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Carcinogenesis Advance Access originally published online on September 8, 2005
Carcinogenesis 2006 27(2):252-261; doi:10.1093/carcin/bgi225
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Carcinogenesis vol.27 no.2 © Oxford University Press 2005; all rights reserved.

7,12-Dimethylbenzanthracene-dependent transcriptional regulation of adenomatous polyposis coli (APC) gene expression in normal breast epithelial cells is mediated by GC-box binding protein Sp3

Aruna S. Jaiswal {dagger}, Ramesh Balusu {dagger} and Satya Narayan *

Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA

* To whom correspondence should be addressed at: UF Shands Cancer Center, Academic Research Building, PO Box 100232, University of Florida, Gainesville, FL 32610, USA. Tel: +1 352 846 1148; Fax: +1 352 392 5802; Email: snarayan{at}ufscc.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present investigation, we have examined the transcriptional regulation of adenomatous polyposis coli (APC) gene expression in the spontaneously immortalized human normal breast epithelial cell line, MCF10A, in response to carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) treatment. The APC mRNA levels and the APC gene's promoter (pAPCP) activity were increased in MCF10A cells after treatment with DMBA. A sequential deletion analysis and site-directed mutagenesis of the pAPCP promoter revealed that the DMBA response is mediated through a GC-box element. Also, the GC-box binding agent mithramycin A, which prevents binding of proteins to the GC-box region, abolished DMBA-mediated increase of the pAPCP promoter activity. The specificity of the proteins binding to the GC-box region was characterized by gel-shift analysis. An increased binding of the GC-box binding proteins was observed in the gel-shift analysis with nuclear extracts from DMBA-treated MCF10A cells, which corresponded to the increased levels of Sp1 and Sp3 proteins. However, a super-shift of the DNA–protein complexes was observed with only anti-Sp3 antibody. Based on the chromatin-immunoprecipitation assay results, the Sp3 appeared to be a genuine protein binding to the GC-box site of the pAPCP promoter. In RNA interference experiments, in which the Sp3 expression was knocked down, the DMBA response on the pAPCP promoter activity was reduced, suggesting that the binding of Sp3 to the GC-box site is critical for DMBA-induced pAPCP promoter activity. From these results we conclude that the increased pAPCP promoter activity in the MCF10A cell line in response to DMBA treatment is mediated by Sp3.

Abbreviations: APC, adenomatous polyposis coli; ChIP, chromatin-immunoprecipitation assay; CAT, chloramphenicol acetyltransferase; DMBA, dimethylbenz(a)anthracene; EMSA, electrophoretic mobility shift assay


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The adenomatous polyposis coli (APC) gene plays an important role in various cellular functions including cell migration and adhesion (1), cell cycle control (2–4), regulation of ß-catenin levels (5–7) and maintaining the proper orientation of chromosomes while serving as a platform between microtubules and chromosomes (8,9). APC also plays a role in apoptosis. In one report it has been shown that overexpression of APC in human colorectal cancer cell lines containing an endogenous inactive APC allele resulted in a substantial diminution of cell growth due to induction of cell death through apoptosis (10). Contrary to the overexpression, the downregulation of APC in mice has been found to increase apoptosis (11). In vitro antisense inhibition of APC has been shown to increase ß-catenin protein levels leading to an incomplete myotube formation due to increased apoptosis (12). This suggests a role for the APC/ß-catenin pathway in myotube development. Our studies support the hypothesis that a reduced level of APC is associated with apoptosis in colon cancer cells. We have shown that C2-ceramide-induced apoptosis in colon cancer cells is linked with reduced levels of APC (3). We have also described that the decreased levels of APC along with ß-catenin and E-cadherin are involved in curcumin-induced apoptosis in colon cancer cells (13).

Mutations in the APC gene are linked with early stage development of colorectal cancer in familial adenomatous polyposis patients. Now, it is well established that the development of colorectal cancer is a multi-step process in which a series of genes are mutated during tumor progression from adenoma to carcinoma (reviewed in ref. 14). This includes tumor suppressors, proto-oncogenes, DNA repair genes, growth factors and their receptor genes, cell cycle checkpoint genes and apoptosis-related genes. The cumulative effect of somatic mutations in these genes is the cause of sporadic colon cancer. About 25–38% of allelic loss in APC/MCC gene locus has been identified in breast cancer samples, but the rate of mutation in the APC gene has been low (15–17). In addition to these, the germ-line and somatic mutations of the APC gene are also found in patients with Turcot syndrome and other brain tumors (18). Based upon these studies it can be concluded that mutations in the APC gene play an important role in the onset of several cancer types, but the biological functions of the wild-type APC gene product are still not clear.

