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Originally published In Press as doi:10.1074/jbc.M704829200 on August 23, 2007

J. Biol. Chem., Vol. 282, Issue 42, 30535-30543, October 19, 2007
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Tamoxifen Induction of CCAAT Enhancer-binding Protein {alpha} Is Required for Tamoxifen-induced Apoptosis*

Jingwei Cheng, David V. Yu, Jian-Hua Zhou1, and David J. Shapiro2

From the Department of Biochemistry, University of Illinois, Urbana, Illinois 61801

Received for publication, June 12, 2007 , and in revised form, August 22, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Low concentrations of tamoxifen or its active metabolite 4-hydroxytamoxifen (OHT) induce estrogen receptor {alpha} (ER{alpha})-dependent apoptosis. To analyze the pathway of OHT-ER{alpha}-induced apoptosis, we developed stably transfected lines of HeLa cells expressing wild-type ER and an inactive mutant ER{alpha} unable to bind estrogen response elements. HeLa cells expressing the mutant ER{alpha} and HeLa cells expressing wild-type ER{alpha} in which the ER was knocked down with an ER-specific small interfering RNA were not killed by Tam or OHT, suggesting that estrogen response element-mediated transcription is required for Tam- and OHT-induced apoptosis. Microarray analysis to identify a gene(s) whose expression is important in OHT-ER-mediated apoptosis identified 19 mRNAs that OHT up-regulated by >1.6-fold and 15 down-regulated mRNAs. Gene function and the time course of induction by OHT-ER{alpha} led us to further investigate CCAAT enhancer-binding protein {alpha} (C/EBP{alpha}), which has roles in cell cycle progression and apoptosis, and p21. Quantitative reverse transcription-PCR, Western blot analysis, and RNA interference knockdown suggest that cell cycle arrest resulting from OHT-ER{alpha} induction of p21 may facilitate apoptosis. OHT-ER{alpha}, but not E2-ER{alpha}, induced C/EBP{alpha} mRNA and protein. RNA interference knockdown of C/EBP{alpha} nearly abolished OHT-ER{alpha}-induced apoptosis. We isolated stable cell lines that were resistant to OHT-induced apoptosis, contain full-length functional ER{alpha}, and undergo apoptosis in response to etoposide. In these OHT-resistant cell lines both before and after OHT treatment, C/EBP{alpha} levels are much lower than in OHT-sensitive cells. These studies establish a novel molecular site responsible for Tam- and OHT-ER{alpha}-induced apoptosis of cancer cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogens, acting through estrogen receptor {alpha} (ER{alpha})3 and ERbeta, exert pleiotropic effects on diverse cells and organ systems. Binding of a potent estrogen such as 17beta-estradiol (E2) to the ER may induce dissociation of ER from a heat shock protein/chaperone complex (1) and enable the ER to dimerize and bind to specific DNA sequences termed estrogen response elements (EREs) (2). Transcription activation by ERs is mediated by two interacting activation functions, AF1 and AF2. AF1 mediates ligand-independent transactivation and is thought to be especially important in transcription by the clinically important selective estrogen receptor modulator, tamoxifen (Tam), and by its active metabolite, 4-hydroxytamoxifen (OHT). AF2 activity depends on estrogen binding to the ER ligand binding domain (LBD) (3–7). When bound to estrogen, the ER LBD assumes a conformation that enables the recruitment of coactivators. The bound coactivators help assemble a multiprotein complex that facilitates both chromatin remodeling and formation of an active transcription complex (8). When bound to Tam or OHT, the ligand binding domain of ER assumes a different conformation that interferes with coactivator binding and appears to facilitate corepressor binding (9).

By competing with E2 and other estrogens for binding to the ligand binding site of ER, Tam and OHT may induce formation of an ER complex that is unable to effectively activate transcription of estrogen-regulated genes important in the growth and development of estrogen-dependent tumors. Tam and OHT can both arrest cell growth by preventing the growth promoting activities of E2 and can induce death of both ER-positive and ER-negative cells (10–12). Tam triggers apoptosis in vitro and shrinks some tumors in vivo. Several mechanisms have been proposed to explain tamoxifen-induced cell death including transcriptional regulation of Bcl-2 family proteins (13), activating mitogen-activated protein kinase and other kinases through nongenomic pathways (11), and triggering an increase of intracellular Ca2+ (14). However, in many of these experiments, very high µM concentrations of Tam or OHT were used, and some of the studies were carried out in cells that lack ER. To analyze these processes in cells that were otherwise identical and differed only in the presence or absence of ER, we analyzed Tamand OHT-induced cell death in ER-negative HeLa cells stably transfected to express hER{alpha} (HeLaER6 (15)). We identified two pathways by which OHT induces cell death. When ER-negative HeLa cells are maintained in medium containing 10–20 µM Tam, OHT, E2, or raloxifene, the cells die within 24 h by a reactive oxygen-based pathway that triggers classical caspase-dependent apoptosis (16). Low (nM) concentrations of OHT and submicromolar concentrations of Tam do not kill or damage ER-negative HeLa cells (Fig. 1). When ER positive HeLaER cells are maintained in medium containing 1–100 nM OHT, they undergo apoptosis over several days. The antagonist ICI 182,780, the selective estrogen receptor modulator, raloxifene, and E2 protect the cells against OHT-ER-mediated cell death, suggesting a role for ER{alpha} (16). However, the most widely accepted current standard of proof for the involvement of a protein in a response, demonstrating an effect using RNAi knockdown was not performed. The mechanism(s) by which low concentrations of OHT induce cell death remains largely unknown. The long time course of OHT-ER{alpha}-induced cell death suggested that genomic processes based on ER-regulated transcription were likely to be important.


Figure 1
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  		FIGURE 1.
ER-dependent and ER-independent pathways for OHT- and Tam-induced apoptosis. Overview of the effects of different concentrations of ER ligands in the absence and presence of ER{alpha}. High concentrations (10–20 µM) of OHT, Tam, RAL, and E2 all induce rapid ER-independent cell death by a classical reactive oxygen-based, caspase-3-dependent, death pathway. In cells containing ER{alpha}, low concentrations of Tam (100–1000 nM) and OHT (10–100 nM) induced apoptosis over several days (based on data from Obrero et al. (16)).

 
In this study we analyzed the role of ER{alpha}-mediated gene transcription in OHT-ER-induced cell death. We initially used RNAi and stable transfection of mutant ERs to examine the requirement for ER{alpha} and for ER{alpha}-mediated gene transcription. Because these experiments supported a role for OHT-ER{alpha}-mediated gene transcription in cell death, we carried out microarray analysis.

