Cancer Research Targets Frontiers in Basic Cancer Research
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 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 Wang, L. H.
Right arrow Articles by Farrar, W. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, L. H.
Right arrow Articles by Farrar, W. L.
[Cancer Research 63, 2046-2051, May 1, 2003]
© 2003 American Association for Cancer Research

Biochemistry and Biophysics

The cis Decoy against the Estrogen Response Element Suppresses Breast Cancer Cells via Target Disrupting c-fos not Mitogen-activated Protein Kinase Activity1

Li Hua Wang2, Xiao Yi Yang, Xiaohu Zhang, Kelly Mihalic, Weihua Xiao and William L. Farrar

Intramural Research Support Program, Science Applications International Corporation-Frederick, National Cancer Institute-Frederick, Frederick, Maryland 21702 [L. H. W., X. Y. Y., X. Z., K. M., W. X.], and Cytokine Molecular Mechanisms Section, Laboratory of Molecular Immunoregulation, National Cancer Institute-Frederick, National Institutes of Health, Frederick, Maryland 21702 [W. L. F.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast cancer, the most common malignancy in women, has been demonstrated to be associated with the steroid hormone estrogen and its receptor (ER), a ligand-activated transcription factor. Therefore, we developed a phosphorothiolate cis-element decoy against the estrogen response element (ERE decoy) to target disruption of ER DNA binding and transcriptional activity. Here, we showed that the ERE decoy potently ablated the 17ß-estrogen-inducible cell proliferation and induced apoptosis of human breast carcinoma cells by functionally affecting expression of c-fos gene and AP-1 luciferase gene reporter activity. Specificity of the decoy was demonstrated by its ability to directly block ER binding to a cis-element probe and transactivation. Moreover, the decoy failed to inhibit ER-mediated mitogen-activated protein kinase signaling pathways and cell growth of ER-negative breast cancer cells. Taken together, these data suggest that estrogen-mediated cell growth of breast cancer cells can be preferentially restricted via targeted disruption of ER at the level of DNA binding by a novel and specific decoy strategy applied to steroid nuclear receptors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast cancer accounts for more deaths of American women than any other malignancy (1) . Current therapy for primary breast cancer includes surgical resection with or without radiation or chemotherapy. Conventional adjuvant chemotherapy is suboptimal because it is associated with significant toxicity, and it may benefit only 20–25% of patients. This has motivated considerable effort toward finding novel therapeutic approaches for breast cancer (2, 3, 4, 5) .

ER,3 which belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors (6, 7, 8) , plays a predominant role in estrogen-dependent breast cancer development and progression so that clinical targeting of the receptor has become an important treatment strategy (9 , 10) . ER can regulate gene transcription either by binding directly to the promoter of target genes or by binding indirectly through a mechanism involving other transcription factors (8 , 11, 12, 13) . Once the ER has bound estrogen and dimerized, it binds to ERE with a consensus sequence of 5'-GGTCAnnnTGACC-3' in the promoter region of genes. The ERE functions by initiating or enhancing the transcription potential of genes. Thus, disrupting DNA binding of ER could have a critical impact on the transcriptional role of ER.

TFDs have been reported as a powerful tool in a new class of antigene strategies for gene therapy (14, 15, 16) . Intracellular delivery of such decoys results in the attenuation of authentic cis-trans interactions, leading to the removal of trans-factors from the endogenous cis-element, with subsequent modulation of gene expression. Therefore, the decoy approach may also permit treatment diseases by the modulation of endogenous transcriptional regulation. Among the TFDs reported to date, E2F decoy has undergone the most extensive evaluation with successful completion of preclinical and toxicology studies, as well as Phase I/II human trials (17 , 18) . Previously, we (19) have identified a cis-element decoy against transcriptional factor Stat6 that potently blocks Stat6 signaling pathway, thus reducing the interleukin 4-induced proliferation of T helper cells in allergic diseases. Cho-Chung et al. (20, 21, 22) reported the CRE-TFD inhibits the growth of MCF-7 cells through the activation of the p53 signaling pathway. However, CRE binding protein is a member of the CRE binding protein/activating transcription factor family that can activate CRE-transcription in response to cyclic AMP, Ca2+, and growth factor stimulation, which can occur in both normal and cancer cells. Piva et al. (23, 24, 25) studied modulation of ER gene expression using the decoy with specific PCR-generated DNA fragment or a decoy against the upstream promoter of the ER gene. In the case of breast cancer, the steroid hormone estrogen and its receptor ER are specifically associated with development and progression. After ERE binding, the liganded ER activates transcription by an as yet unknown mechanism(s), resulting in stimulation of proliferation (26) . It is reasonable to hypothesize that cis-element decoy against the ERE may functionally interfere with ER DNA binding, subsequently suppressing breast cancer cells.

In this study, we developed a phosphorothiolate cis-element decoy against the ERE to target disruption of ER DNA binding and transcriptional activity, thus influencing estrogen-responsive gene expression and cell proliferation or apoptosis of human breast cancer cells. Moreover, the ERE decoy is proving to be an effective molecular tool to directly assess genes that could link ER/DNA interactions with cell growth.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
ODNs were synthesized, and sequence targets were selected. The ERE decoy is a double-stranded phosphorothioate 55 mer that exhibits a high sequence-specific binding affinity to the ER. Sequences used were as following: ERE decoy ODN, 5'-TAATAGGTCACAGTGACCTAGGTCACAGTGACCTAGGTCACAGTGACCTGATTCC-3' and 5'-GGAATCAGGTCACTGTGACCTAGGTCACTGTGACCTAGGTCACTGTGACCTATTA-3'; scramble decoy ODN, 5'-ATGGCATCATGACTAAGCATGTAGCTCGATTCGGCATAGTCAATCCGACATCCGG-3' and 5'-CCGGATGTCGGATTGACTATGCCGAATCGAGCTACATGCTTAG TCATGATGCCAT-3'. Synthetic ODNs were dissolved in sterile TE buffer [10 mM Tris, 1 mM EDTA (pH 8.0)], purified by high-performance liquid chromatography and quantitated by spectrophotometry (Operon Tec., Alameda, CA). Each pair of single-stranded ODN was annealed for 3 h, during which time the temperature was reduced from 90°C to 25°C. Tissue culture materials were purchased from Life Technologies, Inc. (Gaithersburg, MD). E2 and antiactin were obtained from Sigma (St. Louis, MO). The antibodies against c-Fos or ERK1/2 were purchased from Upstate Biotechnology (Lake Placid, NY). Recombinant human ER{alpha} and RXRß were purchased from ABR affinity Bioresearch, INC. (Golden, CO).

