1998.[Medline]
  59. Strahl B. D., Allis C. D. The language of covalent histone modifications. Nature (Lond.), 403: 41-45, 2000.[Medline]
  60. Turner B. M. Histone acetylation and an epigenetic code. Bioessays, 22: 836-845, 2000.[Medline]
  61. Jackson T. A., Richer J. K., Bain D. L., Takimoto G. S., Tung L., Horwitz K. B. The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT. Mol. Endocrinol., 11: 693-705, 1997.[Abstract/Free Full Text]
  62. Shang Y., Hu X., DiRenzo J., Lazar M. A., Brown M. Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell, 103: 843-852, 2000.[Medline]
  63. Underhill C., Qutob M. S., Yee S. P., Torchia J. A novel nuclear receptor corepressor complex, N-CoR, contains components of the mammalian SWI/SNF complex and the corepressor KAP-1. J. Biol. Chem., 275: 40463-40470, 2000.[Abstract/Free Full Text]
  64. Guenther M. G., Barak O., Lazar M. A. The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3. Mol. Cell. Biol., 21: 6091-6101, 2001.[Abstract/Free Full Text]
  65. Nielsen A. L., Ortiz J. A., You J., Oulad-Abdelghani M., Khechumian R., Gansmuller A., Chambon P., Losson R. Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family. EMBO J., 18: 6385-6395, 1999.[Medline]
  66. Ryan R. F., Schultz D. C., Ayyanathan K., Singh P. B., Friedman J. R., Fredericks W. J., Rauscher F. J., III. KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Kruppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing. Mol. Cell. Biol., 19: 4366-4378, 1999.[Abstract/Free Full Text]
  67. Lechner M. S., Begg G. E., Speicher D. W., Rauscher F. J., III. Molecular determinants for targeting heterochromatin protein 1-mediated gene silencing: direct chromoshadow domain-KAP-1 corepressor interaction is essential. Mol. Cell. Biol., 20: 6449-6465, 2000.[Abstract/Free Full Text]
  68. Czermin B., Schotta G., Hulsmann B. B., Brehm A., Becker P. B., Reuter G., Imhof A. Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila. EMBO Rep., 2: 915-919, 2001.[Medline]
  69. Ferreira R., Naguibneva I., Pritchard L. L., Ait-Si-Ali S., Harel-Bellan A. The Rb/chromatin connection and epigenetic control: opinion. Oncogene, 20: 3128-3133, 2001.[Medline]
  70. Lachner M., O’Carroll D., Rea S., Mechtler K., Jenuwein T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature (Lond.), 410: 116-120, 2001.[Medline]
  71. Bannister A. J., Zegerman P., Partridge J. F., Miska E. A., Thomas J. O., Allshire R. C., Kouzarides T. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature (Lond.), 410: 120-124, 2001.[Medline]
  72. Nielsen S. J., Schneider R., Bauer U. M., Bannister A. J., Morrison A., O’Carroll D., Firestein R., Cleary M., Jenuwein T., Herrera R. E., Kouzarides T. Rb targets histone H3 methylation and HP1 to promoters. Nature (Lond.), 412: 561-565, 2001.[Medline]
  73. Vandel L., Nicolas E., Vaute O., Ferreira R., Ait-Si-Ali S., Trouche D. Transcriptional repression by the retinoblastoma protein through the recruitment of a histone methyltransferase. Mol. Cell. Biol., 21: 6484-6494, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Endocrinol.Home page
H. Leong, J. R. Sloan, P. D. Nash, and G. L. Greene
Recruitment of Histone Deacetylase 4 to the N-Terminal Region of Estrogen Receptor {alpha}
Mol. Endocrinol., December 1, 2005; 19(12): 2930 - 2942.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. Z. Fang, D. Chen, Y. Sun, Z. Jin, J. K. Christman, and C. S. Yang
Reversal of Hypermethylation and Reactivation of p16INK4a, RAR{beta}, and MGMT Genes by Genistein and Other Isoflavones from Soy
Clin. Cancer Res., October 1, 2005; 11(19): 7033 - 7041.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Demirpence, E.
Right arrow Articles by Pons, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Demirpence, E.
Right arrow Articles by Pons, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online