The microarray data, more detailed regulatory studies, and what was known about their function led us to focus on CCAAT enhancer-binding protein {alpha} (C/EBP{alpha}) and p21/waf1/CDKN1A/Cip1. C/EBP{alpha} and p21 encoded proteins whose roles in cell growth and cell death and expression patterns were consistent with a possible role in OHT-ER{alpha}-dependent apoptosis.

CEBP/{alpha} was originally reported as a transcriptional factor containing a basic leucine zipper DNA binding motif (17–19). C/EBP{alpha} is exported from the nucleus to the cytoplasm in response to tumor necrosis factor {alpha} (20–22). Roles for C/EBP{alpha} in signal transduction, inhibition of cell cycle progression based on protein-protein interactions through C-terminal interaction with p21 and cyclin-dependent kinase have been proposed (23–28). Rat C/EBP{alpha} increases apoptosis of NCI-H358 cells (29). We analyzed the roles of C/EBP{alpha} and p21 in OHT-ER-induced apoptosis and conclude that although p21 likely contributes to OHT-ER-induced apoptosis, the induction of C/EBP{alpha} by OHT-ER plays the central role in OHT-induced apoptosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Plasmids pIE-LBD and pIE-M-DBD—Restriction enzymes were obtained from Promega. Plasmid pIE-LBD was constructed by subcloning the EcoRI/BamHI fragment of pCMV-hER(EF) (30) into pIRESIneo (Clontech, Palo Alto, CA). Plasmid pIE-M-DBD was constructed by using QuikChange site-directed mutagenesis (Stratagene, San Diego, CA) with primers 5'-GGAGTCTGGTCCTGTGCGGCCTGCAAGGCCTTCTTCAAG-3' and 5'-CTTGAAGAAGGCCTTGCAGGCCGCACAGGACCAGACTCC-3' on pCMV-hER. This results in the mutations E203A and G204A in ER{alpha}. The EcoRI/BamHI fragment was subcloned into pIRESIneo.

Cell Culture—HeLaER cells were grown in Phenol Red-free Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% charcoal-dextran-treated fetal bovine serum and 150 µg/ml G418 (Invitrogen).

Establishment of the HeLaER LBD and HeLaER M-DBD Cell Lines—HeLa cells were plated in 100-mm culture dishes at a concentration of 1.6 x 106 cells per dish. HeLa cells obtained from ATCC were used to construct the HeLaER M-DBD and HeLaER LBD cell lines. The original ER{alpha} M-DBD mutant was a gift from Prof. L. Jameson and was recloned into the pIE plasmid (15). The cells were transfected with 6 µg of SspI-linearized pIE-LBD, pIE-M-DBD using Polyfect (Qiagen, Valencia, CA) liposome following the manufacturer's instructions. The medium was replaced with selection media (50% Dulbecco's modified Eagle's medium, 10% charcoal-dextran-fetal bovine serum, 50% conditioned medium, 500 µg/ml G418 in the HeLaER LBD and HeLaER M-DBD selections) ~24 h post-transfection. After sufficient growth, colonies were isolated and reseeded into 24-well culture plates in selection medium. Cell lines were further expanded to confluence in 6-well plates, then T-75 flasks. For long-term growth, the cells were maintained under selection in medium containing 150~200 µg/ml G418. ER expression was detected by Western blotting. H222 anti-ER antibody (NeoMarkers) was used to detect ER in the HeLaER LBD cells, and NCL-ER-6F11 anti-ER antibody was used to detect ER in the HeLaER M-DBD cells.

Isolation of OHT-resistant Cell Lines—HeLaER cells in Phenol Red-free Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% charcoal-dextran-treated fetal bovine serum and 150 µg/ml G418 were plated in 96-well plates at 0.5 cell/well in medium containing 100 nM OHT. After several weeks, colonies from wells containing a single colony were isolated as OHT-resistant clonal cell lines.

Microarray Analysis—HeLaER cells were treated with 10 nM OHT or ethanol vehicle for 34 h. RNAs were purified with the RNeasy® kit (Qiagen). The mRNAs were reverse-transcribed to cDNAs by using Moloney murine leukemia reverse transcriptase (Invitrogen) and hybridized to Affymetrix GeneChip® Human Genome U133A Arrays (Affymetrix, Santa Clara, CA). The array data were initially processed by the W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois using the GeneChip Robust Multichip Average package in R/Bioconductor. A false discovery rate of <0.05, as determined by Significance Analysis of Microarray software (31), was used to identify genes significantly regulated by ligand treatment. Expression data were then loaded into GeneSpring software and normalized to the control (ethanol-treated) samples. Microarray analysis was carried out in triplicate for OHT-treated samples and for ethanol-treated samples.

Quantitative Real Time PCR—RNA levels in ethanol-treated control samples were set equal to 1. Genes up-regulated or down-regulated by OHT at least 1.6-fold were selected for quantitative RT-PCR analysis for further confirmation. RNAs from HeLaER cells treated with OHT or E2 for 4, 24, or 34 h were extracted with TRIzol reagent (Invitrogen) and purified with the RNeasy® mini kit (Qiagen). One µg of RNA was reverse-transcribed using Moloney murine leukemia reverse transcriptase (Invitrogen), and 1% of the cDNA product was used in quantitative RT-PCR. The forward and reverse primer mix (1 µl of 10 µM) and 12.5 µl of SYBR® green 2x PCR master mix (Applied Biosystems, Carlsbad, CA) in a total volume of 25 µl were put into each well of a 96-welliCycler iQTM PCR plate and assayed using a Bio-Rad iCyclerTM optical system (Bio-Rad). PCR was as follows: 95 °C for 2 min; 45 repeats (95 °C for 25 s, 55 °C for 25 s, 72 °C for 15 s); melting from 55 to 95 °C with 0.5 °C increases per 10 s in each cycle for 80 cycles. The internal standard was 36B4 mRNA.

RNA Interference—siRNA sequences were BLAST-searched against the human genome to ensure that they were sequence-specific for ER{alpha}, C/EBP{alpha}, and p21. The siRNA sequences showed no exact or near exact matches to any other sequences in the human genome. siRNAs were synthesized by Dharmacon (Chicago, IL). ER{alpha} siRNA: 5'-AAGCUACUGUUUGCUCCUAACTT-3' (nucleotides 1201–1223 of hER{alpha} relative to the first nucleotide of the start codon); C/EBP{alpha} siRNA: 5'-ACG AGA CGU CCA UCG ACA U-3' (nucleotides 173–191); p21 siRNA: 5'-CAU ACU GGC CUG GAC UGU UUU-3' (nucleotides 1943–1963 or 2083–2103 of transcript variants 1 and 2, respectively. Variant 2 contains an extra internal sequence segment in the 5'-untranslated region but encodes the same protein as Variant 1.). When cells were ~30–40% confluent, 4 µl of the 20 µM siRNAs were transfected into the HeLaER cells in 12-well plates with OligofectamineTM reagent (Invitrogen).