To understand more precisely the biological functions of the APC protein, we have initiated studies to understand how the APC gene is regulated in normal and cancer cells. Recently, we cloned the promoter region of the APC gene and showed that it is a TATA-less promoter and contains multiple cis-regulatory elements for its regulation (19). We also found that the APC gene is inducible, and its expression can be regulated by DNA-alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which is mediated by p53 (20–22). In the present communication, we are presenting data showing that the APC gene can be regulated by the carcinogen 7,12-dimethylbenzanthracene or equivalent name, 9,10-dimethyl-1,2-benzanthracene (DMBA) in the spontaneously immortalized human normal breast epithelial cell line, MCF10A. DMBA is present from 0.6 to 4 ng in each cigarette in different brands (23,24) and induces mammary carcinogenesis in laboratory animals and causes mutations in the H-ras gene (25). However, the exact mechanism by which DMBA induces mammary carcinogenesis is still not well defined. Our results provide evidence that DMBA induces APC gene expression at the transcriptional level which is mediated through the GC-box binding protein Sp3.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Maintenance of cells and treatment
The spontaneously immortalized human normal breast epithelial cell line, MCF10A, was grown in DMEM/F-12 medium supplemented with 5% horse serum (Sigma Chemical, St Louis, MO), 100 U/ml of penicillin, 100 µg/ml of streptomycin, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin, 10 ng/ml epidermal growth factor and 1% (w/v) L-glutamine at 37°C under a humidified atmosphere of 5% CO2 (26). MCF7 and HeLa cells were grown in RPMI and DMEM medium, respectively, supplemented with 10% FBS. After cells reached 60% confluence, they were treated with different concentrations of DMBA (Sigma Chemical) for indicated periods as shown in the figure legends.

Northern blot analysis
For northern blot analysis, the total RNA from untreated and treated cells was isolated by TRIzol®TM reagent as described by the manufacturer (Invitrogen Life Technologies, Carlsbad, CA). Fifty micrograms of total RNA were separated on 1% formaldehyde–agarose gel and transferred onto a Hybond-N+ membrane (Amersham Biosciences, Piscataway, NJ). The membrane was incubated with 32P-labeled APC probe (EcoRI fragment of APC-HFBCI43; ATCC, Manassas, VA). Later the same membrane was re-probed with 32P-labeled EcoRI fragment of 18S RNA probe for normalization of RNA loading and transfer efficiency (20). The membranes were exposed to X-ray films for detection of specific mRNA signals.

RT–PCR of the Sp1 and Sp3 mRNA levels
RT–PCR was performed with 5 µg of total RNA of untreated and DMBA-treated MCF10A cells. The specific primers used for PCR amplification were Sp1 (sense, 5'-CTACCCCTACCTCAAAGGAAC-3'; antisense, 5'-CTCTCCTTCTTTTTGCTGGCCT-3', length 821 bp), Sp3 (sense, 5'-TTCAGGGAGTTGCAATTGGTG-3'; antisense, 5'-TTCTGTGCCTGTGTCTCTTCA-3', length 448 bp), and ß-actin (internal control) genes (sense, 5'-GGACTTCGAGCAGGAGATGG-3'; antisense, 5'-GCACCGTGTTGGCGTAGAGG-3', length 232 bp).

Western blot analysis
The procedure for western blot analysis was performed as described earlier (20). Changes in Sp1, Sp3 and Sp4 protein levels in nuclear extracts of untreated or DMBA-treated MCF10A cells were determined by western blot analysis using anti-Sp1, Sp3 and Sp4 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) as described above.

