Transcriptional Activation of E2F1 Gene Expression by 17ß-Estradiol in MCF-7 Cells Is Regulated by NF-Y-Sp1/Estrogen Receptor Interactions

Weili Wang, Lian Dong, Brad Saville and Stephen Safe

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
17ß-Estradiol (E2) stimulated proliferation and DNA synthesis in MCF-7 human breast cancer cells, and this was accompanied by induction of E2F1 mRNA and protein levels. Analysis of the E2F1 gene promoter showed that the -146 to -54 region was required for E2-responsiveness in transient transfection assays, and subsequent deletion/mutation analysis showed that a single upstream GC-rich and two downstream CCAAT-binding sites were required for transactivation by E2. Gel mobility shift assays with multiple oligonucleotides and protein antibodies (for supershifts) showed that the -146 to -54 region of the E2F1 gene promoter bound Sp1 and NF-Y proteins in MCF-7 cells. The estrogen receptor (ER) protein enhanced Sp1 interactions with upstream GC-rich sites, and interactions of ER, Sp1, and ER/Sp1 with downstream DNA bound-NF-Y was investigated by kinetic analysis for protein-DNA binding (on- and off-rates), coimmunoprecipitation, and pulldown assays using wild-type and truncated glutathione S-transferase (GST)-Sp1 chimeric proteins. The results showed that Sp1 protein enhanced the Bmax of NF-Y-DNA binding by more than 5-fold (on-rate); in addition, the Sp1-enhanced NF-Y-DNA complex was further stabilized by coincubation with ER and the rate of dissociation (t1/2) was decreased by approximately 50%. Sp1 antibodies immunoprecipitated [35S]NF-YA after coincubation with unlabeled Sp1 protein. Thus, transcriptional activation of E2F1 gene expression in MCF-7 cells by E2 is regulated by multiprotein ER/Sp1-NF-Y interactions at GC-rich and two CCAAT elements in the proximal region of the E2F1 gene promoter. This represents a unique trans-acting protein complex in which ligand-dependent transactivation by the ER is independent of direct ER interactions with promoter elements.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E2F proteins are members of a family of nuclear transcription factors that form heterodimeric complexes with DP proteins and regulate expression of diverse mammalian and viral genes (1, 2, 3, 4, 5, 6, 7). E2F1 plays an important role in cell cycle progression at the G1/S phase transition, and this is related to regulation of several genes involved in DNA synthesis including thymidine kinase, dihydrofolate reductase, and DNA polymerase {alpha} (8, 9, 10, 11, 12). Additionally, E2F1 induces apoptosis in different cancer cell lines; this is separable from effects of this protein on cell cycle progression and does not require p53 (13, 14, 15, 16). Studies with E2F1 knockout mice confirm that E2F1 plays an important role in apoptosis, and increased tumorigenesis in these animals suggests that E2F1 may also function in some tissues as a tumor suppressor gene (17, 18).

17ß-Estradiol (E2) stimulates proliferation and DNA synthesis in estrogen receptor (ER)-positive breast cancer cells, and this is accompanied by an increased percentage of cells in S phase and a decreased fraction in G0/G1. Several studies have shown that this hormone-induced response is accompanied by modulation of several genes/proteins that regulate cell cycle progression (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). For example, treatment of MCF-7 cells with E2 is accompanied by increased cyclin D1 mRNA and protein, cdk7-, cdk2-, and cdk4-dependent kinase activities, cdc25A phosphatase protein, and increased phosphorylation of retinoblastoma (Rb) protein (20, 21, 22, 23, 24, 25). Rb and related proteins physically interact with E2F1 to give a transcriptionally repressed complex (31, 32), and mitogens such as E2 catalyze phosphorylation of Rb and the subsequent release of E2F1 from the protein complex (20, 21, 22). This study shows that treatment of MCF-7 cells with E2 also induces E2F1 protein and gene expression, and deletion analysis of the E2F1 gene promoter has identified a region between -146 to -54 that is required for E2 responsiveness. This sequence contains a GC-rich Sp1-binding site and two CCAAT motifs that bind NF-Y proteins. Previous studies in this laboratory have demonstrated that ER/Sp1 interactions with GC-rich sites are required for E2-mediated transactivation of some genes (33, 34, 35, 36); however, results of this study show that hormone-mediated transactivation of the E2F1 gene requires not only ER/Sp1 binding to GC-rich sites but also interactions with NF-Y proteins bound to downstream CCAAT sites in the E2F1 gene promoter. It has previously been reported that Sp1 and ER physically interact (33), and this study shows that Sp1 also interacts with NF-YA in coimmunoprecipitation experiments, and Sp1 enhanced NF-Y-DNA binding in gel mobility shift assays. These results indicate that DNA-bound Sp1 protein plays a central role in E2-induced E2F1 gene expression by interacting with ER protein, and downstream NF-Y proteins bound to two CCAAT motifs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Induction of E2F1 Gene Expression and Protein by E2 in MCF-7 Cells
Treatment of MCF-7 cells maintained in serum-free medium with 10 nM E2 resulted in modulation of multiple genes/proteins associated with the cell cycle (19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 37, 38, 39), and results illustrated in Fig. 1Go show that immunoreactive E2F1 protein and mRNA levels are induced after treatment with E2. These data are consistent with parallel induction of E2F1-dependent DNA synthesis in this cell line. E2F1 protein levels are maximally induced 12–24 h after treatment with E2, and E2F1 mRNA levels are significantly increased more than 4-fold 12 h after addition of E2. This is the first report showing that estrogen induces E2F1 gene expression. Immunoreactive Sp1 protein levels were unchanged in MCF-7 cells after treatment with E2 for 24 or 48 h (data not shown).