Western Blotting—Cells were resuspended in ice-cold lysis buffer (adapted from Schreiber et al. (32)): 20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 10 mM dithiothreitol, 100 µg/ml phenylmethylsulfonyl fluoride, protease inhibitor mixture (P8340, Sigma) was added right before use). After sonication on ice, the extracts were centrifuged for 5 min at 4 °C, and protein concentration was determined with Coomassie Blue (Bio Rad). Samples were combined with 6x SDS loading buffer, boiled for 5 min, and loaded on to a 12.5% SDS-polyacrylamide gel. After electrophoresis, the gel samples were transferred to nitrocellulose membranes (Schleicher & Schuell). Primary and secondary antibodies were each used at a 1:1000 dilution. Primary antibodies were C/EBP{alpha} (sc-9314), p21 (sc-6246), c-Myc (sc-40), phosphorylated extracellular signal-regulated kinase, actin, and calnexin (Santa Cruz Biotechnology, Santa Cruz, CA) and estrogen receptor {alpha} antibody (Clone 6F11, Biocare Medical, Walnut Creek, CA). Proteins were visualized by reacting with ECL plus reagents (Amersham Biosciences), and band intensity was quantitated using a GE Healthcare Storm PhosphorImager (Amersham Biosciences).

Apoptosis Analysis—Apoptosis was assayed essentially as we described previously (16).

Transient Transfections—Transient transfections of HeLaER, HeLaER LBD, and HeLaER M-DBD cells were performed using Lipofectin Plus reagent (Invitrogen) using the manufacturer's protocol. 100,000 cells per well were seeded in 12-well plates. After 24 h the cells were transfected with 0.4 µg of either (ATL)4-luc or AP1-luc reporter and 50 ng of pRLSV40 per well. E2 or OHT was added to a final concentration of 10 nM after transfection. After 48 h the cells were harvested, and dual luciferase assays (Promega) were performed using the manufacturer's protocol.

Flow Cytometry and Light Phase Microscopy—Flow cytometry assays were performed as previously described (16). Briefly, cells were plated in 6-well plates and incubated 24 h. After treatment with 10–8 M OHT or EtOH vehicle for 3 days, cells were washed with phosphate-buffered saline (PBS) and harvested using PBS-EDTA. Cells were pelleted by centrifugation at 600 rpm at 4 °C for 5 min and resuspended in 1 ml of phosphate-buffered saline. 40 nM of 3,3'-dihexylocarbocyanine iodide was added to the resuspended pellet, and the samples were incubated at 37 °C for 15 min, after which propidium iodide was added to 5 µg/ml. Relative fluorescence intensities were measured using a Coulter XL benchtop flow cytometer with excitation at 488 nm and emission at 520 nm. The cell sorter was also used to ascertain the light scattering properties of the cells. Cells were visualized by light phase microscopy using a TE 300 Micron Eclipse inverted fluorescence microscope.

Statistical Analysis—p values were calculated using the unpaired, two-tailed Student's t test. Data are presented as the mean ± S.E. Significance was established when p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
RNAi Knockdown of ER{alpha} Blocks OHT-mediated Apoptosis—Our observations that low concentrations of Tam and OHT do not induce apoptosis of wild-type ER-negative HeLa cells and that pretreatment with an excess of ICI 182,780/Faslodex (ICI), the selective estrogen receptor modulator raloxifene (RAL), or E2 largely protected HeLaER cells from OHT- and TAM-induced death (16) strongly suggested a role for ER{alpha} in Tam- and OHT-induced cell death. To test the role of ER{alpha} more directly, we carried out an RNAi knockdown of ER{alpha} in the HeLaER cells. The cells were transfected with either the control pGL3 (luciferase) siRNA or the ER-specific siRNA. Western blot analysis showed that the ER{alpha} knockdown lasted at least 5 days (Fig. 2A). The cells were transfected with the control pGL3 siRNA or with the ER{alpha} siRNA and then treated with either EtOH vehicle or 10–8 M OHT (Fig. 2B). Cells transfected with the pGL3 siRNA and treated with OHT became sparse and exhibited the elongated morphology we previously described (16). When displayed using fluorescence-activated cell sorting, the OHT-treated cells transfected with the pGL3 siRNA exhibited a shoulder showing a substantial population of cells undergoing apoptosis. In contrast, in the cells in which ER{alpha} was knocked down with the ER{alpha}-specific siRNA, OHT did not cause a reduction in cell number, a change in cell morphology, or the appearance of a peak of apoptotic cells (Fig. 2B). These data clearly demonstrate that ER{alpha} mediates OHT- and TAM-induced cell death in HeLaER cells.


Figure 2
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  		FIGURE 2.
RNAi knockdown of ER{alpha} blocks OHT-induced apoptosis. A and B, HeLaER cells were transfected with ER{alpha}-specific siRNA (ER) or control pGL3 (luciferase) siRNA. A, whole cell extracts were collected 2–6 days after transfection, and levels of ER{alpha} and the actin control were determined by Western blotting. B, HeLaER cells were transfected with ER or control pGL3 siRNA. After 1 day 10–8 M OHT (OHT) or ethanol vehicle (EtOH) was added to the medium, and the cells were harvested after 3 additional days. Cell images were obtained using phase-contrast microscopy at 40x magnification. Cell viability was determined by using fluorescence activated cell sorting. Transfections, Western blotting, and cell sorting were carried out as described under "Experimental Procedures."

 
OHT Does Not Induce Apoptosis in Stably Transfected Cell Lines Expressing ER Mutants That Do Not Bind to EREs—Although our data show that ER is required for OHT-induced cell death, the functions of ER that are essential were not known. ER{alpha} exerts its actions by rapid nongenomic actions based on modulation of signal transduction pathways (33–38) by regulating gene expression after being tethered to DNA through other transcription factors bound at AP1 (39–41) and SP1 sites (42–44) and by direct binding to DNA at palindromic EREs and dispersed ERE half-sites (45). To identify the ER functions important in OHT-ER{alpha}-induced apoptosis, we isolated stably transfected lines of HeLa cells expressing mutant ERs defective in specific ER functions. Because the ER LBD alone reportedly is sufficient for many non-genomic actions of ER, we isolated a stably transfected cell line expressing only the ER LBD. Previous reports indicated that OHT-ER{alpha} is far more effective than E2-ER{alpha} in activating transcription when ER{alpha} is tethered to DNA through AP1 sites (46). We, therefore, prepared a stably transfected cell line containing two mutations in the ER{alpha} DNA binding domain (E203A and G204A) previously shown to nearly abolish direct binding to EREs while retaining the ability to activate transcription when tethered to an AP1 site (47).