Cell Culture.
The human breast carcinoma cell lines MCF-7 and MDA-MB-231 were obtained from American Type Culture Collection and maintained in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, penicillin-streptomycin (50 IU/ml and 50 µg/ml, respectively). The cells were deprived of serum for 24 h before stimulation. In experiments with E2, cells were cultured in phenol red-free RPMI 1640 (27) .

Proliferation Assays.
Cell proliferation was examined by measuring DNA synthesis using tritiated thymidine (3H-dThd) uptake (28) . After transfection with the ERE or scrambled decoy ODN, the MCF-7 cells (50 x 104/well) were cultured in an 96-well microtiter plate in 200 µl of growth media using 5% FCS in the presence or absence of E2 (50 nM) for 16 h, pulsed for the remaining 4 h with [3H]thymidine (0.5 µCi/200 µl). [3H]Thymidine incorporation was analyzed by liquid scintillation counting.

ELISA to Detect Apoptosis.
The cell death detection ELISA kit (Roche Diagnostics, Mannheim, Germany) was used to measure cytoplasmic histone-associated DNA fragment that result from the induction of apoptosis (29) , according to the manufacturer’s instructions.

EMSA.
The end-labeled [32P] ODN probes correspond to the ERE consensus sequence: 5'-GATCCGTCAGGTCACAG-TGACC-TGATGGATC-3' (27) or the RXRE consensus sequence: 5'-AGCTTCAGGTCAGAGGTCAGA-GAGCT-3', respectively. The probe was incubated with 5 µg of nuclear extracted proteins in 15 µl of binding mixture [50 mM Tris-Cl (pH 7.4), 25 mM MgCl2, 0.5 mM DTT, and 50% glycerol] at 4°C for 15 min. For supershift assay, the nuclear extracts were preincubated with 1 µg of either normal rabbit serum or the specific antibodies to ER or RXRß at 4°C for 30 min. The DNA-protein complexes were resolved on a 5% polyacrylamide gel. The dried gels were exposed to X-ray film (19) .

Transfection of Luciferase Reporter Plasmids.
Using the manufacturer’s instructions, FuGene-6 (Roche Diagnostics) was used to cotransfect the ERE-TK or RXRE-TK reporter plasmid and ERE decoy or scramble into cells (27) . After transfection for 6 h, cells were followed by incubation in phenol red-free medium with or without 50 nM E2 for 16 h. Cell extracts were prepared using the reporter lysis buffer and measured by a luminometer (Monolight 3010; PharMingen, San Diego, CA). All of the experiments were carried out in quadruplicate. The firefly luciferase activity was normalized with the Renilla activity (transfection efficiency) and with the protein content.

RPAs.
Total RNA was isolated using TRIzol from treated and control cells. The mRNA was examined by RPA (27) using 20 µg of total RNA hybridized to 2 x 106 cpm of 33P-labeled probe corresponding to the multiprobe template sets hStress-1 overnight at 56°C. Unhybridized RNA was digested with RNase T1 and RNase A for 45 min at 30°C, then digested with proteinase K for 15 min at 37°C. After phenol/chloroform extraction and sodium acetate/ethanol precipitation, hybridized RNA probes were denatured at 90°C for 3 min and electrophoresed on a 5% polyacrylamide gel. The dried gels were exposed to X-ray film.

Western Blot Analysis.
The cells (108 cells/ml) treated with the ERE or scrambled decoy ODN were solubilized in lysis buffer. Cell lysates were boiled in SDS sample buffer and subjected to 7.5% SDS-PAGE. All proteins were transferred to Immobilion-P (polyvinylidene difluoride) membrane. Western blotting was performed by the mouse antihuman monoclonal c-Fos, antiactin, or ERK1/2 antibody that was diluted 1:1000 in blocking buffer (28 , 30) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The ERE Decoy Inhibits E2-mediated Cell Proliferation and Induces Apoptosis of Human Breast Cancer Cells.
The ER mediates breast cell proliferation and is the principal target for chemotherapy of breast carcinoma (31, 32, 33) . Therefore, we first explored whether the ERE decoy could block estrogen-mediated MCF-7 cell growth. For this assay, the MCF-7 cells were treated with ERE decoy or scramble and stimulated by 50 nM E2. As presented in Fig. 1ACitation , the ERE decoy inhibited the E2-stimulated [3H]thymidine incorporation of MCF-7 cells. In contrast, the scramble did not inhibit this cell growth stimulated by E2. Moreover, fluorescence-activated cell sorting showed the ERE decoy, but not the scramble ODN, also significantly inhibits BrdUrd incorporation and cell cycle progression (data not shown). These findings suggest that the ERE decoy effectively inhibits the E2-stimulated cell proliferation of breast cancer.



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. ERE decoy inhibits E2-mediated MCF-7 cell proliferation. Proliferation of quiescent MCF-7 cells (A) and MDA-MB-231 (B; 5 x 104/well) was examined after treatment with ({blacksquare}) or without () 50 nM E2 for 16 h at 37°C. Cells were then pulsed with [3H]thymidine (0.5 µCi/200 µl) for 4 h and incorporation of radiolabeled probe plotted on the ordinate expressed as total cpm (n = 6). C, ERE decoy induces apoptosis of MCF-7 cells. MCF-7 cells were treated by ERE decoy or scramble ODN in the presence of E2 for 48 h. The formation of cytoplasmic nucleosomal DNA was quantitatively measured by a cell death detection ELISA with cell lysates.

