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Endocrinology Vol. 144, No. 5 1675-1685
Copyright © 2003 by The Endocrine Society

Cell Context-Dependent Differences in the Induction of E2F-1 Gene Expression by 17ß-Estradiol in MCF-7 and ZR-75 Cells

Sharon Ngwenya and Stephen Safe

Department of Veterinary Physiology and Pharmacology (S.S.) and Department of Biochemistry and Biophysics (S.N., S.S.), 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, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
17ß-Estradiol (E2) induces E2F-1 gene expression in ZR-75 and MCF-7 human breast cancer cells. Analysis of the E2F-1 gene promoter in MCF-7 cells previously showed that hormone-induced transactivation required interactions between estrogen receptor {alpha} (ER{alpha})/Sp1 bound to upstream GC-rich sites and NFYA bound to downstream CCAAT sites within the -169 to -54 region of the promoter. This same region of the E2F-1 promoter was also E2 responsive in ER{alpha}-positive ZR-75 cells; however, further analysis of the promoter showed that cooperative ER{alpha}/Sp1/NFY interactions were not necessary for hormone-induced transactivation in ZR-75 cells. The upstream GC-rich motifs (-169 to -111) are activated independently by ER{alpha}/Sp1 in ZR-75 but not MCF-7 cells, and a construct (pE2F-1jm1) containing the -122 to -54 downstream CCAAT site that bound NFYA was also E2 responsive. E2 also induced reporter gene activity in ZR-75 cells transfected with an expression plasmid for a chimeric protein containing the DNA-binding domain of the yeast GAL4 protein fused to NFYA (pM-NFYA) and a construct containing five tandem GAL4 response elements. Subsequent studies showed that hormonal activation of pE2F-1jm1 and pM-NFYA are dependent on nongenomic pathways in which E2 activates cAMP/protein kinase A. Hormone-dependent regulation of E2F-1 gene expression in ZR-75 and MCF-7 involves the same cis elements and interacting transcription factors but different mechanisms, demonstrating the importance of cell context on transactivation pathways, even among ER-positive breast cancer cell lines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CELL CYCLE PROGRESSION and proliferation of tumors and normal tissues/cells are controlled by a constellation of factors that coordinately regulate one or more growth-dependent pathways (1, 2, 3, 4, 5). Many of the critical genes required for cell proliferation modulate progression through different phases of the cell cycle and E2F family members play a critical role in G1->S phase progression (6, 7, 8, 9). Interactions of retinoblastoma (Rb) proteins with E2F suppresses transcription of genes that contain critical E2F-binding sites; however, phosphorylation of Rb results in dissociation of the Rb-E2F complex and subsequent up-regulation of E2F-dependent genes (9, 10, 11, 12). E2F-1 was the first E2F family member identified (13, 14, 15), and several studies have characterized E2F-1-dependent expression of genes required for cell proliferation, and this is related to the oncogenic activity of this transcription factor (6, 7, 8, 9).

Although regulation of E2F-1-dependent transactivation is closely linked to Rb phosphorylation, E2F-1 expression is also modulated by many other nuclear transcription factors and coregulatory proteins (16, 17, 18, 19, 20). For example, p300/CBP-associated factor acetylates E2F-1, and this enhances the transcriptional activity of E2F-1 in U20S cells (16).