To produce the stably transfected cell lines, plasmids containing the constructs coding for the ER LBD or the ER with a mutated DNA binding domain (ER M-DBD) were constructed in the bicistronic expression plasmid that expresses the ER mutant and neomycin phosphotransferase as a bicistronic mRNA transcribed from a single promoter (15). After selection using medium containing G418, we showed that although the HeLa M-DBD did not efficiently activate transcription from an ERE-luciferase plasmid, it retained the capacity for ligand-dependent activation of an AP1-luciferase plasmid as previously reported (data not shown and Ref. 47).


Figure 3
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  		FIGURE 3.
Low nanomolar concentrations of OHT do not kill HeLaER M-DBD cells. HeLaER cells, HeLaER LBD cells, and HeLaER M-DBD cells were maintained in media containing EtOH vehicle or 10 nM-20 µM OHT for 3 days. The percentage of cells exhibiting mitochondrial dysfunction characteristic of early cell death was determined as described under "Experimental Procedures." The data represent the average ± S.E. for three separate experiments.

 
To examine the role of ER-mediated transcription in OHT-ER{alpha}-induced apoptosis, we examined at the ability of OHT to induce apoptosis of the cell lines expressing wild-type ER{alpha}, the ER{alpha} LBD that cannot activate transcription by either direct binding to DNA or through tethering, and the ER{alpha} M-DBD that only activates transcription through tethering. Very high, >10 µM, OHT should induce apoptosis through the ER-independent pathway (Fig. 1). Because all three cell lines were killed by 20µM OHT, they remained sensitive to caspase-dependent apoptosis (Fig. 3, 2 x 10–5 M). OHT at 10–1000 nM should induce apoptosis through the ER-dependent pathway (Fig. 1). The HeLaER, HeLaER LBD, and HeLaER M-DBD cell lines were maintained in medium containing either ethanol vehicle or the indicated concentrations of OHT (10 nM to 20 µM) for 3 days, and cell death was determined (16). As expected, OHT at 10–1000 nM induced apoptosis in the HeLaER cells. Neither the HeLaER LBD nor the HeLaER M-DBD cell lines were killed by 10–1000 nM OHT (Fig. 3). Similar results were obtained with Tam (data not shown). These data support the idea that ERE-mediated transcription is required for OHT-ER{alpha}-induced apoptosis.

ICI Protects HeLaER Cells from OHT-induced Cell Death up to 33 h Post-OHT Treatment—We previously reported that ICI 182,780 does not induce apoptosis. By competing with OHT for binding to ER{alpha}, the addition of excess ICI 182,780 blocks OHT-ER{alpha}-induced apoptosis (16). To determine whether ICI can protect HeLaER6 cells when it is added to the medium after OHT, HeLaER6 cells were treated with 10–8 M OHT and 10–6 M ICI 182,780 0, 2, 12, 24, 33, 36, and 48 h after OHT treatment. Consistent with our earlier report, the addition of an excess of ICI 182,780 at the same time as OHT blocked OHT-induced apoptosis. Surprisingly, the addition of ICI 182,780 up to 33 h after OHT also blocked OHT-induced apoptosis (Fig. 4). By 36 h the cells commit to OHT-ER-induced apoptosis, and ICI begins to lose its ability to block OHT-ER induced apoptosis (Fig. 4). These data suggest that OHT-ER{alpha} mediates a key step in activation of the death pathway 33–36 h after OHT treatment.


Figure 4
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  		FIGURE 4.
ICI 182,780, added up to 33 h after OHT prevents OHT-ER{alpha}-induced apoptosis. OHT (10 nM) was added to the medium at 0 h. At the indicated times after the addition of OHT, 1 µM ICI 182,780 was added. Cell death was assayed as described under "Experimental Procedures" at 72 h after the addition of OHT. The data represent the average ± S.E. for three separate experiments.

 
Because our data indicated that ERE-mediated transcription was required for OHT-ER{alpha}-induced apoptosis, we carried out microarray analysis to identify mRNAs whose expression is regulated by OHT-ER{alpha}. We then analyzed likely mRNAs identified from the microarray analysis to determine whether or not they play a role in OHT-mediated apoptosis. Because the ICI data suggested that a key event in committing the cells to OHT-mediated apoptosis occurs in the 33–36-h time frame, we looked at mRNA levels 34 h after OHT treatment.

Identification of OHT-ER{alpha}-regulated Genes by Microarray Analysis—Microarray studies in ER{alpha} positive MCF-7 cells and in MDA-MB-231 breast cancer cells stably transfected to express ER{alpha} indicated that Tam and OHT act as full or partial agonists on ~20% of E2-regulated genes and also regulate a group of genes not regulated by E2-ER (48). We, therefore, carried out microarray analysis to identify OHT-ER{alpha}-regulated genes. HeLa-ER cells were maintained in medium containing 10 nM OHT for 34 h, the cells were harvested, and RNA was isolated, converted to cDNA, and analyzed on Affymetrix microarrays containing 18,000 human genes. We focused on the 19 genes whose mRNAs were up-regulated by >1.6-fold after OHT treatment and the 15 mRNAs whose levels were down-regulated by >1.5-fold after OHT treatment (Table 1). Approximately 50 additional mRNAs were up-regulated 1.5–1.6-fold after OHT treatment (data not shown). Because none of these mRNAs encoded proteins that seemed likely to mediate OHT-dependent apoptosis, the regulation of these mRNAs was not further characterized. The genes whose mRNA levels were regulated by OHT included two genes (C/EBP{alpha} and p21/CDKN1A/waf1/Cip1) that code for proteins likely to be important in cell growth or cell death (Table 1). C/EBP{alpha} is a transcription factor implicated in cell death in terminally differentiated cells, and p21 is a widely studied cyclin-dependent kinase inhibitor (CDKN1A/waf1/Cip1).


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  		TABLE 1
Genes whose expression is regulated by OHT in HeLaER cells

OHT (10 nM) was added at 0 time, the cells were harvested at 34 h, and mRNA was isolated and analyzed using microarrays as described under "Experimental Procedures." The lane labeled OHT shows mRNAs induced by OHT at least 1.6 fold (up-regulated genes) or down-regulated (down-regulated genes) by at least 1.5-fold. The first row shows the -fold change as seen on the microarray. The second row represents the -fold change of most of the genes as shown by quantitative RT-PCR. A few genes whose expression is unlikely to be related to cell death (such as complement C3) were not further analyzed. Regulation of the expression of some mRNAs by 10 nM E2 and by 10 nM OHT at various times is shown for some mRNAs that were studied in more detail. TFF, transforming growth factor.