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Figure 1. Induction of E2F1 mRNA Levels and Protein by 10 nM E2 in MCF-7 Cells

A, Induction of E2F1 mRNA. MCF-7 cells were treated with 10 nM E2, and E2F1 mRNA levels were determined after 0.5, 1, 2, 4, and 12 h as described in Materials and Methods. E2F1 mRNA levels (relative to untreated cells, 1.0) were 0.84 ± 0.2, 1.3 ± 0.2, 1.7 ± 0.4, 2.1 ± 0.4, and 4.4 ± 1.2 after 0.5, 1, 2, 4, and 12 h, and significant induction (P < 0.05) was observed after 4 and 12 h. B, Induction of E2F1 protein. E2F1 protein levels were determined by Western blot analysis as described in Materials and Methods, and protein levels (relative to untreated cells, 1.0) were 1.9 ± 0.3, 3.1 ± 0.7, and 4.6 ± 1.2 after treatment with E2 for 6, 12, and 24 h, and significant induction (P < 0.05) was observed at all time points.

 
Deletion Analysis of the E2F1 Gene Promoter and Identification of E2-Responsive -146 to -54 Region
Previous studies have identified multiple Sp1- and E2F-binding sites and CCAAT motifs in the E2F1 gene promoter (40, 41) and E2 induced luciferase activity (2.6-fold) in transient transfection studies with pE2F1a, a construct containing the -728/+77 region of E2F1 gene promoter insert linked to a luciferase reporter gene (40). Hormone inducibility was observed using pE2F1b (-242/+77), pE2F1c (-177/+10), and pE2F1d (-122/+77); however, basal activity was significantly decreased with the latter two constructs (Fig. 2AGo). In contrast, both basal and hormone inducibility were restored with pE2F1g (-173/-54) in which the two downstream E2F1-binding sites were deleted. Loss of basal activity was observed in cells transiently transfected with constructs (pE2F1e and pE2F1f) that do not contain the two downstream CCAAT motifs. These results indicate that the -173 to -54 region (pE2F1g) of the E2F1 gene promoter exhibited high basal activity and was E2-responsive. This sequence contains three Sp1-binding sites flanked by one upstream and two downstream CCAAT motifs. Deletion of a downstream CCAAT site (pE2F1i) resulted in loss of hormone responsiveness, whereas E2 induced luciferase activity in MCF-7 cells transfected with pE2F1h (-169/-54) in which the upstream CCAAT site was deleted (Fig. 2AGo). E2 also induced luciferase activity in ER-negative MDA-MB-231 cells transiently transfected with pE2F1 h and ER expression plasmid (data not shown). The potential role of the three GC-rich sites was investigated by mutational analysis (Fig. 2BGo) of pE2F1h; individual mutations of the three upstream GC-rich sites did not markedly affect hormone responsiveness; however, basal activity was lowest with pE2F1hm3 that is mutated in Sp1 site 3. Mutation of individual CCAAT sites (pE2F1hm4 and pE2F1hm5) resulted in loss of basal and hormone-induced luciferase activity. pE2Fj incorporates a single upstream GC-rich site and both downstream CCAAT sites and represents a potential minimal E2-responsive region of the E2F1 gene promoter. Deletion of the upstream GC-rich site (pE2F1jm1) resulted in decreased basal luciferase activity and loss of hormone inducibility, and similar results were observed for an Sp1 mutant construct (data not shown). Mutation of the CCAAT sites (pE2F1jm2) also resulted in decreased basal activity and loss of hormone responsiveness, suggesting that interactions of nuclear factors at the GC-rich and CCAAT sites within the E2F1 gene promoter are required for high basal activity and E2 responsiveness in MCF-7 cells.



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Figure 2. Deletion Analysis of the E2F1 Gene Promoter for Basal Activity and E2-Responsiveness

A, E2-responsiveness of -169 to -54 region. Various constructs were derived from wild-type pE2F1a (-728 to +77), and transient transfection and luciferase assays were carried out as described in Materials and Methods. Luciferase activities were normalized to the reference plasmid (ß-gal) to correct for transfection efficiencies and compared with pE2F1a, where the DMSO-treated group was arbitrarily set at 100%. Activities are expressed as means ± SE for three separate experiments for every treatment group. B, Mutation and deletion analysis of pE2F1h. Mutation and deletion of GC-rich sites and transfection studies were carried out as described in Materials and Methods and panel A. C, Mutation and deletion analysis of pE2F1j. Mutation of GC-rich sites and transfection studies were carried out as described in Materials and Methods and in panel A. pE2F1h was used as a comparative standard in experiments summarized in panels A-C, and there was some variability in luciferase induction by E2 between experiments (3.2-, 3.9- and 3.0-fold, respectively).

 
Sp1 and NF-Y Proteins Bind to Specific Motifs within the -146 to -54 Region of the E2F1 Gene Promoter
Interactions of nuclear extracts and recombinant proteins with E2-responsive regions of the E2F1 gene promoter were investigated in gel electrophoretic mobility shift assays. Results in Fig. 3Go illustrate binding of nuclear extracts from solvent [dimethylsulfoxide (DMSO)] control (lane 1) or cells treated with 10 nM E2 to [32P]-146/-54 oligonucleotide containing the GC-rich and CCAAT sites. Four major high mol wt retarded bands were observed (A–D), and band intensities were slightly increased using nuclear extracts from cells treated with E2 (lane 2). Competition with consensus unlabeled Sp1 oligonucleotide (lane 3) eliminated bands B, C, and D (lane 3), and competition with unlabeled downstream CCAAT-containing oligonucleotide (-115/-54) eliminated bands A, C, and D and decreased the intensity of band B. This downstream sequence contains GC-rich elements that may compete for protein binding to the upstream GC-rich site in the -146/-54 oligonucleotide. Competition with unlabeled estrogen response element (ERE) and mutant Sp1m oligonucleotides (lanes 5 and 6) slightly decreased retarded band formation. A high mobility band (unlabeled) was competitively decreased only by the -115/-54 oligonucleotide. Low molecular weight proteins were observed in assays using nuclear extracts; these bands were not supershifted by antibodies and may be due, in part, to partially degraded proteins. The results suggest that nuclear extracts from MCF-7 cells express proteins that bind GC-rich and CCAAT elements within the -146/-54 region of the E2F1 gene promoter. Based on results of oligonucleotide competition studies, band A contains only a CCAAT-binding protein, band B contains a protein binding a GC-rich site, and bands C and D contain proteins that bind both CCAAT- and GC-rich sequences.



