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Endocrinology Vol. 144, No. 6 2325-2335
Copyright © 2003 by The Endocrine Society

Estrogen Receptor/Sp1 Complexes Are Required for Induction of cad Gene Expression by 17ß-Estradiol in Breast Cancer Cells

Shaheen Khan, Maen Abdelrahim, Ismael Samudio and Stephen Safe

Department of Veterinary Physiology and Pharmacology Texas A&M University, College Station, Texas 77843-4466

Address all correspondence and requests for reprints to: Stephen Safe, Department of Veterinary Physiology and Pharmacology Texas A&M University 4466 TAMU, Veterinary Research Building 409, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cad gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. Cad gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 10 nM 17ß-estradiol (E2) resulted in a 3- to 5-fold increase in cad mRNA levels in both cell lines. The mechanism of hormone-induced cad gene expression was further investigated using constructs containing the growth-responsive -90 to +115 (pCAD1) region of the cad gene promoter. E2 induced reporter gene (luciferase) activity in MCF-7 and ZR-75 cells transfected with pCAD1, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the cad gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2 responsiveness. Results of electrophoretic mobility shift and chromatin immunoprecipitation assays show that both Sp1 and estrogen receptor {alpha} interact with the GC-rich region of the cad gene promoter. Moreover, in transactivation assays with pCAD1, hormone-induced transactivation was inhibited by cotransfection with dominant-negative Sp1 expression plasmid and small inhibitory RNA for Sp1, which silences Sp1 expression in the cells. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the cad gene is also regulated by a nonclassical ER{alpha}/Sp1-mediated pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HORMONE 17ß-ESTRADIOL (E2) and related estrogenic hormones play an important role in several physiological processes, including development of the female and male reproductive tracts as well as bone, vascular, and neuronal function (1, 2, 3, 4). Estrogens are also risk factors for breast cancer in women and for growth of early stage estrogen receptor (ER)-positive tumors (5, 6). Breast cancer cell lines have been extensively used as models for understanding the mechanisms associated with mitogen-induced cell growth and for development of antiestrogenic and anticarcinogenic agents for treatment of this disease (7, 8, 9). MCF-7 cells were among the first ER-positive human breast cancer cell lines characterized as responsive to the mitogenic effects of estrogens and polypeptide growth factors in cell culture and in athymic nude mice bearing MCF-7 cell xenografts (9, 10, 11, 12).

E2 stimulates proliferation of MCF-7 and other ER-positive breast cancer cell lines, and this is accompanied by cell cycle progression and transactivation of multiple genes including several that are involved in the proliferative response. Lippman and co-workers (13, 14, 15, 16, 17, 18, 19) investigated the effects of E2 in MCF-7 cells on several enzymes required for DNA synthesis including those involved in nucleotide biosynthesis. They reported that E2 induced dihydrofolate reductase, thymidylate synthase, and thymidine kinase activities, and these were accompanied by increased DNA synthesis as determined by radiolabeled thymidine uptake. In addition, several genes required for pyrimidine biosynthesis, including carbamylphosphate synthetase, aspartate transcarbamylase, orotidine pyrophosphorylase, and orotidine decarboxylase, were also induced by E2 (14). Research in this laboratory has demonstrated that the mechanisms of hormonal and growth factor regulation of some genes, including those associated with nucleotide biosynthesis and cell growth, are regulated by a nonclassical DNA-independent mechanism that involves ER{alpha}-Sp1 (protein-protein) interactions at E2-responsive GC-rich promoter motifs (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). Genes regulated by ER{alpha}/Sp1 in ER-positive MCF-7 or ZR-75 breast cancer cells include cyclin D1, bcl-2, retinoic acid receptor {alpha}1, IGF binding protein 4, adenosine deaminase, DNA polymerase {alpha}, c-fos, cathepsin D, E2F1, and creatine kinase B. DNA-independent or -dependent interactions of ER{alpha} and Sp1 proteins are also important for expression of TGF{alpha}, progesterone receptor, cathepsin D, heat shock protein 27, low-density lipoprotein lipase, epidermal growth factor receptor, and the receptor for advanced glycation end products in breast and other cancer cell lines (33, 34, 35, 36, 37, 38, 39). This study shows that the trifunctional carbamylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase (cad) gene is induced by E2 in ER-positive MCF-7 or ZR-75 breast cancer cells within 3–12 h after treatment, respectively. Analysis of the proximal growth responsive region of the cad gene promoter showed that basal activity was primarily associated with GC-rich motifs and E2 responsiveness was dependent on the same sites which bound ER{alpha}/Sp1. These data extend the number of E2-responsive genes regulated by ER{alpha}/Sp1 in breast cancer cells and confirm that hormone-induced up-regulation of enzyme activities associated with pyrimidine biosynthesis is accompanied by induction of cad gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and biochemicals
RPMI 1620, PBS, E2, antibiotic/antimycotic solution, and DMEM/F12 (DME/F-12) were purchased from Sigma (St. Louis, MO). Luciferase and ß-galactosidase enzyme assay systems were obtained from Promega Corp. (Madison, WI). Fetal bovine serum were obtained from Intergen (Purchase, NY) and JRH Biosciences (Lenexa, KS). [{gamma}-32P]ATP (3000 Ci/mmol) and [{gamma}-32P]CTP were purchased from Perkin-Elmer Life Sciences (Foster City, CA). Restriction enzymes and T4-polynucleotide were purchased from Promega Corp. ICI 182,780 has been provided by Alan Wakeling (AstraZeneca Pharmaceuticals, Macclesfield, UK). Sp1, ER{alpha}, and other antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cells oligonucleotides and plasmids
MCF-7 and ZR-75 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). MCF-7 cells were cultured in DME/F12 (Sigma) media supplemented with 5% fetal bovine serum (Intergen, Des Plains, IA; or JRH Biosciences) and ZR-75 cells were maintained in RPMI 1620 medium with phenol red and supplemented with 10% fetal bovine serum, sodium pyruvate, sodium bicarbonate, and glucose. Cells were maintained at 37 C with a humidified CO2:air (5:95) mixture. PCR primers were synthesized by Genosys/Sigma (The Woodlands, TX) and sequenced by the Gene Technologies Laboratory, Texas A&M University (College Station, TX). CAD promoter luciferase constructs pCAD1 (-90/+115) and pCAD2 (-90/+25) were kindly provided by Dr. Peggy Farnham (University of Wisconsin, Madison, WI). Human ER{alpha} expression plasmid was kindly provided by Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX). The human ERß expression plasmid was provided by Drs. E. Enmark and J.-A. Gustafsson from the Center for Biotechnology, Novum (Huddinge, Sweden). ER{alpha} deletion constructs HE11, HE15, TAF1, ER null, and HE19C were obtained from Dr. Pierre Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France). Dominant negative Sp1 (pBGENSp1) and the corresponding empty vector (pBGENO) were provided by Drs. Yoshihiro Sowa and Toshiyuki Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan). The following oligonucleotides were prepared by IDT (Coralville, IA) or Promega Corp. and were used in gel mobility shift assays (the mutations are underlined and substituted bases are indicated in bold); CADa1 (-75/-48): 5'-CCC CGC CCC TTA CGT GCC CGG CCC CGC TCA CGC CC-3', CADE (+54/+78): 5'-GCC GTT AGC CAC GTG GAC CGA CTC-3', mutant CADE: 5'-GCC GTT AGC CTG CAG GAC GAC CGA CTC-3', Sp1 (consensus): 5'-AGC TTA TTC GAT CGG GGC GGG GCG AGC G-3' and mutant Sp1 5'-AGC TTA TTC CGA AGC GGG GCG AGC G-3'.