 
To identify if such inhibition of the ERE decoy on the human breast cancer cells may be dependent on the expression of ER, we also tested the effect of the ERE decoy on the ER-negative MDA-MB-231 breast cancer cells. Fig. 1BCitation illustrated the ER ligand E2 could not stimulate [3H]thymidine incorporation of this cell line. In addition, the ERE decoy could not affect [3H]thymidine incorporation. These data indicate such inhibition of the ERE decoy on the human breast cancer cells is dependent on the expression of ER.

To further evaluate whether these cells treated by ERE decoy for longer time periods eventually undergo apoptosis, we used a cell death detection ELISA to assay cytoplasmic histone-associated DNA fragments. Fig. 1CCitation showed treatment of ERE decoy, but not scramble ODNs, for 48 h could induce MCF-7 cell apoptosis in a dose-dependent manner even in the presence of E2, which is consistent with previous observations (34, 35, 36) on MCF-7 cells undergoing apoptosis after estrogen withdrawal.

ERE Decoy Specifically Binds to ER in Vitro.
The ER typically activates gene transcription by binding to ERE (37 , 38) . To examine whether the interaction of the ER with ERE decoy can result in loss of specific binding to ERE, we carried out an EMSA using the recombinant ER{alpha} protein. Fig. 2ACitation illustrates formation of an ER protein:ERE DNA probe complex (Lane a), which could be partially supershifted with anti-ER (Lane b) and 100-fold of the cold probe (Lane f) but not by the normal rabbit serum (Lane c) confirming its identity, indicating that recombinant ER can actively bind to the specific ERE sequences. Notably, incubation of recombinant ER with ERE decoy (Lane d), but not scramble (Lane e), results in substantial decrease in the ability of ER binding to ERE.



View larger version (61K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Binding site specificity of ERE decoy. A, the purified ER{alpha} protein was incubated with anti-ER{alpha} (Lane b), normal rabbit serum (Lane c), ERE decoy (Lane d), scramble (Lane e), or 100-fold of cold ERE ODN (Lane f) in combination with [32P]-labeled ERE ODN probe. Arrows indicate migrational location of each nonsupershifted ER-DNA complex or free probe. B, the purified RXRß protein was incubated with anti-RXRß (Lane b), normal rabbit serum (Lane c), ERE decoy (Lane d), scramble (Lane e), or 100-fold of cold RXRE ODN (Lane f) in combination with [32P]-labeled RXRE ODN probe. Arrows indicate migrational location of each nonsupershifted RXR-DNA complex or free probe.

 
To additionally confirm the specificity of ERE decoy, we also performed an EMSA assay to test if the ERE decoy can affect RXR binding to its response element. Fig. 2BCitation illustrates that recombinant RXRß can specifically bind to the DNA probe containing specific response element sequences (Lane a), which could be partially supershifted with anti-RXRß (Lane b) and 100-fold of the cold probe (Lane f), but not by the normal rabbit serum (Lane c), confirming its identity. Neither an ERE decoy (Lane d), nor a scramble (Lane e) affected the recombinant RXRß protein binding to RXRE. Thus, the double-stranded phosphorothioate ERE decoy has unique ability to specifically bind to ER rather than other nuclear receptors such as RXR.

The ERE Decoy Specifically Blocks E2-induced ER DNA Binding Activity in Breast Cancer Cells in Vivo.
DNA binding and transactivation of ER is critical for ligand-activated ER to regulate gene expression of breast cancer cells. Therefore, to clarify if the ERE decoy alters ER ability to bind to its cognate DNA ERE, we performed EMSA assays to test the effect of ERE decoy on the DNA binding activity of ER in breast cancer cell. Nuclear extracts prepared from MCF-7 cells treated with the ERE decoy in the presence of E2 or control cells were tested for their ability to bind a radiolabeled ERE ODN probe. As shown in Fig. 3ACitation , nuclear extracts obtained from E2-stimulated MCF-7 cells displayed considerable ERE DNA binding activity (Lane b) as compared with equivalent protein samples obtained from non-E2 treated cells (Lane a). These E2-inducible DNA-protein complexes could be partially supershifted with anti-ER (Lane c), but not normal rabbit serum (Lane d), confirming its identity. The E2-induced ERE DNA binding was significantly decreased by the treatment of the ERE decoy (Lane e) but not by the scramble (Lane f). Therefore, the ERE decoy specifically blocks ER DNA binding activity in MCF-7 breast cancer cells.



View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. A, ERE decoy inhibit E2-induced ERE DNA binding in vivo. MCF-7 cells were transfected with an ERE decoy or scramble ODN for 6 h. Cells were then stimulated with medium (-) or 50 nM E2 (+) for 40 min. Nuclear extracts corresponding to 5 µg of protein were incubated in the absence of antibody (Lanes a, e, and f), or in presence of {alpha}-ER (Lane c), normal rabbit serum (Lane d), ERE decoy (Lane e), or scramble (Lane f) in combination with a [32P]-labeled ERE ODN probe. Arrows indicate migrational location of each nonsupershifted ER-DNA complex or free probe. B, ERE decoy inhibits E2-induced ERE transactivation. MCF-7 cells were cotransfected with a 3x ERE-TK luciferase reporter and ERE decoy or scramble ODN for 6 h. Cells were then incubated without or with E2 (50 nM) for 16 h. Luciferase activity of lysed cells was measured and normalized. C, ERE decoy did not inhibit RXR transactivation in cultured MCF-7 cells. MCF-7 cells were cotransfected with a 3x RXRE-TK luciferase reporter and an ERE decoy or control scramble. Cells were then incubated with or without RXR ligands 9-cis-RA for 16 h. Luciferase activity of lysed cells was measured and normalized.