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Figure 3. Binding of Nuclear Extracts from MCF-7 Cells to [32P]-146/-54

Nuclear extracts from DMSO (solvent) or E2-treated MCF-7 cells were incubated with [32P]-146/-54 (lanes 1 and 2) and other competing oligonucleotides as described in Materials and Methods. Four bands (A-D) of increasing molecular weight were formed, and competition with wild-type Sp1 oligonucleotide (lane 3) decreased bands B-D; intensity of all bands was decreased by competition with -115/-54 oligonucleotide (lane 4). Wild-type ERE (lane 5) slightly decreased intensity of all bands, whereas mutant Sp1 oligonucleotide (lane 6) had no effect on retarded band intensities.

 
To simplify the complex protein-DNA interactions, separate binding experiments were carried out utilizing GC-rich upstream (-169/-116) (Fig. 4Go) and CCAAT-containing downstream (-122/-54) (Fig. 5Go) oligonucleotides in gel electrophoretic mobility shift assays. Incubation of nuclear extracts from DMSO (solvent control) and E2-treated cells (lanes 1, 2, and 8) with [32P]-169/-116 gave one major specifically bound retarded band (bound-DNA) that was decreased by coincubation with unlabeled -169/-116 and consensus Sp1 oligonucleotides (lanes 3 and 4). Competition with unlabeled ERE slightly decreased intensity of the retarded band (lane 7), whereas mutant Sp1 and -115/-54 (E2F1 gene promoter) oligonucleotides (lanes 5 and 6) did not competitively decrease intensity of the bound DNA complex. A high mobility band was also observed and only wild-type -169/-116 competitively decreased intensity of this band (lane 3). Supershift experiments (Fig. 4BGo) with Sp1 (lanes 9–11) and Sp3 (lane 12–14) antibodies or nonspecific IgG antibody (lane 15) showed that Sp1 antibodies supershifted the bound DNA complex. In addition, Sp3 antibodies also gave two weak supershifted bands, suggesting that an Sp3-DNA complex may be part of the Sp1-DNA retarded band. Proteins binding to the downstream (-122/-54) region of the E2F1 gene promoter (Fig. 5AGo) gave one major higher molecular weight retarded band (A), two less intense bands (B and C), and two additional intense bands (D and E). Intensity of retarded band A was signfiicantly decreased after competition with wild-type -122/-54 and NF-Y oligonucleotides (lanes 3 and 5). Wild-type and mutant -122/-54 oligonucleotides also decreased the intensity of band E. This suggests that additional nuclear proteins are bound to sites other than the CCAAT motif in this oligonucleotide. The intensity of this band (E), as well as bands A-D, were not decreased after competition with consensus nuclear factor 1 (NF-1), CCAAT-enhancer-binding protein (C/EBP), Sp1, or ERE oligonucleotides. Identities of proteins associated with retarded band E are unknown. Band D appears to be a nonspecific complex that is not competitively decreased by any of the unlabeled oligonucleotides. Supershift experiment with antibodies to NF-YA, NF-1, C/EBP, Sp1, ER{alpha}, and nonspecific goat or rabbit IgG (lanes 11–17, respectively) showed that only NF-YA antibodies formed a ternary supershifted complex (lane 11); in addition, NF-YB antibody eliminated bound-DNA complex but no supershift was observed (data not shown).



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Figure 4. Binding of Nuclear Extracts from MCF-7 Cells to [32P]-169/-116 in Gel Mobility Shift Assays

A, Competitive binding. Nuclear extracts from MCF-7 cells were incubated with [32P]-169/-116 and, in some experiments, coincubated with a 200-fold excess of various unlabeled oligonucleotides and analyzed by gel mobility shift assays as described in Materials and Methods. The specifically bound retarded band is indicated (bound DNA). B, Antibody supershifts. Nuclear extracts from MCF-7 cells were incubated with [32P]-169/-116 (lane 8), Sp1 (lanes 9–11), or Sp3 (lanes 12–14) antibodies and nonspecific IgG (lane 15) and analyzed by gel mobility shift assays as described above in panel A. The retarded band (Sp1-DNA) and Sp1 antibody-supershifted band (Supershift) are indicated with arrows.

 


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Figure 5. Binding of Nuclear Extracts from MCF-7 Cells to [32P]-122/-54

A, Competitive binding. Nuclear extracts from MCF-7 cells were incubated with [32P]-122/-54 in the presence or absence of a 200-fold excess of selected consensus oligonucleotides and analyzed by gel mobility shift assay as described in Materials and Methods. The specifically bound retarded bands are indicated (A-E). B, Antibody supershifts. Nuclear extracts from MCF-7 cells were coincubated with [32P]-122/-54 and antibodies to NF-YA, {alpha}NF-1, C/EBP, Sp1, ER{alpha}, and goat and rabbit IgG (lanes 11–17) and analyzed by gel mobility shift assays as described in Materials and Methods. The retarded band (NF-Y-DNA) and NF-YA-supershifted band (Supershift) are indicated with arrows.