 
OHT-ER Induction of C/EBP{alpha} and p21 mRNAs—We used quantitative RT-PCR to confirm the microarray data and to assay mRNA levels at selected time points. Consistent with other data using this system, in most cases the -fold induction was greater by quantitative RT-PCR than by microarray (48). Because E2 does not induce cell death, we concentrated on mRNAs that were regulated differently by OHT and by E2. Quantitative RT-PCR showed that p21 mRNA is induced by both OHT and by E2 in the 24–48-h post-OHT time frame important for OHT-induced apoptosis (Fig. 5A) and shows little or no induction by RAL and ICI 182,780. C/EBP{alpha} mRNA is strongly induced by OHT, is not induced by E2, and exhibits very weak induction by ICI 182,780 (Fig. 5B). We also tested the effects of E2 and OHT on the levels of C/EBP{alpha} and p21 protein. Western blot analysis shows that E2 does not induce C/EBP{alpha} protein (Fig. 5C, C/EBP, EtOH, and E2) and moderately induces p21 (Fig. 5C, p21, EtOH, and E2), whereas OHT strongly induces both C/EBP/{alpha} and p21 (Fig. 5C). The role of C/EBP{alpha} in apoptosis in other systems (29), the strong induction of C/EBP{alpha} mRNA in the important time period 24–48 h after OHT treatment (Fig. 4), and the induction of C/EBP{alpha} mRNA by OHT, which induces apoptosis of the HeLaER cells, and the absence of a strong induction of C/EBP{alpha} by E2 or ICI, which do not induce apoptosis, all are consistent with the idea that OHT induction of C/EBP{alpha} may be important in OHT-ER induced apoptosis.


Figure 5
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  		FIGURE 5.
Time course of regulation of C/EBP{alpha} and p21 mRNA levels by ER ligands. A and B, the cells were maintained in medium containing ethanol vehicle (EtOH), 10 nM OHT, E2, RAL, or ICI 182,780 for the indicated times, and RNA was isolated and analyzed by quantitative RT-PCR as described under "Experimental Procedures" for the relative abundance of p21 mRNA (A) and C/EBP{alpha} mRNA (B). The level of each mRNA in the ethanol vehicle sample was set equal to 1. C, Western blot analysis of the induction of C/EBP{alpha} and p21 after 48 h in 10 nM E2 or 10 nM OHT. Calnexin was used as the internal standard.

 


Figure 6
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  		FIGURE 6.
OHT-induction and RNAi knockdown of C/EBP{alpha} and P21 proteins. Western blot analysis in untransfected cells and in cells transfected with C/EBP{alpha}-specific siRNA (5'-ACGAGACGUCCAUCGACAU-3'), p21-specific siRNA (5'-CAUACUGGCCUGGACUGUUUU-3'), or control pGL3 (luciferase) siRNA (59). Actin was used as the internal standard.

 
Regulation of C/EBP{alpha} and p21 Protein Levels and Their Knockdown Using RNAi—Because we were going to test the roles of p21 and of C/EBP{alpha} in OHT-ER{alpha}-induced apoptosis using RNAi knockdown and we wanted to determine whether OHT and E2 induce p21 and C/EBP{alpha} proteins, we carried out Western blot analysis. OHT induced p21 protein to a level higher than was seen with E2 (Fig. 6). As expected, OHT, but not E2, induced C/EBP{alpha} protein. C/EBP{alpha} was knocked down by the C/EBP{alpha} siRNA and not by the p21 siRNA. Similarly, p21 was knocked down by the p21 siRNA but not by the C/EBP{alpha} siRNA (Fig. 6).

RNAi Knockdown of C/EBP{alpha} Blocks OHT-ER{alpha}-induced Apoptosis—We then looked at the effect of knockdown of C/EBP{alpha} on apoptosis. To evaluate cell death, we used three fluorescence-activated cell sorter-based assays. These assays target distinct aspects of cell death. Release of a fluorescent dye evaluates loss of mitochondrial membrane potential, uptake of a fluorescent agent that intercalates into DNA targets the loss of plasma membrane integrity, and light scattering detects changes in cell morphology. In preliminary studies, we identified a time after OHT addition that resulted in substantial cell death as measured by all thee assays and showed that transfecting the C/EBP{alpha} and p21 siRNAs into the cells using oligofectamine did not cause cell death (data not shown). In three independent experiments the addition of OHT to the medium induced cell death as measured by all three assays (Fig. 7A, OHT). Transfection with the control pGL3 luciferase siRNA had no effect on cell death (Fig. 7A, PGL3 siRNA + OHT). RNAi knockdown of C/EBP{alpha} nearly abolished OHT-ER{alpha}-induced cell death (Fig. 7A, CEBP{alpha} siRNA + OHT). The difference between the level of cell death in the cells treated with OHT or treated with OHT and transfected with the pGL3 siRNA compared with the level of cell death in the cells transfected with the C/EBP{alpha} siRNA was significant using each of the three assays for cell death (p < 0.05). RNAi knockdown of p21 had a significant but smaller effect on OHT-ER{alpha}-induced cell death (Fig. 7B). There was a statistically significant (p < 0.05) decrease in cell death in the cells treated with the p21 siRNA compared with the cells transfected with the pGL3 siRNA. A second control siRNA recommended by the supplier (Dharmacon) also had no effect on OHT-ER{alpha}-induced cell death (Fig. 7B). These data show that induction of C/EBP{alpha} by OHT plays a key role in the ability of OHT to induce apoptosis.

In Cells Resistant to OHT-ER{alpha}-induced Apoptosis, Expression of C/EBP{alpha} and OHT Induction of C/EBP{alpha} Are Lost—The expression and RNAi knockdown data strongly supported a role for C/EBP{alpha} in OHT-ER{alpha}-induced cell death and suggested a possible role for p21. If OHT induction of C/EBP{alpha} or p21 plays a key role in OHT-ER{alpha}-induced apoptosis, HeLaER cells selected for resistance to OHT-ER{alpha}-induced cell death might show altered expression or regulation of C/EBP{alpha} or p21. The overwhelming majority of HeLaER cells stop growing and die in medium containing 100 nM OHT, but a few cells survive. Based on the RNAi knockdown experiment (Fig. 2), we knew that HeLaER cells that lose ER{alpha} expression would continue to grow in OHT and would be represented among the clonal lines we isolated. Western blot analysis showed that about half of the clonal lines we isolated after growth in OHT-containing medium had lost all or most of their ER{alpha} expression or expressed an ER{alpha} fragment (data not shown). We identified four clonal cell lines that were resistant to OHT-ER{alpha}-induced apoptosis and contained levels of full-length ER{alpha} comparable with two HeLaER cell lines that were sensitive to OHT-ER{alpha}-induced apoptosis (Fig. 8A). Data for all of the ER{alpha}-containing, OHT-resistant clonal cell lines we isolated are shown. No cell lines were excluded. Etoposide, awidely used activator of caspase-dependent apoptosis, retained the ability to kill all 4 of the OHT-resistant cell lines in 24 h (data not shown). Therefore, the four OHT-resistant cell lines are specifically defective in OHT-induced apoptosis but retain functional machinery for caspase-dependent apoptosis activated by the mitochondrial pathway.