Johnson and colleagues (18, 19) investigated cell cycle-dependent activity of constructs containing E2F-1 gene promoter fragments in REF-52 cells treated with serum and based on 5'-deletion analysis; the -204 to -122 region of the promoter was required for maximal responses. The E2F-1-binding sites in the proximal region of the promoter were primarily required for negative control of the E2F-1 promoter in G0 and early G1. Research in this laboratory showed that 17ß-estradiol (E2) induced E2F-1 mRNA and protein levels in estrogen receptor (ER)-positive MCF-7 breast cancer cells, and the induction response was linked to cooperative ER{alpha}/Sp1/NFY interactions that involved three GC-rich (-169 to -116) and two CCAAT (-122 to -54) binding sites (21). The downstream CCAAT sites bound NFYA/NF-YB and were required not only for hormone-dependent activation of E2F-1 but also for basal activity of the E2F-1 promoter. In contrast, these sites play a minimal role in basal activity of the E2F-1 promoter in REF-52 cells (18). Studies in this laboratory have also used ER-positive ZR-75 breast cancer cells for investigating molecular mechanisms of hormone-induced transactivation of E2-responsive genes. In this study, we have compared hormonal activation of constructs containing the GC-rich and CCAAT motifs in the -173 to -54 region of the E2F-1 gene promoter in MCF-7 and ZR-75 cells. The cooperative ER{alpha}/Sp1/NFY interactions required for hormone activation in MCF-7 (21) cells were not necessary in ZR-75 cells in which the GC-rich and CCAAT sites were independently activated by ER{alpha}/Sp1 and cAMP/protein kinase A (PKA)-dependent activation of NFYA, respectively. Previous studies have shown that E2 activates the cAMP/PKA through nongenomic pathways in different mammalian cells including breast cancer cells (22, 23, 24, 25, 26). Thus, our results are consistent with both activation of cAMP/PKA by E2 and the reported cAMP-dependent activation of NFYA in mediating transactivation of human tissue inhibitor of metalloproteinases-2 (TIMP-2) in human breast cancer cells (27).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals, cells, and antibodies
MCF-7 and ZR-75 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in DME/F12 medium with phenol red and supplemented with 2.2 g/liter sodium bicarbonate, 10% fetal bovine serum (FBS), and 10 ml antibiotic-antimycotic solution (Sigma, St. Louis, MO) in an air:carbon dioxide (95:5) atmosphere at 37 C. For transient transfection studies, cells were grown for 1 d in DME/F12 medium without phenol red and 2.5% FBS treated with dextran-coated charcoal, and for Northern blot analysis, the same media without phenol red and FBS-free media were used for at least 48 h before treatment with E2 and other chemicals. E2 was purchased from Sigma. The wild-type ER expression plasmid was provided by Ming-Jer Tsai (Baylor College of Medicine, Houston, TX). Recombinant ER was obtained from Pan Vera Corp. (Madison, WI). The ER deletion mutants HE11, HE15, and HE19 were provided by Pierre Chambon (Institut de Genitique et de Biologie Moleculaire et Cellulaire, Illkirch, France). Dimethyl sulfoxide (DMSO) was used as a solvent for E2, SQ22536, and 8-bromo-cAMP. SQ22536 and 8-bromo-cAMP were purchased from Calbiochem (La Jolla, CA). Sp1, Sp3, and NF-1 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The NFYA antibody was purchased form Rockland, Inc. (Gilbertsville, PA). Luciferase and ß-galactosidase enzyme assay systems are purchased from Promega Corp. (Madison, WI).

Cloning and plasmids
The constructs pE2F-1a, pE2F-1b, and pE2F-1d were kindly provided by Dr. J. R. Nevins (Duke University, Durham, NC). The pE2F-1c constructs were made by RT-PCR (forward primer, 5'-CCGCCATTGGCCGTACCGCCCC-3'; reverse primer, 5'-GATCTTCCCGGCCACTTTTACGCGCCAAA-3') and inserted into pGL2 basic vector (Promega Corp.) at Sac1 and BglII cloning sites. The remaining E2F-1 promoter constructs (pE2F-1e, pE2F-1f, pE2F-1 g, pE2F-1 h, pE2F-1 hm1, pE2F-1 hm2, pE2F-1 hm3, pE2F-1 hm4, pE2F-1 hm5, pE2F-1i, pE2F-1j, pE2F-1jm1, pE2F-1jm2, pE2F-1k, pE2F-1l) were made by inserting synthetic oligonucleotides into the pGL2 basic vector digested with SacI and BglII enzymes at the cloning sites. Oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Resulting plasmids were sequenced at the Gene Technology Laboratory (Texas A&M University, College Station, TX) to confirm appropriate insertion of the oligonucleotide inserts. The sequences of Sp1 (CCGCCCC) and CCAAT protein-binding sites in the E2F-1 promoter have been mutated into CCtttCC and atgcT, respectively, in all constructs containing mutations in these sites. The PKA expression plasmid was provided by Dr. R. Maurer (Oregon Health Sciences University, Portland, OR). The expression plasmids of wild-type NFYA, NF-YB and mutant NFYA ({Delta}4YA13m29), and control plasmid ({Delta}4YA13) were kindly provided by Dr. Roberto Mantovani (Universita di Milano, Milan, Italy). Gal4-luc was provided by Dr. M. Mayo (University of North Carolina, Chapel Hill, NC). The pM-NFYA expression plasmid was made by PCR using primers (forward primer, 5'-GGA ATT CAT GGA GCA GTA TAC GAC A-3'; reverse primer, 5'-GCT CTA GAT TAG GAA ACT CGG ATG A-3') to amplify full-length NFYA using the NFYA expression plasmid as a template. The amplified products were cloned into the pM vector (CLONTECH Laboratories, Palo Alto, CA) between EcoRI and XbaI cloning sites.

Transient transfection and luciferase assays
MCF-7 and ZR-75 cells were seeded in 12-well Falcon plates in DME/F12 medium supplemented with 2.5% dextran-coated charcoal FBS and grown until they were 70% confluent. Plasmids (500 ng) were transiently cotransfected with the ER expression plasmid (500 ng) using the calcium phosphate method. Cells were incubated for 4–6 h and then shocked with 25% glycerol in PBS and treated with DMSO, E2, and kinase inhibitors or their combinations in DMSO for 44–48 h. The cells were harvested in cell lysis buffer (Promega Corp.) Cell lysates were prepared by freeze thawing followed by centrifugation at 14,000 x g for 1 min. Luciferase activity was then determined in a luminometer (Packard Instruments Co., Meriden, CT) with a luciferase assay kit (Promega Corp.) and normalized to ß-galactosidase enzyme activity obtained after transfection with a ß-galactosidase-lacZ plasmid (500 ng) obtained from Invitrogen (Carlsbad, CA). The experiments for each treatment group were carried out at least in triplicate.