 
ERE Decoy Specifically Blocks E2-induced ER Transactivation in Breast Cancer Cells.
To assess whether the ERE decoy blocks the transactivation potential of ER in the human breast cancer cells, we transfected the ERE-luciferase reporter gene construct into MCF-7 cells to quantitatively analyze the effect of the ERE decoy on the E2-stimulated ER transcriptional activation. As shown in Fig. 3BCitation , E2 induced high ERE luciferase activity in MCF-7 cells. ERE decoy substantially reduced transcription activity of ERE stimulated by E2. In contrast, the scramble ODN failed to show the inhibitory effect on transactivation of ERE. These observations suggest that the ERE decoy inhibits ERE DNA binding and subsequent transcriptional activity.

To additionally confirm the specificity of the ERE decoy on the ER transactivation, we also transfected another nuclear receptor, RXR luciferase reporter, into the MCF-7 cells. As shown in Fig. 3CCitation , 9-cis-RA, a RXR-specific ligand, could markedly activate the transcription of RXR gene reporter. However, after treatment of the ERE decoy, inhibition of transactivation of RXR luciferase reporter was not observed. These data suggest the ERE decoy specifically targets inhibition of ER transcription activity.

c-fos Is an Estrogen-responsive Gene Target for ERE Decoy Inhibition of MCF-7 Cell Proliferation.
It has been reported that ERE, a minimum length sequence, has two essential qualities, the ability to bind in a stable complex with ER and to mediate the induction of estrogen-responsive genes (38 , 39) . Several lines of evidence indicate that c-fos proto-oncogene induction by estrogens is a direct effect and, therefore, most likely mediated by the ER. An ERE that binds the ER and confers transcriptional activation to a heterologous promoter has been reported in the 5-flanking region of the human c-fos gene (40 , 41) . Therefore, we investigated the effect of ERE decoy on AP-1 promoter activity and c-fos gene expression. As shown in Fig. 4ACitation , E2 markedly stimulated the c-fos mRNA expression, which is consistent with previous observations (42, 43, 44) . Transfection of ERE decoy resulted in significant inhibition of the c-fos mRNA expression stimulated by estrogen compared with treatment with the scramble ODN. Similarly, decrease in c-Fos protein expression resulting from transfection with ERE decoy is displayed in Fig. 4BCitation . Moreover, we cotransfected ERE decoy or scramble and AP-1 luciferase reporter into MCF-7 cells. As expected, transfection of ERE decoy, but not scramble, significantly reduced estrogen-induced AP-1 luciferase activity (Fig. 4C)Citation . These data suggested c-fos is a target estrogen-responsive gene for ERE decoy suppression of MCF-7 cell proliferation.



View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. c-Fos is a target for ERE decoy inhibition of MCF-7 cell proliferation. A, ERE decoy decreases the gene expression of c-fos. RPA analysis of mRNA was obtained from MCF-7 cells that were transfected with ERE decoy or control scramble at 37°C for 6 h and then stimulated with medium or 50 nM E2 for 1 h. MCF-7 cells mRNA were then hybridized with [33P]-labeled RNA probes corresponding to transcripts for individual human c-fos (hSTRESS-1) according to PharMingen Protocol (see "Materials and Methods"). The autoradiograph of the RNase protected fragments were separated on 5% PAGE is shown. B, MCF-7 cells were transfected with ERE decoy or control scramble at 37°C for 6 h and then stimulated with medium or 50 nM E2 for 6 h. Cells were lysed and Western blotted with anti-c-fos or antiactin. Arrows indicate location of c-fos. (C) ERE decoy inhibits AP-1 luciferase gene reporter activity. MCF-7 cells were cotransfected with a 3x AP-1 binding element-pGL3 promoter luciferase construct and an ERE decoy or control scramble. Cells were then stimulated with or without E2 (50 nM) for 16 h. Luciferase activity of lysed cells was measured and normalized.

 
ERE Decoy Does not Affect Activation of MAPK.
Estrogen activation of the MAPKs (45) , Erk1 and Erk2, occurs in a variety of cell types. These studies indicate an involvement of the MAPK cascade in the proliferative response to E2 stimulation. Activation of MAPK family members Erk1 and Erk2 is triggered by the phosphorylation of a threonine and a tyrosine residue in their regulatory site. To determine whether MAPK inactivation may account for ERE inhibition of the proliferation regulated by E2, we assayed the effect of ERE decoy on the phosphorylation of Erk1 and Erk2 in the MCF-7 cells. As shown in Fig. 5Citation , top panel, ERE decoy does not affect the phosphorylation of Erk1 and Erk2, although estrogen (50 nM) could up-regulate phosphorylation of these kinases. The blot was stripped and total level of Erk1 and Erk2 were detected, which displayed same loading levels (Fig. 5Citation , bottom panel). These results indicate the inhibitory effect of ERE decoy on estrogen-mediated cell proliferation may not be mediated through inactivation of MAPK pathway.