We used quantitative RT-PCR to determine the basal levels of C/EBP{alpha} and p21 in the OHT-sensitive and OHT-resistant cell lines and their regulation by OHT and by E2. The basal level of p21 mRNA in the OHT-resistant cell lines was equal to or higher than the basal level of p21 mRNA in the two OHT-sensitive cell lines. One of the OHT-resistant cell lines (Fig. 8B, clone 5) retained the ability to induce p21 in the presence of OHT. As expected, the two cell lines that are killed by OHT showed strong induction of C/EBP{alpha} mRNA by OHT and little or no induction by E2 (Fig. 8C). In contrast to p21, in all four of the HeLaER lines selected for resistance to OHT-induced cell death, both the basal level of C/EBP{alpha} mRNA and the level of C/EBP{alpha} mRNA after OHT treatment were extremely low (Fig. 8C). Because this was an unbiased selection, the absence of an induced level of C/EBP{alpha} mRNA in all four ER-containing cell lines resistant to OHT-induced apoptosis provides compelling support for a central role of C/EBP{alpha} in cell death mediated by OHT-ER{alpha}.


Figure 7
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  		FIGURE 7.
RNAi knockdown of C/EBP{alpha} or p21 reduces OHT-ER induced apoptosis. HeLaER cells were transfected with the control pGL3 siRNA or a second control siRNA (Dharmacon), C/EBP{alpha} siRNA (A), or p21 (B) siRNA. After 24 h 100 nM OHT was added, and the cells were harvested after an additional 72 h and assayed for mitochondrial dysfunction by leakage of low concentrations fluorescent dye (gray bars) (16) and for plasma membrane integrity by intercalation of a fluorescent dye into DNA (black bars) and for changes in cell morphology by light scattering (open bars). The data represent the average of three independent experiments ± S.E. Comparing C/EBP{alpha} siRNA plus OHT treatment and pGL3 siRNA plus OHT treatment, the p value is 0.01 for the mitochondrial dysfunction assay, 0.01 for the plasma membrane integrity assay, and 0.02 for the cell morphology assay. Comparing p21 siRNA plus OHT treatment and pGL3 siRNA plus OHT treatment, the p value is 0.04 for the mitochondrial dysfunction assay, 0.03 for the plasma membrane integrity assay, and 0.03 for the cell morphology assay. Thus, all of the key data are significant at p < 0.05.

 


Figure 8
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  		FIGURE 8.
HeLaER cells resistant to OHT-induced apoptosis lack regulated C/EBP{alpha}. A, Western blots comparing the level of ER{alpha} in HeLaER cells sensitive to OHT-induced apoptosis (triangles) and selected for outgrowth in OHT and resistance to OHT-induced cell death (asterisks). B and C, levels and regulation of p21 mRNA (B) and C/EBP{alpha} mRNA (C) in the HeLaER6 cells that were used in these studies, for simplicity referred to as HeLaER cells, in HeLaER12 cells, a second clonal line of HeLa cells that is killed by OHT, and in all four ER-containing cell lines selected for resistance to OHT-induced apoptosis (clones 2–5). Cells were maintained in ethanol vehicle (open bars), 100 nM OHT (black bars), or 100 nM E2 (gray bars).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
At least three lines of investigation demonstrate that OHT acts by binding to ER{alpha} to induce apoptosis. Wild-type HeLa cells which lack ER and HeLa cells that contain ER mutants are not killed by 10–1000 nM OHT (Ref. 16 and Fig. 3). High concentrations E2, ICI 182,78, and RAL block OHT-induced apoptosis (16). RNAi knockdown of ER{alpha} abolishes OHT-induced cell death (Fig. 2). Therefore, loss of expression of ER{alpha} or mutational inactivation of ER{alpha} are likely to be observed in some cell lines resistant to OHT-induced cell death. Consistent with a key role for ER{alpha} in OHT-induced cell death, in several cell lines resistant to OHT-induced cell death we detected loss of ER{alpha} expression or production of a truncated ER{alpha} (data not shown).

A central role for C/EBP{alpha} in OHT-ER{alpha}-induced apoptosis is supported by our observations that RNAi knockdown of C/EBP{alpha} nearly abolishes OHT-ER{alpha}-induced apoptosis and by the loss of C/EBP{alpha} and of OHT induction of C/EBP{alpha} in cell lines selected by their ability to grow in concentrations of OHT that kill nearly all of the HeLaER cells. It is striking that all of the cell lines that we isolated that grew out in 100 nM OHT either exhibited reduced expression of full-length ER{alpha} or reduced expression and defective regulation of C/EBP{alpha}.

Because reduced expression of C/EBP{alpha} nearly abolished OHT-induced apoptosis, it seemed likely that expression of increased levels of C/EBP{alpha} enhances OHT-induced cell death. To test this idea, we attempted to transiently transfect wild-type C/EBP{alpha} and C/EBP{alpha} mutants defective in specific functions (28) into the HeLaER cells. However, we were unable to transfect more than ~30% of the cells, and the harsh conditions needed for high transfection efficiency damaged the cells. It was, therefore, not possible to rapidly test the effect of elevated levels of C/EBP{alpha} on OHT-mediated apoptosis.

C/EBP{alpha} is expressed in many cell types including breast and ovary tissues and localizes to the nucleus. C/EBP{alpha} mRNA gives rise to two isoforms of protein, the full-length 42-kDa and 30-kDa with a truncated N terminus. The two isoforms of C/EBP{alpha} show contrasting functions in gene activation and cell proliferation, and the isoform ratio may be important in mediating proliferation and differentiation (49). Our Western blot data indicate that nearly all of the C/EBP{alpha} in these cells is the 42-kDa isoform, and this isoform is responsible for OHT-ER{alpha}-induced apoptosis. Many studies of C/EBP{alpha} focus on its role in lineage determination in hematopoiesis (50). C/EBP{alpha} is a key factor in driving the development of myeloid cells. Our data suggest a quite new but plausible role of C/EBP{alpha} as a critical factor in controlling an apoptosis pathway. Our finding of a role for C/EBP{alpha} in cell death, consistent with several reports describing diverse pathways including p21, and the E2F complex that are impacted by C/EBP{alpha} as it mediates cell cycle arrest and proliferation (51–53). Induction of C/EBP{alpha} expression in breast cancer cells resulted in growth inhibition accompanied by G0-G1 cell cycle arrest. C/EBP{alpha} expression was associated with up-regulation of p21 and peroxisome proliferator-activated receptor {gamma} and down-regulation of c-Myc (52). We suggest that p21 works with C/EBP{alpha} to help determine whether or not cells initiate apoptosis after cell cycle arrest.