Northern blot analysis
MCF-7 and ZR-75 cells were seeded and grown as described above and treated with DMSO or E2. RNA was extracted using an RNA extraction kit (Tel-Test, Friendswood, TX). Total RNA (25 µg) was separated on a 1.2% agarose/1 M formaldehyde gel and transferred onto nylon membrane. The membrane was then exposed to UV light for 5 min to cross-link RNA to the membrane and then baked at 80 C for 2 h. The membrane was prehybridized in a solution containing 0.1% BSA, 0.1% Ficoll, 0.1% polyvinyl pyrollidone, 10% dextran sulfate, 1% sodium dodecyl sulfate, and 5x SSPE (0.75 M sodium chloride, 50 mM NaH2PO4, 5 mM EDTA) for 18–24 h at 60 C and hybridized in the same buffer for 24 h with the [32P]-labeled DNA probe (106 cpm/ml). The E2F-1 cDNA probes (21) were labeled with [{gamma}-32P]dCTP using the random primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis, IN). The resulting blots were visualized using a Storm 860 instrument (Molecular Dynamics, Inc., Sunnyvale, CA) and quantitated using an Instantimager (Packard Instruments Co.). ß-Actin mRNA was used as an internal control to standardize E2F-1 mRNA levels.

Preparation of nuclear extracts
Nuclear extracts were prepared from cells treated with DMSO or E2. Harvested cells were washed twice in 30 ml HEGD buffer (25 nM HEPES, 1.5 mM EDTA, 1 nM dithiothreitol, and 10% glycerol, pH 7.6). The pellet was then incubated for 10 min in 1 ml HED buffer (HEGD without the glycerol). Cells were then transferred to a 2-ml homogenizing tube and homogenized using a Teflon/pestle drill apparatus. The homogenate was transferred to a centrifuge tube, centrifuged at 4000 x g for 10 min, washed twice with HEGD, and finally resuspended in 2 ml HEGD containing 0.5 M sodium chloride and allowed to incubate at 4 C for 1 h.

Gel mobility shift assay with nuclear extracts
Synthetic oligonucleotides were synthesized, purified, annealed, and labeled at the 5'-end using T4-polynucleotide kinase and [{gamma}-32P] ATP. DNA binding was measured using a gel retardation assay. Nuclear extracts were incubated in HEGD with 1 µg poly[d(I-C)] and 1 M ZnCl for 10 min on ice to bind nonspecific DNA-binding proteins. Then 200-fold excess of unlabeled wild-type or mutant oligonucleotide competitors for the competition experiments were incubated with the nuclear extracts for 5 min on ice. The mixture was then incubated for 15 min at 20 C [32P]-labeled DNA probe. Antibodies were added for an additional 15 min for the supershift reactions. The reaction mixture was then loaded onto a 5% polyacrylamide gel and electrophoresed at 150 V for 2.5 h in 0.9 M Tris-borate and 2 mM EDTA, pH 8.0. The gel was dried and protein-DNA complexes were visualized using a Storm 860 instrument (Molecular Dynamics, Inc.).

Statistical analysis
The statistical difference among different groups was determined by ANOVA and t test. The data were expressed as means ± SEs or SDs. At least three determinations were carried out for each data point in the transfection and mRNA studies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparative analysis of the E2F-1 promoter in MCF-7 and ZR-75 cells
E2 induces E2F-1 gene expression in MCF-7 cells, and similar results were obtained in ZR-75 cells in which a more than 2.5-fold increase in E2F-1 mRNA levels were observed after treatment with 10 nM E2 for 6 h (Fig. 1AGo). Previous studies showed that the -173 to -54 region of the E2F-1 gene promoter exhibited high basal activity and, in transient transfection assays in MCF-7 cells, E2-induced reporter gene (luciferase) activity (21). The results in Fig. 1BGo show that E2F-1 h (containing the -169 to -54 E2F-1 promoter insert) was E2 responsive in both MCF-7 and ZR-75 cells. Basal and hormone-induced activity of a series of GC-rich and CCAAT mutants (pE2F-1 hm1-m5) exhibited both similarities and differences in the two cell lines. Compared with pE2F-1 h basal activity among the GC-rich mutants (pE2F-1 hm1, pE2F-1 hm2, and pE2F-1 hm3) varied by more than 4-fold in MCF-7 cells, and this primarily was due to site 2 in which mutation significantly increased basal activity; mutation of site 3 decreased activity. These differences in basal activity were only 2-fold in ZR-75 cells, and this also was due to the GC-rich site 2, suggesting that this motif may preferentially bind inhibitory factors such as Sp3.