Cloning
CAD promoter fragments were synthesized or amplified by PCR as double-stranded DNA and inserted into the pGL2 luciferase reporter plasmid (Promega Corp.) vector between NheI and HindIII polylinker sites. pCAD3 (-67/+115), pCAD4 (-67/+25), pCAD5 (-47/+115), pCAD7 (-20/+115), pCAD8 (1/+115), and pCAD9 (-47/+60) were made by PCR amplification using pCAD1 as the template. pCAD6 (-47/+25) and pCAD10 (-30/+25) were constructed by inserting the oligonucleotides into pGL2 basic vector. pCAD1m1 and pCAD1m2 were made by PCR mutagenesis using 5'-CAG TGC TAG CCC GTG GCT CCG CGG ACC CGC CCC TTA CGT GCC CGG CCC CCA ACC TCA C-3' and 5'-CAG TGC TAG CCC GTG GCT CCG CGG ACC CCA ACC TTA CGT GCC CGG CCC CCA ACC TCA C-3' as the sense primers and 5'-CCA ACA GTA CCG GAA TGC CAA GCT TAC TTA GAT-3' as the antisense primer. All ligation products were transformed into competent Escherichia coli cells. Plasmids were isolated, and clones were confirmed by DNA sequencing.

RT-PCR analysis
CAD PCR primers: CAD (sense primer), 5'-CTAAGCTTAC-TGTGGCCTCAAGTATAAT-3' and CAD (antisense primer), 5'-CTG-GATCCTATGGGAAGAAAATAGACCT-3' were used to amplify 837 bp of human CAD mRNA. RNA was extracted using RNAzol B (Tel-Test, Friendswood, TX), following manufacturer’s protocol. RNA was quantitated by measuring the 260/280-nm absorption ratio, and concentration was adjusted to 100–200 ng/µl RNA for use in RT-PCR. RNA was reverse-transcribed at 42 C for 25 min using oligo-deoxythymidine primer, followed by PCR amplification of RT product using 2 mM MgCl2, 1 µM each gene-specific primer, 1 mM deoxynucleotide triphosphate, and 2.5 U AmpliTaq DNA polymerase (Perkin-Elmer). Primer sets for CAD were added to the mixture, and the gene product were amplified (30 cycles) in a PTC-200 thermal cycler (MJ Research, Inc., Watertown, MA). The resulting 837-bp CAD probe were ligated in pcDNA3. The CAD probe was PCR amplified and then sequenced by the Gene Technologies Laboratory, Texas A&M University.