Both C/EBP{alpha} and p21 are induced by OHT-ER{alpha}, and it is likely that the interplay between these two proteins plays a role in OHT-ER-induced cell death. Although both E2 and OHT induce P21 mRNA in HeLaER cells, induction of p21 protein by OHT is far more effective than induction of p21 by E2. The cyclin-dependent kinase inhibitor p21 exhibits a short half-life of 0.5 to <2 h (54, 55) and is thought to be stabilized by binding to C/EBP{alpha} (56). Because OHT, but not E2, induces C/EBP{alpha}, which stabilizes the p21 protein, OHT elicits a much larger induction of p21 protein than E2.

RNAi knockdown of p21 resulted in a moderate reduction in OHT-induced apoptosis. It is possible that the failure to isolate OHT-resistant cell lines that lack p21 stems from an essential role of p21 in regulating progression through the cell cycle.

Because p21 is a well known cyclin-dependent kinase inhibitor and participates in regulating the cell cycle and C/EBP{alpha} reportedly inhibits progression through the cell cycle (23–27) and contains binding sites for p21 and cyclin-dependent kinase (28), it is quite possible that OHT-induced apoptosis may require C/EBP{alpha} to associate with p21 and initiate downstream pathways leading to cell death. This might involve arresting cell growth. Consistent with this view, in MCF-7 cells tamoxifen acts mainly by arresting cells in the G0/G1 phase of the cell cycle (57, 58). Of course the ability of C/EBP{alpha} to mediate gene expression by acting as a transcriptional factor or to play a role in signal transduction pathway in the cytoplasm may also be important to its role in OHT-induced cell death.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA90371, HD16720, and DKO71909 and by a National Institutes of Health predoctoral traineeship (to D. Y.). 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.

This paper is dedicated to the memory of our late friend and colleague David Yu. Back

1 Present address: Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, China. Back

2 To whom correspondence should be addressed: Dept. of Biochemistry, University of Illinois, 413 RAL, Box B4, 600 S. Mathews, Urbana, IL 61801. Tel.: 217-333-1788; Fax: 217-244-5858; E-mail: djshapir{at}uiuc.edu.