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  		Figure 1. Hormone-induced transactivation of E2F-1 in breast cancer cells. A, Northern blot analysis in ZR-75 cells. Cells were treated with 10 nM E2 for 1, 3, 6, and 24 h, and E2F-1 and ß-tubulin mRNA levels were determined as described in Materials and Methods. E2F-1 mRNA levels in untreated cells (U) is also indicated, and significant (P < 0.05) induction is indicated with an asterisk. B–D, Deletion and mutation analysis of E2F-1 promoter constructs in MCF-7 (left) and ZR-75 (right) cells. Constructs containing wild-type or mutant E2F-1 gene promoter inserts from the hormone-responsive +173 to -54 region were transfected into MCF-7 or ZR-75 breast cancer cells, treated with DMSO (solvent control) or 10 nM E2, and luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) induction by E2 is indicated with an asterisk, and fold induction by E2 is also given. Results in A–D are expressed as means ± SE for three replicate determinations for each treatment group. The 100% control values were the DMSO treatments in cells transfected with pE2F-1 h (B), pE2F-1j (C), and pE2F-1 h (D).

 
Mutation of the upstream (pE2F-1 hm4) or downstream (pE2F-1 hm5) CCAAT sites significantly decreased basal activity in MCF-7 cells; in contrast, loss of basal activity in ZR-75 cells was observed only for the former construct, indicating important differences between contributions of the two CCAAT site in determining basal activity of this region of the E2F-1 gene promoter. The importance of the upstream CCAAT motif was also observed in hormone responsiveness of this series of mutants (Fig. 1AGo). The only major difference in the effects of E2 on this series of constructs was that mutation of the upstream or downstream CCAAT elements resulted in loss of E2 responsiveness in MCF-7 cells transfected with pE2F-1 hm4 or pE2F-1 hm5, whereas the latter (but not the former) construct was hormone responsive in ZR-75 cells. The results observed in MCF-7 cells were consistent with cooperative and ligand (E2)-dependent formation of an ER{alpha}/Sp1/NFYA complex in which both cis elements are required for transactivation (21). However, if this complex is functional in ZR-75 cells, then at least the downstream (3') CCAAT site is not required.

The results in Fig. 1CGo confirm that hormone inducibility in MCF-7 cells transfected with pE2F-1 is lost with constructs in which the GC-rich site is deleted (pE2F-1jm1) or the CCAAT sites are mutated (pE2F-1jm2). In contrast, both wild-type and mutant pE2F-1j constructs were hormone responsive in ZR-75 cells, demonstrating that the CCAAT sites alone were hormone responsive. Figure 1DGo investigates the activity of the three GC-rich sites alone in the -169 to -111 region of the E2F-1 gene (pE2F-1k) or in combination with the downstream (pE2F-1 h), upstream (pE2F-1l), or both upstream/downstream (pE2F-1 g) CCAAT motifs. Results obtained in MCF-7 cells clearly demonstrated that hormone responsiveness required both the GC-rich and downstream CCAAT motifs, whereas the GC-rich site alone was sufficient for E2-induced transactivation in ZR-75 cells.

Comparative activation of E2F-1 promoter constructs by wild-type and variant ER{alpha} expression plasmids
Previous studies (28, 29, 30, 31, 32, 33, 34) have demonstrated that E2 induced reported gene activity in MCF-7 or ZR-75 cells transfected with an E2-responsive GC-rich construct and wild ER{alpha} or a DBD deletion mutant (HE11). The results in Fig. 2AGo show that E2 induced luciferase activity in MCF-7 cells transfected with pE2F-1 h or pE2F-1j and wild-type ER{alpha} but not HE11. These results are consistent with the observation that hormone responsiveness in MCF-7 cells requires both GC-rich and CCAAT motifs to form an ER{alpha}/Sp1/NFYA complex that was not activated in cells transfected with HE11 (21). Thus, formation of the ER{alpha}/Sp1/NFY complex in MCF-7 cells does not allow direct activation of ER{alpha}/Sp1 on GC-rich sites alone. Both ER{alpha} and HE11 activated pE2F-1 h, pE2F-1j, and pE2F-1jm2 in ZR-75 cells, whereas pE2F-1jm1 containing only CCAAT sites was not induced by E2. This was consistent with the hormone responsiveness of the GC-rich sites alone in ZR-75 cells (29, 30, 31, 32, 33, 34) and contrasted to the results obtained in MCF-7 cells. ER{alpha} mutants containing N-terminal (HE19) and C-terminal (HE15) deletions were not hormone responsive in either cell line (data not shown).



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  		Figure 2. Comparative activation of E2F-1 constructs by wild-type and variant ER{alpha} constructs in breast cancer cells. Hormonal activation of constructs in MCF-7 (A) and ZR-75 (B), in breast cancer cells. MCF-7 (A) or ZR-75 (B) cells were cotransfected with constructs containing E2F-1 promoter inserts and ER{alpha} or HE11, treated with DMSO or 10 nM E2, and luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) induction is indicated with an asterisk, and results are expressed as means ± SD for three replicate determinations for each treatment group. The HE19 and HE15 deletion mutants were also used with these constructs; however, hormone inducibility was not observed (data not shown).