Northern blot analysis
Cells were seeded in DME/F12 medium supplemented with 2.5% charcoal-stripped serum and then synchronized in serum-free media for 3 d. Cells were treated with 10 nM E2, and RNA was extracted using RNAzol B (Tel-Test), following the manufacturer’s protocol. Fifteen to 20 µg of RNA were separated on a 1.2% agarose/1 M formaldehyde gel and transferred onto nylon membrane. RNA was cross-linked by exposing the membrane to UV light for 10 min, and the membrane was baked at 80 C for 2 h. The membrane was then prehybridized for 18 h at 55 C and hybridized in the same buffer for 24 h with the [{gamma}32P]-labeled DNA probe. The resulting blots were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). ß-Actin mRNA was used as an internal control to normalize CAD mRNA levels.

Transient transfection assays
Cells were seeded in DME/F12 medium supplemented with 2.5% charcoal-stripped serum. The reporter plasmids were cotransfected with ER{alpha}, ERß, or ER variant expression vectors using the calcium phosphate method for 5–6 h. Cells were then treated with dimethylsulfoxide (Me2SO) or 10 nM E2, and after 36 h cells were harvested in cell lysis buffer (Promega Corp.). Luciferase activities in the various treatment groups were performed on 30 µl of cell extract using the luciferase assay system (Promega Corp.) in a luminometer (Packard Instrument Co., Meriden, CT), and results were normalized to ß-galactosidase enzyme activity.

Gel EMSA
Cells were seeded in DME/F12 medium supplemented with 2.5% charcoal-stripped serum and treated with 10 nM E2 for 30 min. Nuclear extracts were obtained using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Chemical Co.). Oligonucleotides were synthesized, purified, and annealed, and 5 pmol of specific oligonucleotides were 32P-labeled at the 5'-end using T4 polynucleotide kinase and [32{gamma}P]ATP. Nuclear extracts were incubated in HEPES with ZnCl2 and 1 µg poly deoxyinosine-deoxycytidine for 5 min, 100-fold excess of unlabeled wild-type or mutant oligonucleotides were added for competition experiments and incubated for 5 min. The mixture was incubated with labeled DNA probe for 5 min, and antibodies were added for supershift experiments for 30 min on ice. The reaction mixture was loaded onto a 5% polyacrylamide gel and ran at 150 V for 2 h. The gel was dried and protein DNA complexes were visualized by autoradiography.

Chromatin immunoprecipitation (ChIP) assay
ZR-75 or MCF-7 cells were grown in 150-mm tissue culture plates and treated with 20 nm E2 for various times. Formaldehyde was then added to the medium to a final concentration of 1% and incubated with shaking for 10 min at room temperature followed by the addition of glycine (0.125 M) and incubation for 10 min; the media were then removed, and cells were washed with PBS and 1 mM phenylmethylsulfonyl fluoride, scraped, and collected by centrifugation. Cells were then resuspended in swell buffer (85 mM KCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin and aprotinin at pH 8.0) and homogenized. Nuclei was isolated by centrifugation at 1500 x g for 3 min, then resuspended in sonication buffer [1% sodium dodecyl sulfate (SDS); 10 mM EDTA; 50 mM Tris-HCl, pH 8.1], and sonicated for 45 sec. This extract was then centrifuged at 15,000 x g for 10 min at 4 C, aliquoted, and stored at -80 C until used. The cross-linked chromatin preparations were diluted in buffer (1% Triton X-100; 100 mM NaCl; 0.5% SDS; 5 mM EDTA; and Tris-HCl, pH 8.1), and 20 µl of Ultralink protein A or G or A/G beads (Pierce Chemical Co.) were added per 100 µl of chromatin and incubated for 4 h at 4 C. A 100-µl aliquot was saved and used as the 100% input control. Salmon sperm DNA, specific antibodies, and 20 µl of Ultralink beads were added, and the mixture was incubated overnight at 4 C. Samples were then centrifuged; beads were resuspended in dialysis buffer, vortexed for 5 min and centrifuged at 15,000 x g for 3 min. Beads were resuspended in immunoprecipitation buffer (11 mM Tris-HCl; 500 mM LiCl; 1% Nonidet P-40; and 1% deoxycholic acid, pH 8.0) and vortexed for 5 min at 20 C. The procedures with the dialysis and immunoprecipitation buffers were repeated (3–4x), and beads were resuspended in elution buffer (50 nM sodium bicarbonate, 1% SDS, 1.5 µg/m sonicated salmon sperm DNA), vortexed, and incubated at 65 C for 15 min. Supernatants were isolated by centrifugation and incubated at 65 C for 6 h to reverse cross-links. Wizard PCR kits (Promega Corp.) were used to purify DNA, and PCR was used to detect the presence of promoter regions immunoprecipitated with ER or Sp1 antibodies (Santa Cruz Biotechnology, Inc.). The primers -176 5'-CTT GGG GTG GGA GGG ACT-3' and -19 5'-GCG GCA GCA GCA GAG ACT-3' (CAD gene promoter) and +2465 5'-TGT AGT TCT TGA GCA CCT CG-3' and +2605 5'-TGC ACA AGT TCA CGT CCA TC-3' (cathepsin D, exon II) were synthesized and used for PCR analysis of immunoprecipitated DNA.