 
ER{alpha}-Sp1 and Sp1-NF-Y Interactions
Previous studies in this laboratory have demonstrated that coincubation of ER{alpha} protein with Sp1 protein and GC-rich oligonucleotides resulted in enhanced Sp1-DNA retarded band intensity without forming a ternary supershifted complex (33, 34, 35, 36, 42). Results in Fig. 6AGo demonstrate that purified recombinant Sp1 protein binds to the GC-rich [32P]-169/-116 to form a retarded band (bound-DNA) (lane 1), and retarded band intensity is enhanced (2- to 3-fold) after coincubation with recombinant human ER (lanes 2 and 3) but not recombinant transforming growth factor-{alpha} (TGF{alpha}) protein (lane 6) used as a control; intensity of the enhanced band was markedly decreased after coincubation with unlabeled consensus Sp1 oligonucleotide and competition with mutant Sp1 oligonucleotide also decreased intensity of the retarded band (lanes 4 and 5). The intensity of the Sp1-DNA band formed after coincubation of [32P]-169/-116 with nuclear extracts (Fig. 4Go) is also enhanced after coincubation with ER{alpha} (data not shown). These results are consistent with previous studies that show enhanced Sp1-DNA binding by ER{alpha} was associated with an increased on-rate for this complex, whereas dissociation of the Sp1-DNA complex was unchanged by ER{alpha} (33). In contrast, incubation of in vitro translated NF-YA plus NF-YB did not increase Sp1-DNA binding in the presence or absence of ER{alpha} (data not shown). Coincubation of recombinant human ER{alpha} (Fig. 6BGo, lanes 2–4) with nuclear extracts from MCF-7 cells and [32P]-122/-54 does not enhance intensity of the NF-Y-DNA retarded band (B2). However, coincubation of recombinant human Sp1 protein significantly increased intensity of the NF-Y-[32P]-122/-54 (retarded band) and also resulted in formation of a higher molecular weight band (B1) (lanes 5–7). Intensities of both bands were decreased after coincubation with unlabeled -122/-54 oligonucleotide, suggesting that NF-Y binds both CCAAT sites in the higher molecular weight complex (B1). Supershift experiments with NF-YA or Sp1 antibodies indicated that bands B1 and B2 bound NF-YA (data not shown). Further incubation with ER{alpha} does not increase the intensity of the Sp1-enhanced NF-Y-DNA complex (B1 and B2). Incubation of [32P]-122/-54 with nonspecific TGF{alpha} protein does not form a retarded band (data not shown). Interestingly, competition with unlabeled -122/-54 mutated in both CCAAT sites (lane 11) eliminated band B1 but not B2, suggesting that this DNA sequence binds other proteins and Sp1 may enhance formation of this complex. The functional significance of this interaction is doubtful since mutation of both CCAAT sites to give pE2F-1jm2 (Fig. 2CGo) resulted in loss of hormone inducibility. A high-mobility specifically bound complex was also observed; however, this band was not affected by coincubation with ER{alpha} or Sp1 and was not further investigated. Direct interactions of ER{alpha} and Sp1 with NF-YA were investigated in coimmunoprecipitation assays (Fig. 7Go). Sp1 antibodies coimmunoprecipitated [35S]NF-YA after incubation with Sp1 protein (lane 3). In contrast, ER{alpha} antibodies did not precipitate [35S]NF-Ya protein after coincubation with ER{alpha} (lane 5), ER{alpha} plus Sp1 (lane 6), or ER{alpha} plus TGF{alpha} (lane 7). Interactions of [35S]NF-YA with chimeric GST-Sp1 fusion proteins were also investigated (data not shown); repeated experiments were unsuccessful due to high background binding of NF-YA to the GST-Sp1 chimeric protein.



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Figure 6. Interactions of ER{alpha}, Sp1, and NF-Y on Retarded Band Formation

A, Enhanced Sp1-DNA interactions by ER{alpha} protein. [32P]-169/-116 oligonucleotide was incubated with recombinant human Sp1 protein (2 ng) alone (lane 1) and different concentrations of human recombinant ER protein (lanes 2 and 3) or TGF{alpha} protein (lane 6) and analyzed by gel mobility shift assay as described in Materials and Methods. Coincubation with 200-fold excess unlabeled Sp1 oligonucleotide (lane 4) decreased retarded band intensity, whereas minimal effects were observed using mutant Sp1 oligonucleotide. The specifically bound DNA-retarded band is indicated with an arrow, and ER caused a 2- to 3-fold increase in the retarded band intensity (lanes 2 and 3). B, Effects of Sp1 and ER{alpha} on NF-Y-DNA binding. [32P]-122/-54 was incubated with nuclear extracts from MCF-7 cells alone (lane 1), in combination with human recombinant ER protein (lanes 2–4), human recombinant Sp1 protein (lanes 5–7), ER{alpha} + Sp1 protein (lanes 8 and 9), or TGF{alpha} protein (lane 12), and analyzed by gel mobility shift assays as described in Materials and Methods. Specific binding was determined by competition with excess unlabeled wild-type and mutant -122/-54 oligonucleotides (lanes 10 and 11, respectively), and specifically bound DNA complex is indicated with an arrow.

 


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Figure 7. Physical Interactions of Sp1 and NF-YA

[35S]NF-YA was incubated alone or in combination with Sp1, ER{alpha}, or Sp1 + ER{alpha} and treated with various antibodies and analyzed by electrophoresis as described in Materials and Methods. In the coincubation studies, only Sp1 antibody coimmunoprecipitated [35S]NF-YA+Sp1 protein. At least two different ER{alpha} antibodies did not coimmunoprecipitate [35S]NF-YA+ER+Sp1 (lane 5).

 
Effects of Sp1 and ER Proteins on NF-Y-DNA Complex Formation
Previous studies have demonstrated that Sp1 protein affects the kinetics of NF-Y-DNA interactions, and a decreased rate of complex dissociation was observed using oligonucleotides derived from the rat fatty acid synthase and major histocompatibility complex class II-associated invariant gene promoters (43, 44). The results in Fig. 8Go summarize the time-dependent effects of Sp1 and ER{alpha} proteins on NF-Y binding to [32P]-122/-54. NF-Y alone rapidly forms a retarded band within 1 min, and intensity of this complex does not significantly change over 20 min (Fig. 8AGo). Coincubation with Sp1 protein also resulted in rapid (within 1 min) maximal formation of an NF-Y-DNA complex; however, the intensity of the retarded band was five times higher than observed in the absence of Sp1. The t1/2 values for the rate of complex formation were less than 1 min in the presence or absence of Sp1 protein, and the major effects of Sp1 were on the Bmax values for NF-Y-DNA complex formation, which were increased more than 5-fold at all time points. Coincubation with ER{alpha} did not significantly affect retarded band formation (data not shown; Fig. 6BGo). The rate of complex dissociation (off rate) was also determined (Fig. 8BGo), and the t1/2 value for loss of 50% of the retarded band intensity was approximately 0.9 min after incubation with nuclear extracts alone (NF-Y) or in combination with Sp1 protein. In contrast, coincubation of nuclear extracts with Sp1 plus ER{alpha} proteins significantly increased the t1/2 (1.9 min) for complex dissociation, suggesting that ER{alpha} functions to stabilize the Sp1/NF-Y-DNA complex.