 
Role of NFYA in E2-induced transactivation of E2F-1 in MCF-7 and ZR-75 cells
E2 induces luciferase activity in MCF-7 and ZR-75 cells transfected with pE2F-1j (Fig. 3Go, A and B), and cotransfection with NFYA, NF-YB, or 4YA13, which does not exhibit dominant negative activity, did not affect the induction response. In contrast, cotransfection with the dominant negative 4YA13m29 expression plasmid for NFYA blocks hormone activation in MCF-7 cells, and this is consistent with the reported hormone-dependent ER{alpha}/Sp1/NFYA-mediated transactivation, which requires both GC-rich and CCAAT sites (21). In contrast, NFYA dominant negative decreases but does not eliminate hormone-induced transactivation in ZR-75 cells transfected with pE2F-1j (Fig. 3BGo), and this is consistent with E2 responsiveness of the GC-rich sites alone in ZR-75 cells (Fig. 1DGo). The effects of NFYA, NF-YB, 4YA13, and 4YA13m29 expression plasmids on hormone inducibility of pE2F-1k (Fig. 3CGo) and pE2F-1jm1 (Fig. 3DGo) were also investigated in ZR-75 cells. The results indicate that hormone inducibility in cells transfected with the GC-rich construct (pE2F-1k) were unaffected by any of the expression plasmids, whereas the dominant negative NFYA expression plasmid blocked induction of pE2F-1jm1, suggesting that NFYA plays a key role in direct hormone activation through the CCAAT motifs in ZR-75 cells.



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  		Figure 3. Effects of NFYA and transactivation. Transfection of pE2F-1j in MCF-7 (A) and ZR-75 (B) cells. Transfection of pE2F-1j in MCF-7 (A) and ZR-75 (B) cells. Cells were transfected with pE2F-1j and expression plasmids for human ER, NFYA, NF-YB, NFYA + NF-YB, 4YA13m29, or 4YA13, treated with DMSO or 10 nM E2, and luciferase activity was determined as described in Materials and Methods. Transfection of ZR-75 cells with pE2F-1k (C) or pE2F-1jm1 (D). Cells were transfected as described in (A) and (B), and luciferase activity determined as described in Materials and Methods. E, Effects of NFYA on hormonal activation of pERE. ZR-75 cells were transfected with pERE and different amounts (0.25–2.0 µg) of NFYA expression plasmid, treated with DMSO or 10 nM E2, and luciferase activity determined as described in Materials and Methods. Significant (P < 0.05) induction is indicated by an asterisk, and results are expressed as means ± SD for three replicated determinations for each treatment group. The 100% control values were the DMSO treatments in cells transfected pE2F-1j (A and B), pE2F-1k (C), pE2F-1jm1 (D), and pERE (E).

 
The results in Fig. 3CGo illustrate that dominant negative NFYA (4YA13m29) and NFYA expression plasmids do not affect ER{alpha}/Sp1-mediated activation of pE2F-1k, which contains three GC-rich Sp1 binding sites. In contrast, Farsetti et al. (35) showed that NFYA physically interacts with ER{alpha} and inhibits ER{alpha} action in NIH3T3 and HepG2 cells transfected with an ER element (ERE) construct. Moreover, this response was also observed using a form of NFYA that does not bind DNA. Our results show that NFYA does not affect ER{alpha}/Sp1-mediated activation of GC-rich constructs (Fig. 3CGo) and cotransfection with ER{alpha} activates constructs containing only CCAAT motifs (Figs. 1Go, 2Go, and 3DGo). Therefore, we investigated potential inhibitory interactions of NFYA on ER{alpha} in ZR-75 cells transfected with pERE3 (Fig. 3EGo). These results show that NFYA does not inhibit ER{alpha}-mediated transactivation in ZR-75 cells as previously reported in other cell lines (35), demonstrating that inhibitory NFYA-ER{alpha} interactions are dependent on cell context.

Interaction of NFY and Sp proteins with the E2F-1 promoters
Interactions of nuclear extracts from untreated and E2-treated MCF-7 and ZR-75 cells with [32P] -122/-54 oligonucleotide were investigated in gel mobility shift assays (Fig. 4AGo). A major protein-DNA-retarded band was observed with both extracts in MCF-7 (lanes 2 and 3) and ZR-75 (lanes 12 and 13) cells, and it was apparent that treatment with E2 did not affect retarded band intensities. In competitive DNA-binding experiments with unlabeled -122/-54 or consensus NFY oligonucleotides, there were significant decreases in retarded band intensities using extracts from MCF-7 (lanes 4 and 5) or ZR-75 (lanes 14 and 15) cells. In contrast, competition with unlabeled consensus NF-1, Sp1 (GC-rich), or ERE oligonucleotides had minimal effects on retarded band intensities (lanes 6–8 and 16–18). NF-1 antibodies did not supershift the retarded band in MCF-7 (lane 10) or ZR-75 (lane 20) cells. The results demonstrate that despite the differences in the requirements for the CCAAT sites for hormonal activation of constructs derived from the E2F-1 gene promoter, the pattern of retarded bands observed using [32P] -122/-54 was similar for nuclear extracts from MCF-7 or ZR-75 cells. Moreover, the results also indicate that hormonal activation of CCAAT sites in ZR-75 cells was not due to enhanced binding of E2-induced nuclear extracts to [32P] -122/-54.