Inhibition of pCAD1 by small inhibitory RNA (siRNA) for Sp1 protein
Inhibitory RNAs (iRNAs) were prepared by IDT and targeted the coding region 153–173, 672–694, and 1811–1833 relative to the start codon of GL2, lamin B1, and Sp1 genes, respectively. Single-stranded RNAs were annealed by incubating 20 µM of each strand in annealing buffer (100 mM potassium acetate; 30 mM HEPES-KOH, pH 7.4; and 2 mM magnesium acetate) for 1 min at 90 C followed by 1 h at 37 C. Cells were cultured in six-well plates in 2 ml of DME/F12 medium supplemented with 5% fetal bovine serum. After 16–20 h when cells were 50–60% confluent, iRNA duplexes and reporter gene constructs (pCAD1) were transfected using LipofectAMINE Plus Reagent (Invitrogen Life Technologies, Carlsbad, CA). The effects of iSp1 on hormone-induced transactivation of CAD gene was investigated in ZR-75 cells treated with 15 nM E2 and transfected with pCAD1 (500 ng) and ER{alpha} expression plasmid (200 ng). iRNA duplex (0.75 µg) was transfected in each well to give a final concentration of 50 nM. Cells were harvested 48 h after transfection by manual scraping in 1x lysis buffer (Promega Corp.).

Statistical analysis
Experiments were repeated two or more times, and data are expressed as the mean ± SE for at least three replicates for each treatment group. Statistical differences between treatment groups were determined by Scheffé’s test. Treatments were considered significantly different from controls if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
1. Hormone-induced regulation of cad gene expression in breast cancer cells
E2-responsive MCF-7 and ZR-75 cells were maintained in serum-free media before treatment with 10 nM E2 for 1, 3, 6, 12, and 24 h. Cad mRNA levels were determined at all time points in both cell lines (Fig. 1Go) and significant induction was observed in MCF-7 cells after 3 and 6 h (5- and 3-fold increase) and after 12 h (>4.5-fold) in ZR-75 cells. Although the time course induction of Cad mRNA levels by E2 was different in MCF-7 and ZR-75 cells, the induced mRNA appeared to be transient in both cell lines. The proximal growth-responsive region of the human and hamster cad gene promoter are similar and contain two upstream GC-rich binding sites and 1 (hamster) or 2 (human) downstream E box motifs. pCAD1 contains the -90 to +115 region of the human CAD gene promoter linked to the firefly luciferase reporter gene (40). Treatment of MCF-7 or ZR-75 cells with E2 alone (10–100 nM) did not induce reporter gene activity (Fig. 1Go, C and D) suggesting that nongenomic hormonal activation of kinases may not be required for transactivation of the growth-responsive cad gene promoter. Studies in several laboratories have demonstrated that hormonal activation of E2-responsive promoters containing estrogen-responsive elements, activator protein-1, and GC-rich motifs is minimal in ER-positive breast cancer cells without cotransfection with ER{alpha} (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50). This has been attributed to overexpression of the construct in the transfected cells (47) where ER becomes limiting and E2-responsiveness is restored by cotransfection with ER{alpha} or ERß expression plasmids. The results in Fig. 1Go, C and D, show that E2 induced luciferase activity only in MCF-7 or ZR-75 cells cotransfected with ER{alpha} and pCAD1, whereas hormone-induced transactivation was not observed in cells cotransfected with ERß. Further analysis of domains of ER{alpha} required for activation of pCAD1 were investigated using ER{alpha} variants containing mutations in helix 12 of activation function 2 (AF2; taf1) and deletions of AF2 (HE15), AF1 (HE19), and the DNA-binding domain/hinge (HE11) regions. ER{alpha}-null contains both mutations in helix 12 and deletion of AF1 (Fig. 2Go). MCF-7 and ZR-75 cells were transfected with pCAD1 (Fig. 2Go, A and B) plus wild-type or variant ER{alpha} expression plasmids. Treatment with 10 nM E2 induced luciferase activity only in cells transfected with ER{alpha}, HE11 or taf1 in ZR-75 cells. In MCF-7 cells, hormone inducibility was observed in cells cotransfected with ER{alpha} and HE11; however, transactivation by HE19 and taf1 was dependent on promoter context. The fold-inducibility of pCAD constructs was higher in ZR-75 cells (>8-fold) transfected with ER{alpha} compared with MCF-7 cells (<3.5-fold); inducibility in MCF-7 and ZR-75 cells transfected with HE11 or TAF1 was 2- to 3-fold. These data are consistent with results previously observed for other genes regulated by ER{alpha}/Sp1 where transactivation is observed for wild-type ER{alpha} and HE11, demonstrating that DNA binding by ER{alpha} is not required (23, 24, 51, 52).