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Figure 8. Kinetic Effects of ER and Sp1 on NF-Y-DNA Binding

A, Effects of Sp1 protein on NF-Y-[32P]-122/-54 complex formation (on-rate). [32P]-122/-54 oligonucleotide was incubated with nuclear extracts from MCF-7 cells alone ({circ}) or in combination with Sp1 protein ([{blacktriangleup}), and the time-dependent formation of the NF-Y-DNA complex was determined by gel mobility shift assays as described in Materials and Methods. The results for each time point are means ± SE for three separate determinations. Coincubation with Sp1 protein significantly (P < 0.05) increased the Bmax value for intensity of the specifically bound retarded band by more than 5-fold at every time point. Coincubation with ER{alpha} protein did not affect the rate of complex formation (data not shown). B, Effects of Sp1 and ER{alpha}+Sp1 proteins on dissociation of the NF-Y-DNA complex (off-rate). [32P]-122/-54 oligonucleotide was incubated with nuclear extracts from MCF-7 cells alone ({circ}) or in the presence of Sp1 protein alone ({blacktriangleup}) or Sp1+ER{alpha} proteins (•), and dissociation of the NF-Y-[32P]-122/-54 complex was analyzed by gel mobility assays as described in Materials and Methods. Results are expressed as percent of initial binding (set at 100%), and results for every time point is a mean ± SE for three separate determinations. The dissociation curves for nuclear extracts alone and in the presence of Sp1 protein were indistinguishable; however, in the presence of Sp1+ER{alpha} proteins, there was a significant (P < 0.05) decrease in the off-rate at all time points less than 9 min.

 
Role of NF-Y on ER/Sp1 Transactivation
E2 induces luciferase activity in MCF-7 cells cotransfected with pE2F1j, and wild-type ER (Figs. 2Go and 9Go) and cotransfection with wild-type expression plasmids for NF-YA or NF-YB do not significantly affect E2 responsiveness; in cells cotransfected with NF-YA and NF-YB, there was a slight increase in the induction response (6.2-fold) (Fig. 9Go). The {Delta}4YA13m29 construct encodes for a dominant negative NF-YA mutant protein (45), and {Delta}4YA13 does not exhibit dominant negative activity. In MCF-7 cells cotransfected with pE2F1j plus {Delta}4YA13m29, there was a marked decrease in basal CAT activity and loss of E2 inducibility, thus confirming the role of NF-YA in ER{alpha}-mediated induction of E2F1 gene expression. The control plasmid ({Delta}4YA13) did not affect E2 responsiveness. Thus, ER{alpha}/Sp1 in combination with NF-Y proteins are required for induction of E2F1 gene expression by E2, and these data describe a new multiprotein complex required for ER{alpha} action.



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Figure 9. Effects of {Delta}4YA13m29 (Dominant Negative NF-YA) on E2 Responsiveness

MCF-7 cells were transiently transfected with pE2F1j and ER{alpha} plus expression plasmids for NF-YA, NF-YB, NF-YA + NF-YB, {Delta}4YA13m29 (dominant negative NF-YA), and {Delta}4YA13 (an NF-YA mutant, not dominant negative) and treated with E2, and luciferase activity was determined as described in Materials and Methods. Only the dominant negative NF-YA expression plasmid significantly decreased (P < 0.05) E2-induced luciferase activity.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E2F1 gene expression is tightly regulated during the cell cycle; only low levels are present in quiescent or growth-arrested cells, and after mitogen stimulation, E2F1 accumulates in late G1. Johnson and co-workers (40) used E2F1 promoter-luciferase constructs transiently transfected into rat embryo fibroblast cell line and showed that time-dependent induction of luciferase activity after addition of serum was primarily dependent on the -204 to -122 region of the promoter. Maximal responses were observed after 24 h, and this correlated with increased E2F1 mRNA levels. In growth-arrested ER-positive breast cancer cells, treatment with E2 increases the percentage of cells in S phase and decreases cells in G0/G1, and this is accompanied by up-regulation of several cell cycle enzymes (19, 20, 21, 22, 23, 24, 25, 26, 37, 38). The results summarized in Fig. 1Go also demonstrate that both E2F1 protein and mRNA levels also significantly increased after treatment of growth-arrested MCF-7 cells with E2, suggesting that E2-mediated cell cycle progression is due, in part, to E2F1 gene activation. This study further investigates the molecular basis of hormone inducibility by analysis of the E2F1 gene promoter.

Maximal basal activity in MCF-7 cells was observed for pE2F1g (-173/-54), and this includes some overlap with results in rat embryo fibroblasts showing that the -204 to -122 region of the E2F1 gene promoter was important for basal activity (40). The results in MCF-7 cells also show that downstream regions of the promoter (i.e. -122 to -54) are also required for high basal activity (Fig. 2Go). The pE2F1g construct contains an upstream and two downstream CCAAT sites separated by a GC-rich region, and further deletion of the upstream or downstream CCAAT motif (pE2F1h and pE2F1i) resulted in a 40% or 94% decrease in basal activity, suggesting that at least one of the downstream CCAAT sites plays an important role in basal activity of the E2F1 gene promoter. This was confirmed by comparing luciferase activities of cells transfected with pE2F1h (-169/-54) or pE2F1j (-146 to -54) and several mutant constructs; deletion of either of the downstream CCAAT motifs resulted in a more than 75% loss of basal luciferase activity. This high basal activity conferred by the two CCAAT sites was also dependent on the presence of at least one upstream GC-rich site. For example, a comparison of luciferase activities of wild-type and mutant pE2F1h (-169/-54) and pE2F1j shows that the two CCAAT sites alone are not sufficient for high basal luciferase activity, and one or more of the upstream GC-rich sites are also required (Fig. 2Go, B and C). These results are consistent with previous studies (40) showing that the E2F1-luc construct (-122) containing only the downstream CCAAT motifs exhibited low basal and serum-inducible reporter gene activity, indicating that Sp1 and NF-Y proteins function as trans-activators in regulating basal E2F1 gene expression.