3 The abbreviations used are: ER, estrogen receptor; hER, human ER; ERE, estrogen response element; Tam, tamoxifen; OHT, 4-hydroxytamoxifen; E2, 17beta-estradiol; AF-1, activation function 1; AF-2, activation function 2; siRNA, small interfering RNA; RNAi, RNA interference; RAL, raloxifene; LBD, ligand binding domain; C/EBP{alpha}, CCAAT enhancer-binding protein {alpha}; DBD-M, mutated DNA binding domain. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Klinge, C. M., Brolly, C. L., Bambara, R. A., and Hilf, R. (1997) J. Steroid Biochem. Mol. Biol. 63, 283–301[CrossRef][Medline] [Order article via Infotrieve]
  2. Nilsson, S., Makela, S., Treuter, E., Tujague, M., Thomsen, J., Andersson, G., Enmark, E., Pettersson, K., Warner, M., and Gustafsson, J. A. (2001) Physiol. Rev. 81, 1535–1565[Abstract/Free Full Text]
  3. Kumar, V., Green, S., Stack, G., Berry, M., Jin, J. R., and Chambon, P. (1987) Cell 51, 941–951[CrossRef][Medline] [Order article via Infotrieve]
  4. Webster, N. J., Green, S., Jin, J. R., and Chambon, P. (1988) Cell 54, 199–207[CrossRef][Medline] [Order article via Infotrieve]
  5. Webster, N. J., Green, S., Tasset, D., Ponglikitmongkol, M., and Chambon, P. (1989) EMBO J. 8, 1441–1446[Medline] [Order article via Infotrieve]
  6. Lees, J. A., Fawell, S. E., and Parker, M. G. (1989) Nucleic Acids Res. 17, 5477–5488[Abstract/Free Full Text]
  7. Tora, L., White, J., Brou, C., Tasset, D., Webster, N., Scheer, E., and Chambon, P. (1989) Cell 59, 477–487[CrossRef][Medline] [Order article via Infotrieve]
  8. Robyr, D., Wolffe, A. P., and Wahli, W. (2000) Mol. Endocrinol. 14, 329–347[Free Full Text]
  9. Shang, Y., and Brown, M. (2002) Science 295, 2465–2468[Abstract/Free Full Text]
  10. Ferlini, C., Scambia, G., Marone, M., Distefano, M., Gaggini, C., Ferrandina, G., Fattorossi, A., Isola, G., Benedetti Panici, P., and Mancuso, S. (1999) Br. J. Cancer 79, 257–263[Medline] [Order article via Infotrieve]
  11. Mandlekar, S., and Kong, A. N. (2001) Apoptosis 6, 469–477[CrossRef][Medline] [Order article via Infotrieve]
  12. Mandlekar, S., Hebbar, V., Christov, K., and Kong, A. N. (2000) Cancer Res. 60, 6601–6606[Abstract/Free Full Text]
  13. Zhang, G. J., Kimijima, I., Onda, M., Kanno, M., Sato, H., Watanabe, T., Tsuchiya, A., Abe, R., and Takenoshita, S. (1999) Clin. Cancer Res. 5, 2971–2977[Abstract/Free Full Text]
  14. Kim, J. A., Kang, Y. S., Jung, M. W., Lee, S. H., and Lee, Y. S. (1999) Cancer Lett. 147, 115–123[CrossRef][Medline] [Order article via Infotrieve]
  15. Zhang, C. C., Krieg, S., and Shapiro, D. J. (1999) Mol. Endocrinol. 13, 632–643[Abstract/Free Full Text]
  16. Obrero, M., Yu, D. V., and Shapiro, D. J. (2002) J. Biol. Chem. 277, 45695–45703[Abstract/Free Full Text]
  17. Landschulz, W. H., Johnson, P. F., and McKnight, S. L. (1989) Science 243, 1681–1688[Abstract/Free Full Text]
  18. Vinson, C. R., Sigler, P. B., and McKnight, S. L. (1989) Science 246, 911–916[Abstract/Free Full Text]
  19. Agre, P., Johnson, P. F., and McKnight, S. L. (1989) Science 246, 922–926[Abstract/Free Full Text]
  20. Xu, L. X., Sui, Y. F., Wang, W. L., Liu, Y. F., and Gu, J. R. (1994) Chin. Med. J. (Engl. Ed.) 107, 596–599
  21. Buck, M., Zhang, L., Halasz, N. A., Hunter, T., and Chojkier, M. (2001) EMBO J. 20, 6712–6723[CrossRef][Medline] [Order article via Infotrieve]
  22. Williams, S. C., Angerer, N. D., and Johnson, P. F. (1997) Gene Expr. 6, 371–385[Medline] [Order article via Infotrieve]
  23. Umek, R. M., Friedman, A. D., and McKnight, S. L. (1991) Science 251, 288–292[Abstract/Free Full Text]
  24. Hendricks-Taylor, L. R., and Darlington, G. J. (1995) Nucleic Acids Res. 23, 4726–4733[Abstract/Free Full Text]
  25. Porse, B. T., Pedersen, T. A., Xu, X., Lindberg, B., Wewer, U. M., Friis-Hansen, L., and Nerlov, C. (2001) Cell 107, 247–258[CrossRef][Medline] [Order article via Infotrieve]
  26. Wang, H., Iakova, P., Wilde, M., Welm, A., Goode, T., Roesler, W. J., and Timchenko, N. A. (2001) Mol. Cell 8, 817–828[CrossRef][Medline] [Order article via Infotrieve]
  27. McKnight, S. L. (2001) Cell 107, 259–261[CrossRef][Medline] [Order article via Infotrieve]
  28. Harris, T. E., Albrecht, J. H., Nakanishi, M., and Darlington, G. J. (2001) J. Biol. Chem. 276, 29200–29209[Abstract/Free Full Text]
  29. Halmos, B., Huettner, C. S., Kocher, O., Ferenczi, K., Karp, D. D., and Tenen, D. G. (2002) Cancer Res. 62, 528–534[Abstract/Free Full Text]
  30. Kraus, W. L., McInerney, E. M., and Katzenellenbogen, B. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12314–12318[Abstract/Free Full Text]
  31. Tusher, V. G., Tibshirani, R., and Chu, G. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5116–5121[Abstract/Free Full Text]
  32. Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Free Full Text]
  33. McEwen, B. S., and Alves, S. E. (1999) Endocr. Rev. 20, 279–307[Abstract/Free Full Text]
  34. McEwen, B., Akama, K., Alves, S., Brake, W. G., Bulloch, K., Lee, S., Li, C., Yuen, G., and Milner, T. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 7093–7100[Abstract/Free Full Text]
  35. McEwen, B. S. (2001) J. Appl. Physiol. 91, 2785–2801[Abstract/Free Full Text]
  36. Toran-Allerand, C. D., Singh, M., and Setalo, G., Jr. (1999) Front. Neuroendocrinol. 20, 97–121[CrossRef][Medline] [Order article via Infotrieve]
  37. Toran-Allerand, C. D. (2000) Novartis Found. Symp. 230, 56–73[Medline] [Order article via Infotrieve]
  38. Linford, N., Wade, C., and Dorsa, D. (2000) J. Neurocytol. 29, 367–374[CrossRef][Medline] [Order article via Infotrieve]
  39. Feng, W., Webb, P., Nguyen, P., Liu, X., Li, J., Karin, M., and Kushner, P. J. (2001) Mol. Endocrinol. 15, 32–45[Abstract/Free Full Text]
  40. Bjornstrom, L., and Sjoberg, M. (2002) J. Biol. Chem. 277, 48479–48483[Abstract/Free Full Text]
  41. Gaub, M. P., Bellard, M., Scheuer, I., Chambon, P., and Sassone-Corsi, P. (1990) Cell 63, 1267–1276[CrossRef][Medline] [Order article via Infotrieve]
  42. Petz, L. N., and Nardulli, A. M. (2000) Mol. Endocrinol. 14, 972–985[Abstract/Free Full Text]
  43. Qin, C., Singh, P., and Safe, S. (1999) Endocrinology 140, 2501–2508[Abstract/Free Full Text]
  44. Wang, F., Porter, W., Xing, W., Archer, T. K., and Safe, S. (1997) Biochemistry 36, 7793–7801[CrossRef][Medline] [Order article via Infotrieve]
  45. Koszewski, N. J., and Notides, A. C. (1991) Mol. Endocrinol. 5, 1129–1136[Abstract]
  46. Webb, P., Lopez, G. N., Uht, R. M., and Kushner, P. J. (1995) Mol. Endocrinol. 9, 443–456[Abstract]
  47. Jakacka, M., Ito, M., Weiss, J., Chien, P. Y., Gehm, B. D., and Jameson, J. L. (2001) J. Biol. Chem. 276, 13615–13621[Abstract/Free Full Text]
  48. Frasor, J., Stossi, F., Danes, J. M., Komm, B., Lyttle, C. R., and Katzenellenbogen, B. S. (2004) Cancer Res. 64, 1522–1533[Abstract/Free Full Text]
  49. Calkhoven, C. F., Muller, C., and Leutz, A. (2000) Genes Dev. 14, 1920–1932[Abstract/Free Full Text]
  50. Ramji, D. P., and Foka, P. (2002) Biochem. J. 365, 561–575[Medline] [Order article via Infotrieve]
  51. Muller, C., Calkhoven, C. F., Sha, X., and Leutz, A. (2004) J. Biol. Chem. 279, 7353–7358[Abstract/Free Full Text]
  52. Gery, S., Tanosaki, S., Bose, S., Bose, N., Vadgama, J., and Koeffler, H. P. (2005) Clin. Cancer Res. 11, 3184–3190[Abstract/Free Full Text]
  53. D'Alo, F., Johansen, L. M., Nelson, E. A., Radomska, H. S., Evans, E. K., Zhang, P., Nerlov, C., and Tenen, D. G. (2003) Blood 102, 3163–3171[Abstract/Free Full Text]
  54. Coleman, M. L., Marshall, C. J., and Olson, M. F. (2003) EMBO J. 22, 2036–2046[CrossRef][Medline] [Order article via Infotrieve]
  55. Timchenko, N. A., Wilde, M., Nakanishi, M., Smith, J. R., and Darlington, G. J. (1996) Genes Dev. 10, 804–815[Abstract/Free Full Text]
  56. Timchenko, N. A., Harris, T. E., Wilde, M., Bilyeu, T. A., Burgess-Beusse, B. L., Finegold, M. J., and Darlington, G. J. (1997) Mol. Cell. Biol. 17, 7353–7361[Abstract]
  57. Otto, A. M., Paddenberg, R., Schubert, S., and Mannherz, H. G. (1996) J. Cancer Res. Clin. Oncol. 122, 603–612[CrossRef][Medline] [Order article via Infotrieve]
  58. Perry, R. R., Kang, Y., and Greaves, B. R. (1995) Br. J. Cancer 72, 1441–1446[Medline] [Order article via Infotrieve]
  59. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Nature 411, 494–498