View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. ERE decoy does not affect activation of MAPK. Top panel, cell lysates were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against dually phosphorylated Erk1 and Erk2 (pErk1 and pErk2). Bottom panel, the blot was stripped, and total levels of Erk1 and Erk2 were detected using an antibody recognizing Erk1/2. Arrows indicate location of Erk1 and Erk2.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The ERE Decoy Provides a Novel Molecular Intervention Approach to Estrogen-mediated Cell Growth and Apoptosis of Breast Cancer.
Synthetic ODNs with high affinity for a target transcription factor can be introduced into target cells as decoy cis-elements to bind the factor and alter gene transcription (14, 15, 16, 17 , 46, 47, 48) . Morishita et al. (49 , 50) demonstrated the usefulness of the decoy approach in a rat model of restenosis. ER is a typical steroid nuclear receptor and functions as a ligand-activated transcriptional factor. Penolazzi et al. (23 , 24) and Piva et al. (25) analyzed putative regulatory sequences localized inside P1 canonical promoter and P3 upstream promoter of ER gene by using the decoy strategy. Using the human breast cancer cell line ER-positive MCF-7, we first identified the effect of a cis-element decoy against the DNA binding element of ER on estrogen-mediated cell growth or apoptosis of human breast cancer cells. The specificity of ERE decoy on ER DNA binding and transactivation was characterized by using gel shift analysis and reporter gene assays. ERE decoy, but not scramble ODN, blocked the purified recombinant ER protein specifically bind to the ERE sequence in vitro. This inhibitory effect of ERE decoy on ER DNA binding and transactivation was also confirmed in the human breast cancer cells in vivo. However, ERE decoy failed to show significant inhibition on DNA binding and transactivation of the different class of nuclear receptor RXR. More importantly, specificity of ERE decoy on ER DNA binding domain was supported by its functional inability to affect cell growth of MDA-MB-231, an ER-negative cell line. Therefore, we believe that the ERE decoy may specifically react with the ERE sequences of target genes, leading to the significant loss of DNA binding and transactivation of ER. This event subsequently inhibits the ER-mediated gene expression and cell growth of the human breast cancer. The effectiveness of ERE decoy in breast cancer cells prompted the consideration of its use in therapy. The duration of TFD action is dependent, to a large extent, on the intracellular stability of the decoy ODN (14) . The ERE decoy we used here is a double-stranded linear DNA with phosphothioation, which increases resistance to nuclease digestion. Moreover, the decoy at high concentration after direct exposure indeed inhibited MCF-7 cell proliferation (data not shown). Careful and systemic planning and improvement of this technology by addressing the method and timing of delivery, ODN stability and specificity of action (14 , 46 , 48) will facilitate the development of the ERE decoy as an important therapeutic modality for human breast cancer.

ERE Decoy Inhibits Breast Cancer Cell Proliferation via Down-Regulating c-fos Gene Expression but not Inactivation of MAPK.
EREs have been identified in hormone-responsive genes such as c-fos (41 , 42) and c-jun (51 , 52) but not c-myc (53) , which bind the ER and are well suited to amplify tissue responses emanating from the initial ER-ERE interactions (54) . Here, we provide the direct evidence that expression of c-fos gene is linked with ERE transcription by specifically blocking ERE DNA binding. Moreover, ERE decoy significantly reduced AP-1 luciferase activity. c-fos plays a key role in regulating cell proliferation and cell cycle entry through activation of cyclin D1. Crowe et al. (55) reported that c-fos function (i.e., cell cycle progression) is mediated at least, in part, by transcriptional down-regulation of p21Cip1/WAF1using a c-fos/ER fusion construct in which this transcription factor is conditionally activated by E2.

Recently, several studies have indicated an involvement of the MAPK cascade in the proliferative response to E2 stimulation (56 , 57) . However, Lobenhofer and Marks (58) demonstrated that E2 treatment of MCF-7 cells stimulates cell cycle progression in the absence of detectable MAPK activation regardless of E2 or serum concentrations, cell density, or the method of hormone delivery. We have here found the ERE decoy does not affect activation of MAPK signal pathway, although estrogen (50 nM) marked stimulates phosphorylation of MAPK (Fig. 5)Citation . Our observations are in support of the concept that ERE decoy inhibition of E2-induced proliferation is based on the ability of this specific transcriptional factor decoy to attenuate ER binding to estrogen-responsive genes (c-fos) essential for mitogenesis rather than inactivation of the established mitogenic signaling pathway.

It is worthwhile to mention that PPAR{gamma} may also be involved in the effect of ERE decoy on breast cancer cells because the PPRE also contains an AGGTCA half-site. The ERE and PPRE differ in the topological arrangement of these half-sites. The ERE is a palindrome, whereas the PPRE is a direct repeat. Moreover, the affinity of the PPAR/RXR heterodimer for the ERE is lower than for the PPRE (59) . Wahli (59 , 60) and Wang (61) observed signaling cross-talk between PPAR/RXR and ER through ERE. More importantly, PPAR activation significantly inhibits cell growth of breast cancer cells (62) . Therefore, whether the ERE decoy holds up against PPAR{gamma} in breast cancer cells awaits additional investigation.

Taken together, this is the first demonstration that transaction factor decoy may affect steroid-dependent cell growth or apoptosis of the human breast cancer and provides the proof of principle for a possible molecular intervention.


   ACKNOWLEDGMENTS
 
We thank Dr. Joost Oppenheim for his critical review of the manuscript. We also thank Dr. Taosheng Chen for kindly providing us with critical plasmids that made this work possible.


   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 This project has been funded in whole or in part with federal funds from the National Cancer Institute/NIH Grant NO1-CO-12400. Back

2 To whom requests for reprints should be addressed, at National Cancer Institute-Frederick, P. O. Box B, Building 560, Room 31-76, Frederick, MD 21702. Phone: (301) 846-6870; Fax: (301) 846-6187; E-mail: lhwang{at}ncifcrf.gov Back

3 The abbreviations used are: ER, estrogen receptor; cAMP, CRE, cyclic AMP-responsive element; E2, 17ß-estradiol; EMSA, electrophoretic mobility shift assay; ERE, estrogen response element; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; ODN, oligodeoxynucleotide; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; PPRE, peroxisome proliferator-activated receptor response element; RPA, RNase protection assay; RXR, retinoic X receptor; RXRE, retinoic X receptor response element; TFD, transcription factor decoy; TK, thymidine kinase. Back