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  		Figure 1. Hormone induced activation of cad gene/reporter gene expression. Northern analysis of cad mRNA from MCF-7 (A) and ZR-75 (B) cells. The cells were treated with Me2SO for 24 h (lane 1) and 10 nM E2 for 1, 3, 6, 12, and 24 h (lanes 2–5, respectively). Cell extracts were obtained, and total RNA was isolated and subjected to Northern analysis as described in Materials and Methods. The intensity values were quantified using a PhosphorImager and were normalized to the values of ß-tubulin mRNA. Significant induction (P < 0.05) is indicated by an asterisk. Activation of pCAD1 in MCF-7 [C] and ZR-75 [D] cells. Cells were transfected with pCAD1 and 50 ng of ER{alpha} or ERß expression plasmids, and the effects of E2 on luciferase activity were determined as described in Materials and Methods. Results are expressed as means ± SD for at least three replicate determinations for each treatment group, and significant (P < 0.05) induction is indicated by an asterisk.

 


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  		Figure 2. Effect of wild-type and variant ER{alpha} on pCAD1 [A, B] and pCAD2 [C, D] in MCF-7 and ZR-75 cells. Cells were transfected with pCAD1 or pCAD2 and ER{alpha} or HE11, HE15, HE19, ER null, and TAF1. pSp13 is used as a positive control for HE11 in ZR-75 cells. Luciferase activity was determined as described in Materials and Methods. Results are expressed as means ± SD for at least three replicate determinations for each treatment group and significant (P < 0.05) induction is indicated with an asterisk.

 
2. Deletion analysis of the CAD gene promoter
The E2-responsive -90 to +115 region of the human CAD gene promoter contains three GC-rich Sp1 binding sites and two E-boxes. E2 induced reporter gene activity in MCF-7 (Fig. 3AGo) and ZR-75 (Fig. 3BGo) cells transfected with pCAD1 which contains both GC-rich and E-box motifs. Deletion analysis of the growth-responsive region of the CAD gene promoter gave a unique pattern of responses in both cell lines. The effects of successive 5'-deletions of the upstream GC-rich sites on basal activity and E2-responsiveness was determined in transient transfection studies using pCAD1, pCAD3, pCAD5, pCAD7, and pCAD8. Basal activity observed with these constructs in MCF-7 cells decreased with increasing deletion of GC-boxes 1–3; however, GC-box 2 appeared to be the most essential element for high basal activity. Moreover, E2 responsiveness of pCAD1, pCAD3, pCAD5, pCAD7, and pCAD8 in MCF-7 cells was dependent on GC-boxes 1 and 2 but not 3, and GC-box 2 was primarily responsible for E2-mediated transactivation. Constructs containing GC-box 3 alone (pCAD9, pCAD5, pCAD6, or pCAD10) or in combination with the two E-box motifs (pCAD5) were E2 nonresponsive. Transfection of constructs containing the E-box motifs alone (pCAD7 and pCAD8) or comparison of activities associated with deletion of the E-boxes (e.g. pCAD1 vs. pCAD2; pCAD3 vs. pCAD4) indicated that in MCF-7 cells these motifs were not E2-responsive and inhibited basal activity in MCF-7 cells. These results are in contrast to previous studies with both the human and hamster CAD gene promoter constructs in other cell lines where the E-box regions were primarily responsible for high basal activity (40, 53, 54, 55). In ZR-75 cells transfected with the same constructs (Fig. 3BGo), there was a higher level of E2-inducibility and less variability in basal activity compared with MCF-7 cells. Deletion of the E-boxes in constructs containing GC-boxes 1–3 (pCAD1 vs. pCAD2) or GC-boxes 2–3 (pCAD3 vs. pCAD4) resulted in increased basal activity and hormone-inducibility in ZR-75 cells, whereas in MCF-7 cells (Fig. 3AGo), only increased basal activity was observed. In ZR-75 cells, a comparison of a series of constructs containing successive deletion of GC-boxes 1–3 (pCAD1/pCAD3/pCAD5 and pCAD2/pCAD4/pCAD9, pCAD6 and pCAD10) indicated that E2 responsiveness was primarily associated with GC-boxes 1 and 2. The hormone inducibility observed for pCAD9 (-47 to +60), which contains GC-box 3 was not observed for pCAD6 (-47 to +25) or pCAD10 (-30 to +25), suggesting that a GC-box-independent region between +60 and +25 retained some hormone responsiveness. Constructs containing the E-boxes (pCAD7 and pCAD8) or GC-box 3 and promoter sequences up to the first E-box were also E2 responsive. These results suggest that the E-box and/or +25 to +60 region of the CAD gene promoter exhibited E2-inducibility in ZR-75 but not MCF-7 cells; however, GC-boxes 1 and 2 were the major E2-responsive sites in the human CAD gene promoter. The effects of mutation analysis of the CAD promoter GC-boxes on E2-responsiveness were also investigated in ZR-75 cells (Fig. 3CGo). Mutation of GC box 2 (pCAD1m1) did not eliminate E2 responsiveness; however, deletion of both GC boxes 1 and 2 (pCAD1m2) resulted in the loss of hormone responsiveness. These results further confirm that cooperative binding to both GC-rich motifs contribute to hormone-dependent activation of the Cad gene promoter constructs.