The E2F1 gene promoter does not contain perfect or imperfect palindromic EREs, and results of deletion analysis (Fig. 2Go) showed that promoter regions required for E2 responsiveness were similar to those observed for basal activity. Maximal activity required at least one of the three GC-rich sites (-169 to -116) and both downstream CCAAT motifs (e.g. pE2F1h and mutants and pE2F1j). Several studies have reported that E2 responsiveness of constructs derived from the cathepsin D, retinoic acid receptor {alpha}1, and c-fos gene promoters are associated with ER{alpha}/Sp1 interactions at GC-rich sites (34, 35, 36, 42). The upstream GC-rich sequence in the E2F1 gene promoter exhibits some similarities to these functional enhancer sequences; for example, Sp1 protein binds [32P]-169/-116 and ER{alpha} enhances Sp1-DNA interactions (Fig. 6AGo) but does not form a supershifted ternary complex as previously reported for E2-responsive and nonresponsive GC-rich promoter elements (33, 34, 35, 36, 42). In contrast, deletion analysis of the E2F1 gene promoter shows that the upstream GC-rich sites alone are not sufficient for transactivation in transient transfection studies (Fig. 2Go). Thus, hormone responsiveness of the E2F1 gene promoter (-169 or -146/-54) in transfection studies in MCF-7 cells not only requires ER{alpha}/Sp1 binding to GC-rich sites but also interactions with downstream CCAAT-binding proteins, suggesting possible interactions between these transcription factors.

Gel mobility shift assays using [32P]-146/-54 and nuclear extracts from MCF-7 cells gave a complex pattern of at least four retarded bands that contain Sp1- and CCAAT-binding proteins (Fig. 3Go), and these interactions could be simplified using [32P]-labeled GC-rich upstream (-169/-116, Fig. 4Go) or CCAAT-downstream (-122/-54, Figs. 5Go and 6BGo) oligonucleotides. Direct and competitive binding and antibody supershifts show that Sp1 and NF-Y proteins interact with upstream and downstream sequences, respectively (Figs. 4Go and 5Go). These results suggest that E2-mediated transcriptional activation of E2F1 involves binding of Sp1 and NF-Y proteins to their respective enhancer elements, coupled with interactions of these proteins with ER{alpha} protein, that does not bind promoter DNA. Previous studies have demonstrated that ER{alpha} and Sp1 physically interact (33), and ER{alpha} enhanced Sp1-DNA binding in gel mobility shift assays without forming a ternary supershifted complex (33, 34, 35, 36, 42). Similarly, ER{alpha} enhanced binding of Sp1 to [32P]-169/-116 (Fig. 6AGo), and this type of interaction has been observed in other studies showing that human T cell leukemia virus type-1 Tax, sterol-regulatory element-binding protein, and cyclin D1 enhanced bZIP, Sp1, and ER binding to their respective cognate DNA enhancer sequences (46, 47, 48).

Interactions between Sp1 and NF-Y or other CCAAT-binding proteins have been observed on other gene promoters (43, 44, 49, 50, 51), and results in Fig. 6BGo demonstrate that the NF-Y-DNA (-122/-54) complex forms a retarded band that is not affected after coincubation with ER{alpha}, whereas Sp1 protein markedly enhanced intensity of the NF-Y-DNA band. In contrast, NF-Y did not enhance Sp1-DNA ([32P]-169/-116) complex formation or form a ternary supershifted complex (data not shown). Previous studies showed that ER{alpha}-Sp1 physically interacted in coimmunoprecipitation and GST pull-down assays (33). Direct interactions of ER and NF-YA were not observed in coimmunoprecipitation assays, whereas Sp1 and NF-YA proteins were coimmunoprecipitated (Fig. 7Go). Thus, Sp1 binds both ER{alpha} and NF-YA proteins, and this is consistent with their cooperative interactions on the E2F1 gene promoter observed in this study.

Stabilities of NF-Y binding to CCAAT regions in the fatty acid synthetase and major histocompatibility complex class II gene promoters are enhanced by Sp1 protein, and this has been related to increased half-lives of NF-Y-DNA complexes (43, 44). Binding of NF-Y to [32P]-122/-54 in the presence or absence of Sp1 protein was maximal within 1 min, and differences in on-rate t1/2 values could not be determined (Fig. 8Go). However, results showed that the Bmax for binding was increased by more than 5-fold, and coincubation with ER did not significantly affect the on-rate of NF-Y-DNA binding (data not shown). In contrast, dissociation of the NF-Y-DNA complex was not significantly affected by Sp1, and differences between this and other studies (42, 43) may be promoter dependent; however, in the presence of Sp1 plus ER{alpha}, there was a 2-fold increase in stability of the DNA-NF-Y complex (Fig. 8Go). These in vitro results suggest that transcriptional activation of E2F1 gene expression by E2 may be due, in part, to stabilization of the Sp1-NF-Y-DNA complex by ER{alpha}, and this is consistent with increased retarded band intensities using extracts from E2-treated cells compared with solvent controls ( Figs. 3–5GoGoGo). CCAAT-binding sites are frequently observed in TATA-less promoters (including the E2F1 gene promoter) (49), and ER{alpha}, Sp1, and NF-Y can induce conformational changes in chromatin structure. These effects could result in recruitment of general transcriptional machinery via both protein-protein and protein-DNA interactions (Fig. 10Go). However, other possible mechanisms may also play a role in ER-dependent transactivation, and this includes activation of kinases and modulation of other proteins that affect NF-Y or Sp1 action.