Received 9/13/02. Accepted 2/27/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Menck H. R., Cunningham M. P., Jessup J. M., Eyre H. J., Winchester D. P., Scott-Conner C. E., Murphy G. P. The growth and maturation of the National Cancer Data Base. Cancer (Phila.), 80: 2296-2304, 1997.[Medline]
  2. Jordan V. C. Selective estrogen receptor modulation: a personal perspective. Cancer Res., 61: 5683-5687, 2001.[Free Full Text]
  3. Mueller E., Sarraf P., Tontonoz P., Evans R. M., Martin K. J., Zhang M., Fletcher C., Singer S., Spiegelman B. M. Terminal differentiation of human breast cancer through PPAR{gamma}. Mol. Cell, 1: 465-1470, 1998.[Medline]
  4. Bentrem D. J., O’Regan R. M., Jordan V. C. New strategies for the treatment of breast cancer. Breast Cancer, 8: 265-274, 2001.[Medline]
  5. Carroll J. S., Swarbrick A., Musgrove E. A., Sutherland R. L. Mechanisms of growth arrest by c-myc antisense oligonucleotides in MCF-7 breast cancer cells: implications for the antiproliferative effects of antiestrogens. Cancer Res., 62: 3126-3131, 2002.[Abstract/Free Full Text]
  6. Joffe M. Activation of estrogen receptors. Science (Wash. DC), 274: 1285-1286, 1996.[Free Full Text]
  7. McDonnell D. P., Norris J. D. Connections and regulation of the human estrogen receptor. Science (Wash. DC), 296: 1642-1644, 2002.[Abstract/Free Full Text]
  8. Shang Y., Brown M. Molecular determinants for the tissue specificity of SERMs. Science (Wash. DC), 295: 2465-2468, 2002.[Abstract/Free Full Text]
  9. Norris J. D., Paige L. A., Christensen D. J., Chang C. Y., Huacani M. R., Fan D., Hamilton P. T., Fowlkes D. M., McDonnell D. P. Peptide antagonists of the human estrogen receptor. Science (Wash. DC), 285: 744-746, 1999.[Abstract/Free Full Text]
  10. Brzozowski A. M., Pike A. C., Dauter Z., Hubbard R. E., Bonn T., Engstrom O., Ohman L., Greene G. L., Gustafsson J. A., Carlquist M. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature (Lond.), 389: 753-758, 1997.[Medline]
  11. Shiau A. K., Barstad D., Loria P. M., Cheng L., Kushner P. J., Agard D. A., Greene G. L. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell, 95: 927-937, 1998.[Medline]
  12. Wang W., Dong L., Saville B., Safe S. Transcriptional activation of E2F1 gene expression by 17 ß-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. Mol. Endocrinol., 13: 1373-1387, 1999.[Abstract/Free Full Text]
  13. Safe S. Transcriptional activation of genes by 17ß-estradiol through estrogen receptor-Sp1 interactions. Vitam. Horm., 62: 231-252, 2001.[Medline]
  14. Dzau V. J. Transcription factor decoy. Circ. Res., 90: 1234-1236, 2002.[Free Full Text]
  15. Tomita N., Morishita R., Tomita T., Ogihara T. Potential therapeutic applications of decoy oligonucleotides. Curr. Opin. Mol. Ther., 4: 166-170, 2002.[Medline]
  16. Ahn J. D., Morishita R., Kaneda Y., Lee S. J., Kwon K. Y., Choi S. Y., Lee K. U., Park J. Y., Moon I. J., Park J. G., Yoshizumi M., Ouchi Y., Lee I. K. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ. Res., 90: 1325-1332, 2002.[Abstract/Free Full Text]
  17. Mann M. J., Whittemore A. D., Donaldson M. C., Belkin M., Conte M. S., Polak J. F., Orav E. J., Ehsan A., Dell’Acqua G., Dzau V. J. Ex vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial. Lancet, 354: 1493-1498, 1999.[Medline]
  18. Ehsan A., Mann M. J., Dell’Acqua G., Dzau V. J. Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy oligonucleotide gene therapy. Thorac. Cardiovasc. Surg., 121: 714-722, 2001.
  19. Wang L. H., Yang X. Y., Kirken R. A., Resau J. H., Farrar W. L. Targeted disruption of stat6 DNA binding activity by an oligonucleotide decoy blocks IL-4-driven T(H)2 cell response. Blood, 95: 1249-1257, 2000.[Abstract/Free Full Text]
  20. Lee Y. N., Park Y. G., Choi Y. H., Cho Y. S., Cho-Chung Y. S. CRE-transcription factor decoy oligonucleotide inhibition of MCF-7 breast cancer cells: cross-talk with p53 signaling pathway. Biochemistry, 39: 4863-4868, 2000.[Medline]
  21. Park Y. G., Park S., Lim S. O., Lee M. S., Ryu C. K., Kim I., Cho-Chung Y. S. Reduction in cyclin D1/Cdk4/retinoblastoma protein signaling by CRE-decoy oligonucleotide. Biochem. Biophys. Res. Commun., 281: 1213-1219, 2001.[Medline]
  22. Cho Y. S., Kim M. K., Cheadle C., Neary C., Park Y. G., Becker K. G., Cho-Chung Y. S. A genomic-scale view of the cAMP response element-enhancer decoy: a tumor target-based genetic tool. Proc. Natl. Acad. Sci. USA, 99: 15626-15631, 2002.[Abstract/Free Full Text]
  23. Penolazzi L., Lambertini E., Aguiari G., del Senno L., Piva R. Modulation of estrogen receptor gene expression in human breast cancer cells: a decoy strategy with specific PCR-generated DNA fragments. Breast Cancer Res. Treat., 49: 227-235, 1998.[Medline]
  24. Penolazzi L., Lambertini E., Aguiari G., del Senno L., Piva R. Cis element ’decoy’ against the upstream promoter of the human estrogen receptor gene. Biochim. Biophys. Acta, 1492: 560-567, 2000.[Medline]
  25. Piva R., del Senno L., Lambertini E., Penolazzi L., Nastruzzi C. Modulation of estrogen receptor gene transcription in breast cancer cells by liposome delivered decoy molecules. J. Steroid Biochem. Mol. Biol., 75: 121-128, 2000.[Medline]