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  		Figure 3. Deletion and mutation analysis of pCAD constructs in MCF-7 [A, C] and ZR-75 [B] cells. Cells were transiently transfected with various pCAD deletion/mutation constructs, 500 ng of human ER{alpha} expression plasmid and treated with Me2SO or 10 nM E2; luciferase activity was determined as described in Materials and Methods. Results are expressed as means ± SD for at least three replicate determinations for each treatment and significant (P < 0.05) induction is indicated by an asterisk.

 
3. Protein interactions with the cad gene promoter
Interactions of nuclear extracts from MCF-7 and ZR-75 cells treated with Me2SO (lanes 6 and 12) or 10 nM E2 (lanes 1–5 and 7–11) with the GC-rich [32P]CADa1 and [32P]Sp1 oligonucleotides were investigated in gel mobility shift assays (Fig. 4Go, A and B). Nuclear extracts from both cell lines bound [32P]Sp1 to give several bands which have previously been identified as Sp1 and Sp3 proteins (27, 28, 29, 30, 31). A major low mobility Sp1-[32P]Sp1 complex (indicated with an arrow) was observed (lanes 1, 3, 5, and 6) and this band was unaffected by nonspecific IgG antibody (lane 1) or competition with unlabeled mutant Sp1 oligonucleotide (lane 3); in contrast, Sp1 antibody supershifted (SS) this complex (lane 2), and all bands were competitively decreased using unlabeled wild-type Sp1 oligonucleotide (lane 4). There was an increase in intensity of the Sp1-DNA complex band using nuclear extracts from MCF-7 cells treated with E2 (Fig. 4AGo, lane 5) compared with Me2SO (Fig. 4AGo, lane 6); comparable differences in band intensities were not observed in ZR-75 cells (Fig. 4BGo, lanes 5 and 6). The patterns of low mobility nuclear extract-[32P]CADa1 bands were similar to those observed using [32P]Sp1; however, there were several additional higher mobility complexes observed using [32P]CAD1 (lanes 6–12). The Sp1-[32P]CADa1 and Sp1-[32P]Sp1 complexes exhibited similar mobilities (lanes 7–12 vs. 1–6; complex indicated with an arrow). The specifically bound Sp1-[32P]CADa1 complex (lanes 7–9, 11, and 12) was unaffected after coincubation with nonspecific IgG (lane 7) or mutant unlabeled Sp1 oligonucleotide (lane 9), whereas Sp1 antibody supershifted (SS) the complex to give a band (lane 8) with similar mobility to the supershifted complex observed in lane 2; and coincubation with consensus Sp1 oligonucleotide (lane 10) decreased intensities of the higher mobility complexes. The lower mobility complexes were not affected by coincubation with Sp1 antibody or consensus oligonucleotide and were not further investigated.



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  		Figure 4. Sp1 and USF-1 binding to CAD promoter. Nuclear extracts from MCF-7 (A) or ZR-75 (B) cells treated with Me2SO (lanes 6 and 12) or 10 nM E2 (lanes 1–5 and 7–11) were incubated with [32P]Sp1 or [32P]CADa1 [containing the proximal GC-rich (-75 to -39) region of the cad promoter] and unlabeled wild-type or mutant Sp1 oligonucleotides, Sp1 antibody, or nonspecific IgG as described in Materials and Methods. C, Binding of USF-1 to [32P]CADE (+54 to +78) or [32P]mutCADE in MCF-7 cells. Nuclear extracts from MCF-7 cells treated with Me2SO or 10 nM E2 were incubated with wild-type or mutant [32P]-labeled CADE and unlabeled wild-type or mutant E-box oligonucleotides, USF-1 antibody, or nonspecific IgG as described in Materials and Methods.

 
Nuclear extracts from MCF-7 cells were also incubated with [32P]CADE which contained the E-box motif in the +54 to +78 region of the human CAD gene promoter. Incubation of [32P]CADE with nuclear extracts gave a retarded band (lane 1, indicated with an arrow), which was competitively decreased by a 50-fold excess of unlabeled wild-type CADE (lane 3) but not mutant CADE oligonucleotide (lane 4). This specifically bound band was supershifted by USF1 antibody (lane 5) but not by nonspecific IgG (lane 6). The specifically bound complex was not observed using radiolabeled mutCADE (lane 7); however, two more mobile bands were observed and only the more mobile band was formed with wild-type [32P]CADE. Proteins interacting with [32P]CADE to form the less mobile band were not identified. These results demonstrate that USF1/2 in nuclear extracts of MCF-7 cells bind the E-box motifs in the CAD gene promoter, and interactions of USF1/2 in MCF-7 cell nuclear extracts with the E-box major late promoter element in the cathepsin D gene promoter has previously been reported (33, 56).