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Figure 10. Proposed Model of E2-Dependent Transcriptional Activation of the E2F1 Gene in MCF-7 Cells in Which Sp1 Protein Binds Both ER{alpha} and NF-YA to Mediate the Hormonally Induced Response

 
Transcriptional activation of several genes by E2 may require interaction of ER with other DNA-bound transcription factors such as AP-1 or Sp1 (33, 34, 35, 36, 42, 52, 53). ER{alpha}/Sp1 action is complex and gene promoter specific. For example, GC-rich motifs in the c-fos and retinoic acid receptor {alpha}1 promoters are sufficient for E2-induced signaling in MCF-7 cells via ER{alpha}/Sp1 interactions (34, 36). In contrast, ER{alpha}/Sp1 interactions at GC-rich sites in cathepsin D, Hsp27, and uteroglobin gene promoters require, in addition, ERE or ERE half-site motifs for ER-DNA binding (54, 55, 56, 57). Results of this study demonstrate that ER{alpha}/Sp1 binding to GC-rich sites in the E2F1 gene promoter are necessary but not sufficient for transactivation of E2F1, and cooperative interactions with NF-Y are required for a hormone-induced response (Fig. 10Go). Previous studies have shown that hormone-induced responses can be mediated through interactions of their receptors with DNA-bound Sp1 protein (33, 34, 35, 36, 42, 58), and the results of this study also show that both ER{alpha} and Sp1 proteins are required for transcriptional activation of E2F1 in MCF-7 cells. However, our results also demonstrate that interactions with NF-Y are also required for E2 responsiveness, and ER/Sp1 interactions with NF-Y and other transcription factors are currently being investigated in this laboratory.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals, Cells, and Antibodies
MCF-7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in DMEM/F12 medium without phenol red and supplemented with 5% FBS plus 10 ml antibiotic-antimycotic solution (Sigma Chemical Co., St. Louis, MO) in an air-carbon dioxide (95:5) atmosphere at 37 C. E2 was purchased from Sigma Chemical Co. Sp1, Sp3, ER{alpha}, NF-1, and C/EBP antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). NF-YA antibody was purchased from Rockland, Inc. (Gilbertsville, PA). Luciferase and ß-galactosidase enzyme assay systems were obtained from Promega Corp. (Madison, WI). All other chemicals and biochemicals were the highest quality available from commercial sources. Oligonucleotides were synthesized by the Gene Technology Laboratory, Texas A&M University (College Station, TX).

Plasmids and Cloning
The human ER{alpha} expression plasmid was a generous gift from Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX). The expression plasmids of wild-type NF-YA and NF-YB and mutant NF-YA ({Delta}4YA13m29) and control plasmid ({Delta}4YA13) were kindly provided by Dr. Roberto Mantovani (Universita di Milano, Milan, Italy) (45). Constructs pE2F1a, pE2F1b, and pE2F1d were kindly provided by Dr. J. R. Nevins, Duke University (Durham, NC). The pE2F1c construct was made by the RT-PCR method (forward primer: 5'-CCGCCATTGGCCGTACCGCCCC-3'; reverse primer: 5'-GATCTTCCCGGCCACTTTTACGCGCCAAA-3') and inserted into pGL2 basic vector (Promega Corp.) at SacI and BglII cloning sites. The remaining E2F1 promoter-reporter constructs were made by inserting synthetic oligonucleotides into pGL2 basic vector (Promega Corp.) digested with SacI and BglII enzymes at the cloning sites. Resulting plasmids were sequenced by the Gene Technology Laboratory, Texas A&M University, to confirm appropriate insertion of the oligonucleotide inserts. The sequences of Sp1-binding sites (CCGCCCC) and CCAAT protein-binding sites in the E2F1 promoter were mutated into CCtttCC and atgcT, respectively, in all constructs containing mutation in these sites.

Transient Transfection Assay
MCF-7 cells were seeded in 60-mm petri dishes in DME/F12 medium supplemented with 2.5% dextran-coated charcoal FBS and grown until 70% confluence was reached. Plasmids (2–3 µg) were transiently cotransfected with ER expression plasmid (2 µg) using the calcium phosphate method. After transfection, cells were grown overnight in serum-free medium and treated with DMSO [0.1% (vol/vol) as control] or 10 nM E2 for 44 h. Cells were then washed with PBS and harvested with cell lysis buffer (Promega Corp.). Cell lysates were prepared by freeze-thawing (one time) followed by centrifugation at 10,000 x g for 5 min. Luciferase activity was determined in a luminometer (Parkard Instrument 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 (2.0 µg) obtained from Invitrogen (Carlsbad, CA).

Preparation of Whole-Cell Extracts
Cells were seeded in 100-mm petri plates and grown in DME/F12 media plus 5% dextran-coated charcoal-FBS. After cells reached 70% confluence, they were synchronized in serum-free medium for 3 days and then treated with DMSO (0.1% vol/vol as a control) and 10 nM E2 for 6, 12, and 24 h, respectively. Cell monolayers were then washed once in ice-cold PBS and scraped into lysis buffer [50 mM HEPES (pH 7.5), 150 mM sodium chloride, 10% (vol/vol) glycerol, 1% Triton X-100, 1.5 mM magnesium chloride, 1 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride, 200 µM sodium orthovanadate, 10 mM pyrophosphate, and 100 nM sodium fluoride]. Cells were incubated for 30 min, and then centrifuged at 10,000 x g for 5 min. Supernatants were precleared by addition of 20 µl of protein A-agarose beads for 30 min followed by centrifugation for 5 min at 10,000 x g. Lysates used for Western blot analysis were stored at -80 C until required. All procedures were carried out at 4 C.

Western Immunoblot Analysis
Cell lysates, prepared as described above, were loaded on SDS polyacrylamide gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane using an electroblotting apparatus overnight at 4 C. Membranes were blocked with TBS [10 mM Tris-HCl, (pH, 8.0); 150 mM sodium chloride] plus 5% milk (blotto bluffer) for 1 h and then incubated in primary antibody at 0.1 to 1.0 µg/ml in the blotto buffer for 1–2 h at 20 C. Membranes were then rinsed with water and washed for 5 min (two times) in TBS buffer. Membranes were incubated in enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 min, excess ECL reagent was removed by dabbing with a Kimwipe, and the membrane was sealed in plastic wrap. Membranes were then exposed to ECL hyperfilm for visualization of immunoreactive bands. E2F1 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the assay was carried out as described in treatment protocols provided by the company. Protein levels were quantitated using a Sharp JX-330 densitometer and a Scanalytics Zero D software package (Scanalytics, Billerica, MA).