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  		Figure 4. Gel EMSAs. A, Binding to -122/-54. Nuclear extracts from MCF-7 or ZR-75 cells were incubated with [32P] -122/-54 and various unlabeled oligonucleotides or antibodies (NFYA or NF-1) and analyzed by gel mobility shift assays as described in Materials and Methods. B, Binding to -169/-111. Nuclear extracts from MCF-7 or ZR-75 cells were incubated with [32P] -169/-111 and unlabeled nucleotides or antibodies (Sp1 or Sp3), and analyzed by gel mobility shift assays as described in Materials and Methods. Results in these assays were observed in duplicate experiments.

 
Interaction of nuclear extracts from both cell lines with the GC-rich -169 to -111 region of the E2F-1 promoter were also comparable (Fig. 4BGo). Extracts from both cell lines formed multiple bands including a higher molecular weight complex previously associated with DNA interactions with Sp1 proteins (Ref. 21 ; bound DNA; lanes 2, 3, 11, and 12). Intensity of this "bound band" was decreased after competition with unlabeled -169/-111 and consensus Sp1 oligonucleotides (lanes 4, 5, 13, and 14) but not after competition with unlabeled mutant Sp or ER oligonucleotides (lanes 6, 7, 15, and 16). Coincubation with Sp1 or Sp1 antibodies gave supershifted bands with extracts from MCF-7 (lanes 8 and 9) or ZR-75 (lanes 17 and 18) cells. Thus, extracts from both cell lines form Sp1 and Sp3 complexes with the GC-rich -169 to -111 sequence from the E2F-1 gene promoter. In this study and previous reports (21, 28), we did not observe ER{alpha} associated with Sp1 or NFYA in gel mobility shift assays, even though ER{alpha} physically interacts with both proteins. However, ER{alpha} enhances Sp1-DNA complex formation (on-rate) and stabilized the NFA-DNA complex, which was also enhanced by Sp1 (21). The failure to observe ternary protein-protein-DNA complexes in gel shift assays has also been reported for other interacting transcription factors. For example, cyclin D1, sterol regulatory element-binding protein, and the human T cell lymphotropic virus type 1 Tax protein enhance binding of ER, Sp1, and c-jun to their cognate response elements, respectively, but do not form a supershifted complex (36, 37, 38).

Hormonal activation of CCAAT sites through nongenomic pathways
The PKA inhibitor SQ22536 (400 µM) blocked E2-induced luciferase activity in cells transfected with pE2F-1jm1 (Fig. 5AGo), and this inhibitor did not affect cell viability. Previous studies showed that SQ22536 inhibited cAMP/PKA activation of a cAMP response element in ZR-75 cells (39), suggesting that this pathway may be required for activation of the CCAAT sites. This observation was consistent with the known activation of this pathway by E2 (22, 23, 24, 25, 26) as was a recent report showing that cAMP inducers activate TIMP-2 through CCAAT sites (27). Results illustrated in Fig. 5BGo show that higher concentrations of E2 (>10 nM) in the absence of cotransfected ER{alpha} induce luciferase activity; PKA expression plasmid and 8-BrcAMP also induce reporter gene activity in ZR-75 cells transfected with pE2F-1jm1. Moreover, hormonal or PKA-induced transactivation in ZR-75 cells transfected with this construct was not inhibited after cotransfection with 4YA13, whereas dominant negative NFYA (4YA13m29) significantly blocked both induction responses (Fig. 5CGo). These data are consistent with nongenomic activation of cAMP-PKA by E2 and subsequent downstream activation of NFYA. We also investigated hormonal activation of NFYA in ZR-75 cells transfected with an expression plasmid for a chimeric protein containing the DNA binding domain of the yeast GAL4 protein fused to NFYA (full length) (pM-NFYA) and a construct containing five tandem GAL 4 response elements linked to a bacterial luciferase reporter gene (pGAL45). E2 induced a 16-fold increase in reporter gene activity that was inhibited in cells cotreated with E2 plus SQ22536 (Fig. 5DGo). These results confirm that hormonal activation of the cAMP/PKA pathway in ZR-75 cells directly activates NFYA, and this is consistent with the observed hormonal activation of constructs containing the CCAAT sites. Thus, hormonal activation of E2F-1 in ZR-75 cells involved both genomic ER{alpha}/Sp1 and nongenomic pathways and clearly differed from the genomic ER{alpha}/Sp1/NFY mechanism previously described in MCF-7 cells (21).