The results in Fig. 5AGo investigate the role of dominant negative Sp1 expression plasmid (pBGENSp1) vs. empty vector (pBGENO) on hormone inducibility in MCF-7 and ZR-75 cells. The empty vector alone slightly decreased hormone inducibility; however, the results show that dominant negative Sp1 significantly inhibited E2-induced transactivation in MCF-7 and ZR-75 cells transfected with pCAD1, thus confirming the role of Sp1 in this response. We have further investigated the role of Sp1 in mediating hormone-induced cad gene expression using an siRNA duplex that targets Sp1 mRNA resulting in down-regulation of both Sp1 mRNA and protein (57). Transfection of siRNA into MCF-7 or ZR-75 cells significantly decreases Sp1 protein in whole cell extracts (50–70%) and both basal and hormone inducibility in cells transfected with pSp13 (57). The results in Fig. 5BGo show that E2 induced luciferase activity in ZR-75 cells transfected with pCAD1, and both basal and induced responses were inhibited 60% by siRNAs targeted to the luciferase reporter gene (iGL2) and Sp1 (iSp1), whereas siRNA targeted to lamin B had no effect. These results further confirm the role of Sp1 in hormone-induced transactivation of CAD through interaction of the ER{alpha}/Sp1 complex with GC-rich motifs.



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  		Figure 5. Role of Sp1 in hormonal activation of pCAD1. A, Dominant negative Sp1. Cells were transfected with pCAD1, treated with E2, cotransfected with dominant negative Sp1 expression plasmid (pBGENSP1) or empty Vector pBGEN0, and luciferase activity was determined as described in Materials and Methods. The ratio of pCAD1:pBGENSP1 (1:1) resulted in significant (P < 0.05) *, Inhibition of E2-induced luciferase activity in both ZR-75 and MCF-7 cells. [B] siRNA for Sp1. ZR-75 cells were transfected with pCAD1 and siRNAs for lamin C (iLMN), luciferase (iGL2), and Sp1 (iSp1) treated with 10 nM E2 or Me2SO and luciferase activity determined as described in Materials and Methods. Results are expressed as means ± SD for at least three replicate determinations for each treatment group and significant (P < 0.05) inhibition in cells transfected with iGL2 and iSp1 compared with cells transfected with control or iLMN is indicated with an asterisk.

 
Confirmation of ER{alpha}/Sp1 interactions with the CAD gene promoter were investigated using the chromatin immunoprecipitation assays (Fig. 6Go) and PCR primers directed to the -176 to -19 region of the promoter. Sp1 antibodies immunoprecipitated the CAD promoter in untreated ZR-75 cells and in cells treated with 10 nM E2 for 30, 60, and 90 min (Fig. 6Go). ER{alpha} (H184) antibodies gave weak to nondetectable bands where the ER{alpha} (G20) antibody showed a hormone-induced increase in ER{alpha} interactions with the CAD gene promoter. Previous studies have reported interaction of the Brahma-related gene 1 (BRG-1) with hormone-responsive gene promoters (58, 59), and in this study we detected BRG-1 associated with the CAD gene promoter in the presence or absence of hormone. These data confirm interactions of ER{alpha} and Sp1 with the human CAD gene promoter in ZR-75 cells, and current studies are investigating interaction of other nuclear cofactors required for ER{alpha}/Sp1 action.



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  		Figure 6. Analysis of ER{alpha} and Sp1 interactions with the Cad gene promoter by ChIP. A, CAD gene promoter. ZR-75 cells were treated with 20 nM E2 for different time points and after immunoprecipitation of cross-linked complexes, the chromatin was analyzed by PCR as described in Materials and Methods. Single PCR products were obtained for all antibodies, and the identity of these bands was confirmed using Southern analysis and radiolabeled probes derived from the CAD promoter (data not shown). B, Cathepsin D gene promoter. As a control, we also investigated the interactions of ER{alpha}, Sp1, and Brg-1 with the +2465 to +2605 region [E2 nonresponsive (30 32 )] of the cathepsin D gene promoter by PCR as described in Materials and Methods. Only minimal to nondetectable bands were observed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The CAD gene encodes enzymes required for the first three steps in de novo pyrimidine synthesis, and previous studies in MCF-7 cells showed that E2 activated two of these enzyme activities, carbamylphosphate synthetase and aspartate carbamyltransferase (14). Hormonal activation of CAD is consistent with the mitogenic activity of E2 and the increase of purine and pyrimidine pools required for DNA synthesis and cell division. Previous studies have reported that serum or mitogenic stimulation of various cancer cell lines was accompanied by a parallel increase in CAD gene expression at the G1/S phase boundary of the cell cycle (40, 53, 54, 55, 60). Farnham and co-workers (40, 53, 54, 55) have extensively investigated regulation of the hamster cad gene and the growth-responsive proximal region of the cad gene promoter. Their results indicate that growth-dependent regulation of the hamster cad gene promoter is linked to the proto-oncogene c-myc and formation of Myc-Max heterodimers that bind to the E-box motif. The human and hamster CAD gene promoters are similar and both contain upstream GC-rich sites; however, the human promoter contains two E-boxes (5' and 3'), whereas only the 5' E-box is expressed in the hamster promoter. Recent studies in NIH 3T3 cells indicate that the 5' E-box in the human promoter is the major growth-responsive element and transfected c-Myc preferentially activates CAD gene promoter constructs through this motif (40).