Northern Blot Analysis
MCF-7 cells were seeded and grown as described above. Cells were then treated with DMSO (0.1% ol/vol) or 10 nM E2 for 0.5, 1.0, 2.0, 4.0, and 12 h, respectively. RNA was extracted using an RNA extraction kit from Tel-Test (Friendswood, TX). Twenty-five micrograms of total RNA obtained from each treatment group were separated by electrophoresis on 1.2% agarose gel, transferred onto a nylon membrane, bound to the membrane by UV cross-linking, and baked at 80 C for 2 h. The membrane was then prehybridized in a solution containing 0.1% BSA, 0.1% Ficoll, 0.1% polyvinyl pyrolidone, 10% dextran sulfate, 1% SDS, 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 E2F1 cDNA probes were labeled with [{gamma}-32P]dCTP. Gels were exposed to film Eastman Kodak Co., Rochester, NY) and quantitated via densitometry as described above. ß-Actin mRNA was used as an internal control to standardize E2F1 mRNA levels.

Nuclear Extract Preparation and Gel Mobility Shift Assay
Nuclear extracts were prepared from MCF-7 cells treated with DMSO (0.1% vol/vol) or 10 nM E2 for 4 h utilizing cells maintained in serum-free medium for 3 days. Nine picomoles of oligonucleotides were labeled at the 5'-end using T4-polynucleotide kinase and [{gamma}-32P]ATP. Nuclear extracts from control (DMSO) or E2-treated cells or recombinant human Sp1 (Promega Corp.) or ER (Pan Vera Co., Madison, WI) protein were incubated for 15 min at 0 C in HEGD [2 mM HEPES, 1.5 mM EDTA, 1.0 mM dithiothreitol, 10% glycerol (vol/vol), pH 7.6] buffer with 1 µg poly[d(I-C)] to bind nonspecific DNA-binding proteins and 200-fold excess of unlabeled wild-type or mutant oligonucleotide competitors for the competition experiments. After addition of [32P]-labeled DNA, the mixture (final volume, 20 µl) was incubated for an additional 20 min at 20 C. Reaction mixtures were loaded onto a 5% polyacrylamide gel and electrophoresed at 200 V in 0.9 M Tris-borate and 2 mM EDTA, pH 8.0, at 4 C. Gels were dried and protein-DNA complexes were visualized by autoradiography and quantitated using a Sharp JX-330 densitometer and a Scanalytics Zero-D software package as described above. For gel supershift experiments, antibodies were added after standard gel mobility shift assay procedure, and reactions were further incubated for 20–30 min at 20 C. For the off-rate assay, a 5-fold scale up of the standard gel shift assay (described above) was carried out and incubated for 20 min at 20 C. A 60-fold molar excess of the competitor (NF-Y DNA-binding element) was added to the binding reaction mixtures. Fifteen micoliters of the reaction samples were loaded onto a continuously electrophoresing gel at 0, 1, 3, 5.5, 9, and 13 min after addition of the competitor. For on-rate assay, [32P]-labeled probes were added to the reaction for 1, 2, 4, 8, 15, and 20 min after incubation with poly[d(I-C)]. The samples were then loaded onto a 5% polyacrylamide gel. Intensities of the bound bands were quantitated as described above. Oligonucleotides (sense strand) used for the competition of CCAAT-binding proteins are given below:

NF-Y: 5'-GGT AGG AAC CAA TGA AAT GCG AGG TAA-3'

C/EBP: 5'-TGC AGA TTG CGC AAT CTG CA-3'

NF-1: 5'-TTT TGG ATT GAA GCC AAT ATG ATA A-3'

wt-ERE: 5'-GTC CAA AGT CAG GTC ACA GTG ACC TGA TCA AAG TT-3'

mt-ERE: 5'-GTC CAA AGT CAG GaC ACA GTG tCC TGA TCA AAG TT-3'

wt-Sp1: 5'-AGC TTA TTC GAT CGG GGC GGG GCG AGC G-3'

mt-Sp1: 5'-AGC TTA TTC GAT CGa aGC GGG GCG CAG CG-3'

Coimmunoprecipitation Assays
[35S]NF-YA was synthesized using [35S]methionine and a rabbit reticulocyte lysate kit (TNT-coupled reticulocyte lysate system; Promega Corp.). Four microliters of in vitro translated [35S]NF-YA was incubated with 30 ng Sp1, 30 ng ER, 30 ng Sp1 plus 30 ng ER, or 30 ng TGF{alpha} plus 30 ng ER proteins at 4 C for 1 h in the same buffer system used for gel mobility shift assays. One microgram of anti-NF-YA, anti-Sp1, anti-ER antibodies, or preimmune serum was added and incubated for 1.5 h at 4 C with gentle rocking and 15 µl Agarose A/G PLUS beads were then added and further incubated at 4 C for another 1.5 h. The beads were washed with cell lysis buffer (8x, as described above), boiled for 5 min in 2x SDS loading buffer, and resolved on 10% SDS-PAGE. [14C]-Labeled protein molecular weight markers (Amersham Pharmacia Biotech) were used to determine the molecular weight of precipitated [35S]NF-YA protein.

Statistics
Results are expressed as means ± SE for at least three independent (replicate) experiments for each treatment group. Statistical significance was determined by ANOVA and Student’s t test, and the levels of probability are noted for each experiment.


    FOOTNOTES
 
Address requests for reprints to: Stephen H. Safe, Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, Texas 77843-4466.

This work was supported by the Welch Foundation, the NIH (Grants CA-76636 and ES-09106), and the Texas Agricultural Experiment Station. S.S. is a Sid Kyle Professor of Toxicology.

Received for publication March 12, 1999. Revision received April 20, 1999. Accepted for publication May 3, 1999.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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