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  		Figure 5. Kinase-dependent activation of CCAAT sites or NFYA in ZR-75 cells. Transfection of ZR-75 cells with pE2F-1jm1 and treatment with kinase inhibitors (A) or activators of cAMP/PKA (B). ZR-75 cells were treated with E2 (+ cotransfected ER{alpha}) ± 400 µM SQ22536 (A) or 150–350 nM E2 alone (no cotransfected ER{alpha}), 800 µM 8-bromo-cAMP or a PKA expression plasmid (1 µg; B), and luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) induction, compared with DMSO, is indicated by an asterisk. C, Inhibition of PKA-dependent activation of pE2F-1jm1 by dominant negative NFYA ({Delta}4YA13m29). ZR-75 cells were transfected with pE2F-1jm1 treated with DMSO, PKA expression plasmid, or E2 alone or in combination with {Delta}4YA13 or {Delta}4YA13m29 expression plasmid, and luciferase activity determined as described in Materials and Methods. Significant (P < 0.05) induction (*) or inhibition (**) is indicated. D, Activation of pM-NFYA. ZR-75 cells were transfected with pM-NFYA/pGAL45, treated with DMSO, 10 nM E2, SQ22536, or E2 + SQ22536, and luciferase activity determined as described in Materials and Methods. Significant (P < 0.05) induction (*) or inhibition (**) is indicated. Results (A–D) are means ± SD of three replicate determinations for each treatment group. The DMSO treatments in each experiment serve as a 100% control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
E2F transcription factors play a critical role in cell growth and are key regulators of G1->S phase progression of the cell cycle (6, 7, 8, 9). E2F plays a paradoxical role in carcinogenesis and exhibits characteristics consistent with oncogenic and tumor suppressor activity (5, 6, 7, 8). E2F-1-/- and E2F-1+/- mice are viable and, surprisingly, exhibit age-dependent hyperplastic and neoplastic responses, suggesting some tumor suppressor activity (40, 41). E2F-1 also exhibits oncogenic activity, and E2F-1 enhances formation of some tumors in mice lacking a functional Rb1 allele (42, 43). Using a transgenic murine model in which overexpression of E2F-1 is controlled by the keratin 5 promoter, it was reported that E2F-1 alone or in combination with other factors enhanced development of skin carcinomas and tumors in many other tissues (44, 45, 46). In contrast, these transgenic mice were resistant to development of skin tumors in a two-stage initiation-promotion model (46). Thus, E2F-1 overexpression can result in both oncogenic and tumor suppressive activity in the same tissue.

MCF-7 and ZR-75 are ER-positive breast cancer cell lines that are extensively used as models for investigating cell context-dependent differences in the molecular mechanisms of hormone-induced transactivation. For example, in ZR-75 cells E2 induces transactivation in cells transfected with constructs containing GC-rich proximal promoter inserts from the vascular endothelial growth factor gene, whereas E2 decreases activity in MCF-7 and HEC1A cells (47) transfected with the same constructs. Previous studies have reported that E2 induces E2F-1 mRNA levels in MCF-7 cells (21), and similar results were observed in ZR-75 cells (Fig. 1AGo). Deletion and mutation analysis of a series of constructs containing the -169/-173 to -54 region of the E2F-1 clearly demonstrates cell context-dependent differences in hormonal activation of these constructs in MCF-7 vs. ZR-75 cells (Figs 1Go, B–D). In the former cell line, one or more of the GC-rich motifs (-169 to -111) and both CCAAT elements (-122 to -54) are required for ER{alpha}/Sp1/NFYA interactions on this promoter. This same complex may play some role in hormone-induced transactivation in ZR-75 cells; however, the results clearly demonstrate that the GC-rich sites alone (pE2F-1k) or the CCAAT sites alone (pE2F-1jm1) are sufficient for hormone activation.

Gel mobility shift assays exhibit similar patterns of Sp1 and NFYA binding to the proximal region of the E2F-1 gene promoter in MCF-7 and ZR-75 cells (Figs. 3Go and 4Go); however, there were clear differences in transactivation by ER{alpha} mutants in the two cell lines (Fig. 2Go). In ZR-75 cells, hormone-induced transactivation was observed in cells transfected with the DBD-mutant HE11 and constructs containing GC-rich sites and mutated CCAAT sites (-146/-54) alone or in combination with CCAAT motifs (Fig. 2BGo). These results are consistent with activation of GC-rich constructs by ER{alpha}/Sp1 observed for other E2-responsive gene promoters in ER-positive breast cancer cells. In contrast, transfection with HE11 does not activate pE2F-1 h or pE2F-1j (Fig. 2AGo) in MCF-7 cells, and this differentiates between the transcriptionally active ER{alpha}/Sp1/NFYA complex formed in MCF-7 cells in which the GC-rich site alone is not hormone responsive, whereas in ZR-75 cells, both GC-rich and CCAAT sites alone are hormone responsive.

ER{alpha}/Sp1-mediated activation of GC-rich motifs has been characterized in several E2-responsive gene promoters (26, 28, 29, 30, 31, 32, 33, 34), and hormone-dependent activation of constructs containing only the GC-rich sequences (i.e. pE2F-1jm2 and pE2F-1k) in ZR-75 cells is not surprising. The failure to activate the GC-rich constructs from the E2F-1 gene promoter in MCF-7 cells (Fig. 1Go) suggests that the cell context-dependent differences between ZR-75 and MCF-7 cells must be related in part to specific regions within this GC-rich promoter. Current studies are focused on identifying motifs within the GC-rich region of the E2F-1 promoter that determine cell context-dependent differences in their hormone responsiveness.