E2 activates CAD mRNA levels in MCF-7 and ZR-75 cells (Fig. 1Go) and also reporter gene activity in cells transfected with pCAD1, which contains the growth-responsive -90 to +115 region of the CAD gene promoter (Fig. 2Go). Previous studies have demonstrated that E2 transiently induces c-myc gene expression in MCF-7 cells, and a synthetic antisense c-myc phosphorothioate oligonucleotide inhibited c-myc protein expression and partially inhibited E2-induced growth of MCF-7 cells (61, 62). Another study indicated the c-myc and other protooncogenes (c-fos and c-jun) were not growth rate limiting in MCF-7 cells (63). Deletion and mutation analysis of the CAD gene promoter in MCF-7 and ZR-75 cells clearly demonstrates that E-box motifs that bind Myc-Max are not essential for basal or hormone-induced transactivation (Fig. 3Go). Previous studies have demonstrated that the E-box motif within the major late promoter element in the proximal -120 to -101 region of the cathepsin D gene promoter binds USF1/2, which are highly expressed in nuclear extracts of MCF-7 cells (35, 56). Not surprisingly, the +54 to +78 E-box in the cad gene promoter also forms a USF1/2-DNA retarded band complex after incubation with nuclear extracts from MCF-7 cells (Fig. 4CGo). Therefore, the high expression of USF1/2 in MCF-7 cells and subsequent binding to the cad promoter E-boxes may competitively inhibit hormone-induced myc complexes from binding and activating cad gene expression from the E-box motif. Sp1 protein interacts with the GC-rich motifs within the Cad gene promoter (Fig. 4Go), and further analysis by ChIP confirms interaction of both ER{alpha} and Sp1 with the proximal region of the cad promoter. ChIP has previously shown that ER{alpha} and Sp1 proteins also bind GC-rich regions of other E2-responsive genes (30, 32), and we are currently investigating the temporal interactions of ER{alpha}, Sp1, and other cofactors with their respective GC-rich motifs.

Deletion analysis of the CAD gene promoter demonstrates that the GC-rich motifs are required for hormone-induced transactivation in ER-positive MCF-7 and ZR-75 cells. The pattern of activation by wild-type and variant ER{alpha} was comparable in both cell lines in which deletion of the DNA binding domain (HE11) did not result in loss of hormone inducibility in both cell lines transfected with pCAD1 (Fig. 2Go). These results are consistent with previous studies on other GC-rich promoters activated by ER{alpha}/Sp1 because transactivation does not require the DNA binding domain of ER{alpha} (23, 24, 51). The role of ER{alpha}/Sp1 in activation of Cad gene expression was further supported by the inhibitory effects of both dominant negative Sp1 and siRNA for Sp1 (Fig. 5Go). Recent studies in the laboratory have demonstrated that iSp1 selectively decreases Sp1 protein in MCF-7 and ZR-75 cells and blocks basal and hormone-induced transactivation in cells transfected with a GC-rich (pSp13) complex (57). The pattern of responses for activation/inactivation of pSp13 (57) and pCAD1 in MCF-7 cells cotransfected with iSp1 were identical (Fig. 5Go), thus confirming that Sp1 protein is required for hormone-induced transactivation in cells transfected with pCAD1.

In summary, results of this study have demonstrated that the reported hormone-dependent increase in cad activity in breast cancer cells (14) is accompanied by induced gene expression (Fig. 1Go) that is linked to ER{alpha}/Sp1 interactions with GC-rich motifs. Many of the genes regulated by ER{alpha}/Sp1 in ER-positive breast cancer cells play a role in purine/pyrimidine biosynthesis (cad, thymidylate synthase) and metabolism (adenosine deaminase) and cell proliferation (cyclin D1, E2F1, c-fos, and bcl-2). These observations are consistent with a recent report showing that siRNA for Sp1 inhibits hormone-induced cell cycle progression in MCF-7 cells (57). Activation of ER{alpha} through interaction with estrogen-responsive element motifs is primarily AF2 dependent, whereas ER{alpha}/Sp1 depends, in part, on the AF1 domain of ER{alpha} (51). Most AF2-dependent coactivators do not enhance ER{alpha}/Sp1-mediated transactivation; however, Brg-1 which interacts with the GC-rich region of the Cad gene promoter (Fig. 5Go) also coactivates ER{alpha}/Sp1 (data not shown). Current studies are investigating molecular mechanisms of ER{alpha}/Sp1 action and coactivator/coregulatory proteins required for this hormone-regulated pathway.


   Footnotes
 
The financial assistance of the NIH (CA-76636 and ES-09106), the Department of Defense Breast Cancer Research Program, and the Texas Agricultural Experiment Station is gratefully acknowledged.

Abbreviations: AF, Activation function; ChIP, chromatin immunoprecipitation; E2, 17ß estradiol; ER, estrogen receptor; iRNA, inhibitory RNA; Me2SO, dimethylsulfoxide; SDS, sodium dodecyl sulfate; siRNA, small inhibitory RNA.

Received December 16, 2002.

Accepted for publication February 5, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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