  26. Zwijsen R. M. L., Wientjens E., Klompmaker R., van der Sman J., Bernards R., Michalides R. J. A. M. CDK-independent activation of estrogen receptor by cyclin D1. Cell, 88: 405-415, 1997.[Medline]
  27. Wang L. H., Yang X. Y., Mihalic K., Xiao W., Li D., Farrar W. L. Activation of estrogen receptor blocks interleukin-6-inducible cell growth of human multiple myeloma involving molecular cross-talk between estrogen receptor and STAT3 mediated by co-regulator PIAS3. J. Biol. Chem., 276: 31839-31844, 2001.[Abstract/Free Full Text]
  28. Wang L. H., Kirken R. A., Erwin R. A., Yu C. R., Farrar W. L. JAK3, STAT, and MAPK signaling pathways as novel molecular targets for the tyrphostin AG-490 regulation of IL-2-mediated T cell response. J. Immunol., 162: 3897-3904, 1999.[Abstract/Free Full Text]
  29. Frankfurt O. S., Krishan A. Enzyme-linked immunosorbent assay (ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening. J. Immunol. Methods, 253: 133-144, 2001.[Medline]
  30. Yang X. Y., Wang L. H., Mihalic K., Xiao W., Chen T., Li P., Wahl L. M., Farrar W. L. Interleukin (IL)-4 indirectly suppresses IL-2 production by human T lymphocytes via peroxisome proliferator-activated receptor {gamma} activated by macrophage-derived 12/15-lipoxygenase ligands. J. Biol. Chem., 277: 3973-3978, 2002.[Abstract/Free Full Text]
  31. Prall O. W., Rogan E. M., Sutherland R. L. Estrogen regulation of cell cycle progression in breast cancer cells. J. Steroid Biochem. Mol. Biol., 65: 169-174, 1998.[Medline]
  32. Seth P., Porter D., Lahti-Domenici J., Geng Y., Richardson A., Polyak K. Cellular and molecular targets of estrogen in normal human breast tissue. Cancer Res., 62: 4540-4544, 2002.[Abstract/Free Full Text]
  33. MacGregor J. I., Jordan V. C. Basic guide to the mechanisms of antiestrogen action. Pharmacol. Rev., 50: 151-196, 1998.[Abstract/Free Full Text]
  34. Kyprianou N., English H. F., Davidson N. E., Isaacs J. T. Programmed cell death during regression of the MCF-7 human breast cancer following estrogen ablation. Cancer Res., 51: 162-166, 1991.[Abstract/Free Full Text]
  35. Pratt M. A., Krajewski S., Menard M., Krajewska M., Macleod H., Reed J. C. Estrogen withdrawal-induced human breast cancer tumour regression in nude mice is prevented by Bcl-2. FEBS Lett., 440: 403-408, 1998.[Medline]
  36. Detre S., Salter J., Barnes D. M., Riddler S., Hills M., Johnston S. R., Gillett C., A’Hern R., Dowsett M. Time-related effects of estrogen withdrawal on proliferation- and cell death-related events in MCF-7 xenografts. Int. J. Cancer, 81: 309-313, 1999.[Medline]
  37. Klinge C. M. Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res., 29: 2905-2919, 2001.[Abstract/Free Full Text]
  38. Driscoll M. D., Sathya G., Muyan M., Klinge C. M., Hilf R., Bambara R. A. Sequence requirements for estrogen receptor binding to estrogen response elements. J. Biol. Chem., 273: 29321-29330, 1998.[Abstract/Free Full Text]
  39. Klein-Hitpass L., Schorpp M., Wagner U., Ryffel G. U. An estrogen-responsive element derived from the 5' flanking region of the xenopus vitellogenin A2 gene functions in transfected human cells. Cell, 46: 1053-1061, 1986.[Medline]
  40. Weisz A., Rosales R. Identification of an estrogen response element upstream of the human c-fos gene that binds the estrogen receptor and the AP-1 transcription factor. Nucleic Acids Res., 18: 5097-5106, 1990.[Abstract/Free Full Text]
  41. Hyder S. M., Stancel G. M., Nawaz Z., McDonnell D. P., Loose-Mitchell D. S. Identification of an estrogen response element in the 3'-flanking region of the murine c-fos protooncogene. J. Biol. Chem., 267: 18047-18054, 1992.[Abstract/Free Full Text]
  42. Loose-Mitchell D. S., Chiappetta C., Stancel G. M. Estrogen regulation of c-fos messenger ribonucleic acid. Mol. Endocrinol., 2: 946-951, 1988.[Abstract/Free Full Text]
  43. Weisz A., Bresciani F. Estrogen induces expression of c-fos and c-myc protooncogenes in rat uterus. Mol. Endocrinol., 2: 816-824, 1988.[Abstract/Free Full Text]
  44. Duan R., Xie W., Li X., McDougal A., Safe S. Estrogen regulation of c-fos gene expression through phosphatidylinositol-3-kinase-dependent activation of serum response factor in MCF-7 breast cancer cells. Biochem. Biophys. Res. Commun., 294: 384-394, 2002.[Medline]
  45. Collins P., Webb C. Estrogen hits the surface. Nat. Med., 5: 1130-1131, 1999.[Medline]
  46. Mann M. J., Dzau V. J. Therapeutic applications of transcription factor decoy oligonucleotides. J. Clin. Investig., 106: 1071-1075, 2000.[Medline]
  47. Mann M. J., Gibbons G. H., Hutchinson H., Poston R. S., Hoyt E. G., Robbins R. C., Dzau V. J. Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues. Proc. Natl. Acad. Sci. USA, 96: 6411-6416, 1999.[Abstract/Free Full Text]
  48. Morishita R., Aoki M., Kaneda Y. Oligonucleotide-based gene therapy for cardiovascular disease: are oligonucleotide therapeutics novel cardiovascular drugs?. Curr. Drug Targets, 1: 15-23, 2000.[Medline]