Dominant negative NFYA (4YA13m29) interacts with NFYB, but the resulting complex does not bind CCAAT sites (48, 49). In MCF-7 cells transfected with pE2F-1j, dominant negative NFYA inhibited E2-induced transactivation, whereas inducibility is decreased but not lost in ZR-75 cells (Fig. 3Go). Overexpression of 4YA13m29 in ZR-75 cells also blocked activation of a construct containing the CCAAT sites (pE2F-1jm1) but not the GC-rich sites (pE2F-1k; Fig. 3Go), confirming the hormone inducibility of the CCAAT motifs in this cell line. Transfection with NFYA or {Delta}4A13 (a long form of NFYA) did not affect hormone responsiveness. Interactions between NFYA and ER{alpha} have previously been reported on the human coagulation factor XII promoter in which NFYA inhibits ER{alpha}-mediated transactivation from motifs that contain an overlapping CCAAT/nonconsensus ERE site (35). NFYA also inhibits hormone-induced transactivation in NIH3T3 and human HepG2 cells transfected with ER{alpha} and a construct containing a consensus ERE promoter (35). In contrast, our results in ZR-75 cells show that NFYA does not inhibit ER{alpha}/Sp1 action in cells transfected with a GC-rich construct (pE2F-1k; Fig. 3CGo), and NFYA does not inhibit hormone-induced transactivation from an ERE promoter in ZR-75 cells (Fig. 3EGo). Thus, inhibitory NFYA-ER{alpha} interactions are also cell context dependent.

The unique hormone-dependent activation of constructs containing CCAAT motifs that bind NFYA was not accompanied by increased binding to these sites as determined in gel mobility shift assays (Fig. 4AGo). A recent report showed that cAMP induced transactivation of human TIMP-2 through activation of NFYA bound to a CCAAT site (27). Because E2 activates the cAMP/PKA pathway in breast cancer cells (22, 23, 24, 25, 26), we further investigated the role of this nongenomic pathway in E2-dependent activation of an E2F-1-derived construct containing CCAAT motifs (pE2F-1jm1). The results in Fig. 5AGo show that hormonal activation of pE2F-1jm1 is inhibited by the adenylate cyclase inhibitor SQ22536; E2 (in the absence of cotransfected ER{alpha}), 8-bromo-cAMP and constitutively active PKA also activate pE2F-1jm1 (Fig. 5BGo), and dominant negative NFYA inhibits E2 and PKA induction of the same construct (Fig. 5CGo). Moreover, E2-dependent activation of the GAL4-NFYA fusion protein is also inhibited by SQ22536 in ZR-75 cells, and this was consistent with comparable inhibition of pE2F-1jm1. This represents a novel nongenomic pathway for activation of NFYA by E2 and is consistent with reports in other cell lines showing cAMP/PKA-dependent activation of NFYA (27).

Nongenomic pathways activated by E2 have been characterized in multiple cell lines including breast cancer cells (50, 51, 52, 53, 54). The mechanisms associated with these pathways are complex and may be dependent on several factors including cell context and ER subtype (55, 56). Results of this study clearly demonstrate that cell context (MCF-7 vs. ZR-75) is an important factor in hormonal regulation of E2F-1 gene expression, and in ZR-75 cells, a combination of both genomic (ER{alpha}/Sp1) and nongenomic (cAMP/PKA) signaling is required. Interestingly, a combination of these pathways has also been reported for induction of c-fos, cyclin D1, and bcl-2 in MCF-7 or ZR-75 cells (32, 57, 58). These gene promoters all contain E2-responsive GC-rich motif as well as cAMP response elements (bcl-2 and cyclin D1) or a serum response element (c-fos) activated through MAPK and phosphatidylinositol-3-kinase (57, 58, 59). Because inhibitors of MAPK and phosphatidylinositol-3-kinase pathways block E2-induced proliferation of MCF-7 cells (57, 58, 59), the identification of downstream E2-responsive gene targets such as E2F-1, cyclin D1, c-fos, and bcl-2 is consistent with the contributions of nongenomic pathways of estrogen action. Current studies are investigating the mechanisms of nonclassical genomic and nongenomic pathways on growth-regulatory genes in breast cancer cells and determining cellular factors that influence cell context-dependent mechanistic differences, even among ER-positive breast cancer cell lines.


   Footnotes
 
This work was supported by NIH (Grants ES-09253 and ES-09106) and the Texas Agricultural Experiment Station.

Abbreviations: DMSO, Dimethyl sulfoxide; E2, 17ß-estradiol; ER{alpha}, estrogen receptor {alpha}; ERE, ER element; FBS, fetal bovine serum; HEGD, HEPES, EDTA, glycerol, and dithiothreitol; PKA, protein kinase A; Rb, retinoblastoma, TIMP-2, tissue inhibitor of metalloproteinases-2.

Received November 5, 2002.

Accepted for publication January 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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