Site-directed mutagenesis
Site-directed mutagenesis of the GC-box binding sequence of the promoter region of the APC gene was carried out using the Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). The pAPCP promoter construct as a template and the selected primers (forward primer 5'-GCAGGCTGTGCGGTTGGGTCAGGCCCTGTGCCCCACTGCG-3' and backward primer 3'-CGTCCGACACGCCAACCCAGTCCGGGACACGGGGTGACGC-5') were used in PCR as per manufacturer's protocol. Changing the sequence from GGGCGGG to GGGTCAG created mutation in the GC-box binding site. The GC-box mutant promoter construct was named pAPCP(GC-mut).

pAPCP promoter activity assays
The pAPCP promoter activity was either assayed by luciferase- or chloramphenicol acetyltransferase (CAT)-reporter assays as indicated in figure legends. MCF10A cells were grown to 60% confluence in 60 mm tissue culture dishes. The transfection of the plasmids into cells was performed with FuGENE-6 reagent as described by the manufacturer (Roche Diagnostics, Indianapolis, IN). A 9 µg of the luciferase- or CAT-reporter plasmid containing pAPCP or pAPCP(GC-mut) promoter sequence and 0.5 µg of the pCMV-ß-galactocidase (ß-gal) plasmid were mixed with 9 µl of the FuGENE-6. The DNA–lipid complex was assembled for 45 min and slowly added to the plates with proper mixing. After 24 h post-transfection, cells were treated with DMBA for an additional 48 h. Then cells were harvested, cellular lysates prepared and luciferase- or CAT-reporter assays performed. The activity of each assay was normalized with ß-gal activity in order to correct the differences in the transfection efficiency. Quantitative analysis of the data was done by Sigma Plot software (SPSS, Chicago, IL).

Electrophoretic mobility shift assay
The DNA–protein binding reactions were assembled in a final volume of 20 µl containing 20 mM HEPES, pH 7.9, 1 mM DTT, 5 mM MgCl2, 80 mM KCl, 10% (v/v) glycerol, 0.5 µg poly(dI·dC) and 5 µg nuclear extract. Reactions were carried out for 10 min, followed by the addition of 1 ng of 32P-labeled double-stranded GC-box consensus oligonucleotide of the pAPCP promoter (GC-box, 5'-GCGGTTGGGCGGGGCCCTGT-3') or the consensus GC-box oligonucleotide (Con-GC-box, 5'-ATTCGATCGGGGCGGGGCGAGC-3', Santa Cruz Biotechnology) and incubated further for 20 min at 22°C. For super-shift analysis, anti-Sp1, anti-Sp3 and anti-Sp4 antibodies were added prior to the addition of 32P-labeled probe and incubated for 30 min at 22°C. Then, the entire reaction mixture was loaded directly on a 4% non-denaturing polyacrylamide gel. After electrophoresis, the DNA–protein complexes were visualized by autoradiography. For competition experiments, a molar excess of unlabeled double-stranded wild-type, GC-box mutant (5'-GCGGTTGACTGCGGCCCTGT-3') or non-specific GAGA-binding site oligonucleotides (5'-GGGAAGCGGAGAGAGAAGCAGCTGTG-3') were added to the reaction mixture 20 min before the addition of 32P-labeled probe, as indicated in figure legends. To further characterize the GC-box binding proteins, we added 0.1 µM mithramycin A to 32P-labeled probe before the addition of the nuclear extract. Mithramycin A binds to the GC-box sequence and thus blocks the interaction of the protein binding to this region (27,28).

RNA interference
A vector-based overexpression of the double-stranded RNA (dsRNA) that is homologous to the Sp3 gene was used in these studies. A double-stranded oligonucleotide designed to contain a nucleotide sequence specific for the Sp3 mRNA (sense, 5'-GAGTCTCAGCAGCCAACCATTCAAGAGATGGTTGCTGCTGAGACTCTTTTT-3' and its complimentary) was cloned in human U6 promoter at SalI and XbaI sites of the transilent shRNA vector (Panomics, Redwood City, CA). The plasmid harboring the insert, named pSiRNA-Sp3, was transfected into MCF10A and HeLa cells using FuGENE-6 or Lipofectamine 2000 reagents as described above. The use of HeLa cells in these experiments served as a control since the Sp3-knockdown conditions have been standardized in this cell line. Three micrograms of pAPCP or pAPCP(GC-mut), 4 µg of pSiRNA-Sp3 and 0.5 µg of ß-gal plasmids were mixed with 9 µl of FuGENE-6 or 14 µl of Lipofectamine 2000. The DNA–lipid complex was assembled for 30 min at room temperature and slowly added to the plates with proper mixing. After 24 h post-transfection, cells were treated with DMBA for an additional 48 h. The Sp1 and Sp3 protein levels and pAPCP promoter activity were determined by western blot analysis and CAT-reporter assay, respectively, as previously described (19,20).