  49. Morishita R., Gibbons G. H., Horiuchi M., Ellison K. E., Nakama M., Zhang L., Kaneda Y., Ogihara T., Dzau V. J. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Proc. Natl. Acad. Sci. USA, 92: 5855-5859, 1995.[Abstract/Free Full Text]
  50. Morishita R., Sugimoto T., Aoki M., Kida I., Tomita N., Moriguchi A., Maeda K., Sawa Y., Kaneda Y., Higaki J., Ogihara T. In vivo transfection of cis element "decoy" against nuclear factor-{kappa}B binding site prevents myocardial infarction. Nat. Med., 3: 894-899, 1997.[Medline]
  51. Hyder S. M., Nawaz Z., Chiappetta C., Yokoyama K., Stancel G. M. The protooncogene c-jun contains an unusual estrogen-inducible enhancer within the coding sequence. J. Biol. Chem., 270: 8506-8513, 1995.[Abstract/Free Full Text]
  52. Paech K., Webb P., Kuiper G. G., Nilsson S., Gustafsson J., Kushner P. J., Scanlan T. S. Differential ligand activation of estrogen receptors ER{alpha} and ERß at AP1 sites. Science (Wash. DC), 277: 1508-1510, 1997.[Abstract/Free Full Text]
  53. Dubik D., Watson P. H., Venditti M., Shiu R. P. Mechanism of estrogen activation of c-myc oncogene expression. Oncogene, 7: 1587-1594, 1992.[Medline]
  54. Hyder S. M., Shipley G. L., Stancel G. M. Estrogen action in target cells: selective requirements for activation of different hormone response elements. Mol. Cell. Endocrinol., 112: 35-43, 1995.[Medline]
  55. Crowe D. L., Brown T. N., Kim R., Smith S. M., Lee M. K. A c-fos/estrogen receptor fusion protein promotes cell cycle progression and proliferation of human cancer cell lines. Mol. Cell. Biol. Res. Commun., 3: 243-248, 2000.[Medline]
  56. Lee H. W., Eghbali-Webb M. Estrogen enhances proliferative capacity of cardiac fibroblasts by estrogen receptor- and mitogen-activated protein kinase-dependent pathways. J. Mol. Cell. Cardiol., 30: 1359-1368, 1998.[Medline]
  57. Castoria G., Barone M. V., Di Domenico M., Bilancio A., Ametrano D., Migliaccio A., Auricchio F. Non-transcriptional action of oestradiol and progestin triggers DNA synthesis. EMBO J., 18: 2500-2510, 1999.[Medline]
  58. Lobenhofer E. K., Marks J. R. Estrogen-induced mitogenesis of MCF-7 cells does not require the induction of mitogen-activated protein kinase activity. J. Steroid Biochem. Mol. Biol., 75: 11-20, 2000.[Medline]
  59. Keller H., Givel F., Perroud M., Wahli W. Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements. Mol. Endocrinol., 9: 794-804, 1995.[Abstract/Free Full Text]
  60. Nunez S. B., Medin J. A., Braissant O., Kemp L., Wahli W., Ozato K., Segars J. H. Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor. Mol. Cell. Endocrinol., 127: 27-40, 1997.[Medline]
  61. Wang X., Kilgore M. W. Signal cross-talk between estrogen receptor {alpha} and ß and the peroxisome proliferator-activated receptor gamma1 in MDA-MB-231 and MCF-7 breast cancer cells. Mol. Cell. Endocrinol., 194: 123-133, 2002.[Medline]
  62. Yin F., Wakino S., Liu Z., Kim S., Hsueh W. A., Collins A. R., Van Herle A. J., Law R. E. Troglitazone inhibits growth of MCF-7 breast carcinoma cells by targeting G1 cell cycle regulators. Biochem. Biophys. Res. Commun., 286: 916-922, 2001.[Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. L. Nott, Y. Huang, X. Li, B. R. Fluharty, X. Qiu, W. V. Welshons, S. Yeh, and M. Muyan
Genomic Responses from the Estrogen-responsive Element-dependent Signaling Pathway Mediated by Estrogen Receptor {alpha} Are Required to Elicit Cellular Alterations
J. Biol. Chem., May 29, 2009; 284(22): 15277 - 15288.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
S. Deurveilher, E. M. Cumyn, T. Peers, B. Rusak, and K. Semba
Estradiol replacement enhances sleep deprivation-induced c-Fos immunoreactivity in forebrain arousal regions of ovariectomized rats
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2008; 295(4): R1328 - R1340.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
H. Gao, J. Xiao, Q. Sun, H. Lin, Y. Bai, L. Yang, B. Yang, H. Wang, and Z. Wang
A Single Decoy Oligodeoxynucleotides Targeting Multiple Oncoproteins Produces Strong Anticancer Effects
Mol. Pharmacol., November 1, 2006; 70(5): 1621 - 1629.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. L. Temple and S. Wray
Bovine Serum Albumin-Estrogen Compounds Differentially Alter Gonadotropin-Releasing Hormone-1 Neuronal Activity
Endocrinology, February 1, 2005; 146(2): 558 - 563.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
G. Castoria, A. Migliaccio, M. Di Domenico, M. Lombardi, A. de Falco, L. Varricchio, A. Bilancio, M. V. Barone, and F. Auricchio
Role of Atypical Protein Kinase C in Estradiol-Triggered G1/S Progression of MCF-7 Cells
Mol. Cell. Biol., September 1, 2004; 24(17): 7643 - 7653.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. L. Temple, E. Laing, A. Sunder, and S. Wray
Direct Action of Estradiol on Gonadotropin-Releasing Hormone-1 Neuronal Activity via a Transcription-Dependent Mechanism
J. Neurosci., July 14, 2004; 24(28): 6326 - 6333.
[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 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 Wang, L. H.
Right arrow Articles by Farrar, W. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, L. H.
Right arrow Articles by Farrar, W. L.

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