Chromatin immunoprecipitation assay
chromatin-immunoprecipitation assay (ChIP) assay was performed using MCF10A cells. MCF10a cells were grown to 50% confluence and treated with 25 µM of DMBA for different periods. Cells were fixed directly by adding 270 µl of 37% formaldehyde to 10 ml of culture media and harvested. Cells were washed with ice-cold phosphate-buffered saline containing protease inhibitors (1 mM phenylmethyl sulfonyl fluoride, 1 µg of aprotinin/ml and 1 µg of pepstatin A/ml) and lysate prepared for immunoprecipitation using a ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). Cells were lysed with 200 µl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.1) containing the protease inhibitors. This lysate was sonicated three times for 30 s with 30 s time intervals using a Branson Sonifer 450 at 20% constant maximal power. After clarification, the supernatant fraction was diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl) with protease inhibitors to a final volume of 2 ml. Samples were pre-cleared incubating with 75 µl of salmon sperm DNA/protein A–agarose slurry for 30 min at 4°C and then centrifuged at 110xg for 1 min in an Eppendorf 5417 c/R centrifuge. Immunoprecipitations were performed by incubating 2 ml of sample at 4°C overnight with 5 µg of anti-Sp1 and 5 µg anti-Sp3 antibodies (Santa Cruz Biotechnology). Protein A agarose/antibody/chromatin complexes were washed in the following order with 1 ml each of the low salt-immunecomplex wash buffer, high salt-immunecomplex wash buffer, lithium chloride-immunecomplex wash buffer and TE for 5 min with rotation. Protein A agarose/antibody/chromatin complex was eluted with 250 µl of elution buffer (1% SDS, 0.1 M NaHCO3) twice and pooled the two aliquots. Reverse cross-linking was done with 20 µl of 5 M NaCl for 4 h at 65°C followed by the addition of 10 µl of 0.5 M EDTA, 20 µl of 1 M Tris–HCl, pH 6.5 and 2 µl of 10 mg/ml Proteinase K to the combined aliquots and incubated for 1 h at 45°C. DNA was recovered by phenol–chloroform extraction and ethanol precipitation. The primers 5'-CAACTTCCTTGCTTGCTGGG-3' (sense) and 3'-GCGGAGAGAGAAGCAGCTG-5' (antisense) were used to amplify a 147 bp (–178 to –35) region of the pAPCP promoter. The PCR was carried out as follows: 1 cycle at 95°C for 30 s, 37 cycles at 94°C for 30 s, 56°C for 30 s and 72°C for 1 min and final extension 1 cycle at 72°C for 8 min.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
APC mRNA levels and cloned pAPCP promoter activity is increased in MCF10A cells treated with DMBA
The focus of the present study was to examine the mechanism by which DMBA regulates the transcription of the APC gene expression in normal breast epithelial cells. For these studies we used the spontaneously immortalized human normal breast epithelial cell line, MCF10A (26). Cells were treated with different concentrations of DMBA for 48 h, and APC mRNA levels were determined by northern blot analysis. We found an increased level of APC mRNA in these cells treated with DMBA (Figure 1A and B). The increase in the APC mRNA level was found up to 25 µM of DMBA treatment; however, at higher concentrations such as 50 and 100 µM of DMBA treatment the APC mRNA level was decreased (Figure 1A and B). Then, to examine whether the increased APC mRNA levels were due to increased transcriptional activity, we determined the promoter activity of the APC gene in MCF10A cells. We used our previously cloned APC promoter (pAPCP) in these studies (19,22). The pAPCP promoter was transfected into MCF10A cells and then treated with 25 µM of DMBA. The pAPCP promoter activity was determined from extracts of control and DMBA-treated cells. Results showed a significantly increased pAPCP promoter activity in a time-dependent manner with the highest at the 48 h period (Figure 2A). The concentration of 25 µM of DMBA treatment was found optimum for these studies (Figure 2B). Concentrations >25 µM of DMBA treatment caused a decrease in the pAPCP promoter activity. Thus, in subsequent experiments 25 µM of DMBA concentration and 48 h treatment period were used. So far, these results suggest that the increased APC mRNA levels in MCF10A cells are due to increased transcriptional regulation of APC gene expression.


Figure 1
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  		Fig. 1. APC mRNA levels in MCF10A cells treated with DMBA. (A) Shows the APC mRNA levels. Cells were treated with different concentrations of DMBA for 48 h and then processed for northern blot analysis. A representative autoradiogram of APC mRNA and 18S RNA is shown here. (B) Shows a quantitative analysis of the northern blots of two representative experiments.

 

Figure 2
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  		Fig. 2. pAPCP promoter activity is increased in MCF10A cells after treatment with DMBA. MCF10A cells were transfected with pAPCP plasmid and then either treated with 25 µM of DMBA for different periods (A, time course) or with different concentrations of DMBA (B, concentration curve) for 48 h. Results were normalized with ß-gal activity. Time course and concentration curve of the pAPCP promoter was determined by a luciferase-reporter assay. Data are the mean ± SE of three different experiments. Asterisk indicates significantly different from untreated control.

 
GC-box cis-regulatory element of the pAPCP promoter is responsive to DMBA treatment
Once we determined that the pAPCP promoter activity in MCF10A cells is increased after DMBA treatment, we examined the mechanism by which the pAPCP promoter activity is increased in MCF10A cells after DMBA treatment. Since DMBA-induced signals will pass on through transcription factors to the APC gene, it is important to analyze whether the pAPCP promoter contains a DMBA-responsive cis-regulatory element. The pAPCP promoter contains several cis-regulatory elements including potential binding sites for Oct-1, AP2, p53, Sp, E-box A, E-box B and E-box M. In addition, it has a GC-box and a CAAT-box (Figure 3A) (19,22). To examine which one of these cis-regulatory element(s) might be involved in DMBA response, sequential deletion mutants of the pAPCP promoter were generated as described in earlier studies (19,22). These mutants were transfected into MCF10A cells and then treated with 25 µM of DMBA for 48 h. The promoter activity was determined by CAT-reporter assay. The DMBA responsiveness to the pAPCP promoter diminished after the deletion of Oct-1, AP2 or p53-binding sites, which reappeared after the deletion of these sites from the promoter. A decrease in the pAPCP promoter activity after the deletion of the Oct-1, AP2 and p53-binding sites indicates that these elements could be potential sites for DMBA response. However, further deletion experiments showed a significant increase in the pAPCP(592) promoter activity which was comparable to the wild-type pAPCP promoter activity (Figure 3B). The pAPCP(592) promoter construct retains a GC-box, which seems to be responsible for the increased APC gene expression after DMBA treatment (Figures 3B and 4A). This appears to be true, since the activity of the GC-box less pAPCP(622) plasmid derived from the GC-box containing pAPCP(592) plasmid did not show the response of DMBA treatment. These results indicate that the GC-box is critical for DMBA-induced transcriptional upregulation of APC gene expression. Since, from the deletion experiments, the Oct-1, AP2 and p53-binding sites also appear to play a role in DMBA-induced pAPCP promoter activity, it became necessary to further clarify whether DMBA responsiveness is mediated through GC-box alone or with Oct-1, Ap2 and p53-binding sites. To address this concern, we introduced a site-directed mutagenesis at the GC-box site and changed its DNA sequence from GGGCGGG to GACTGCG. Both wild-type and GC-box mut [pAPCP(GC-mut)] plasmids were transfected into MCF10A cells, treated with 25 µM of DMBA for 48 h, and then the CAT-reporter activity was determined. Results showed no change in the pAPCP(GC-mut) promoter activity after DMBA treatment (Figure 4B), suggesting that the GC-box is the DMBA-responsive cis-regulatory element in the pAPCP promoter.


Figure 3
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  		Fig. 3. Determination of the DMBA-responsive cis-regulatory element of the pAPCP promoter. (A) Structure of the pAPCP wild-type and deletion mutation constructs. (B) CAT-reporter activity. MCF10A cells were transfected with different deletion mutation constructs, treated with 25 µM of DMBA for 48 h, and then processed for CAT-reporter activity. Data were normalized with ß-gal activity in the same experiment and are the mean ± SE of three different experiments. Asterisk indicates significantly different from untreated control.

 

Figure 4
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  		Fig. 4. GC-box of the pAPCP promoter is responsive to DMBA treatment. (A) Cells were transfected with pAPCP(592) plasmid and treated with different concentrations of DMBA. (B) Cells were transfected with either pAPCP or pAPCP(GC-mut) plasmids and then treated with 25 µM of DMBA. (C) Cells were transfected with pAPCP plasmid and pre-treated with 1 µM of mithramycin A for 2 h before the treatment with 25 µM of DMBA. In all these experiments, the DMBA treatment was for 48 h. The effect of DMBA on pAPCP promoter was determined by CAT-reporter activity. Data are mean ± SE of three different experiments. Asterisk indicates significantly different than untreated control. Double asterisk indicates significantly different from mithramycin A treated control.

 
To further confirm the role of the GC-box cis-regulatory element in DMBA-induced transcriptional regulation of APC gene expression, we used mithramycin A, which is known to specifically bind to the GC-box in DNA and inhibit the transcriptional regulation of target genes in which the GC-box binding proteins play a major regulatory role (27,28,29). The pre-treatment of MCF10A cells with mithramycin A before DMBA treatment reduced DMBA response on the pAPCP promoter activity (Figure 4C). These results further suggest that DMBA-induced expression of the APC gene is regulated by GC-box in MCF10A cells.

GC-box binding protein Sp3 is involved in DMBA-induced transcriptional regulation of pAPCP promoter
Electrophoretic mobility shift assays (EMSA) were carried out to analyze the interactions of proteins binding to the GC-box region of the pAPCP promoter that might be involved in DMBA-induced transcriptional regulation of APC gene expression. To characterize the binding specificity of proteins to the GC-box, we incubated 32P-labeled double-stranded GC-box oligonucleotide probe with nuclear extracts of MCF10A cells as described in Materials and methods. We observed four different DNA–protein complexes in EMSA. They might originate from homo- or heterodimer formation of different nuclear proteins binding to the GC-box oligonucleotide. All four DNA–protein complexes formed with the 32P-labeled probe were eliminated by competition with a 25-fold excess of the unlabeled GC-box oligonucleotide (Figure 5A, lanes 1–4), but were unaffected by a similar fold-excess of GC-box mutant or unrelated GAGA-binding site oligonucleotides (Figure 5A, lanes 5–7 and 8–10, respectively). To further characterize the binding specificity of the nuclear factors to the GC-box oligonucleotide, we incubated mithramycin A with the 32P-labeled GC-box oligonucleotide before mixing with the nuclear extract. Since mithramycin A binds with GC-box DNA, it is expected that it will block the binding of GC-box binding proteins to this site. The results showed a dose-dependent decrease in the DNA–protein complex formation in the presence of mithamycin A (Figure 5B, lanes 11–14). Taken together, these results suggest that proteins present in the MCF10A nuclear extract bind tightly and specifically to the GC-box region of the pAPCP promoter.


Figure 5
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  		Fig. 5. Characterization of binding of GC-box site of the pAPCP promoter to MCF10A cell nuclear extract. (A) 32P-labeled GC-box oligonucleotide was incubated with nuclear extract as described in Materials and methods. A 5-, 10- and 25-fold excess of unlabeled wild-type or mutant GC-box or unrelated GAGA-binding site oligonucleotides were present in the lanes where indicated. (B) Effect of mithramycin A on the binding of nuclear proteins to the GC-box oligonucleotide. The 32P-labeled GC-box oligonucleotide was pre-incubated with 1 µM mithramycin A before mixing with the nuclear extract. The DNA–protein complexes were separated on a 4% non-denaturing acrylamide/bis-acrylamide gel. The positions of the shifted DNA–protein complexes and the free probes are marked in the autoradiogram. Oligo, oligonucleotide; w/t, wild-type; mut, mutant.

 
Then we examined whether DNA–protein complexes were changed in nuclear extracts of DMBA-treated MCF10A cells as compared to untreated cells. The nuclear extract was prepared from MCF10A cells treated with different concentrations of DMBA for 48 h and used for EMSA. The results showed an increased level of DNA–protein complex formation with nuclear extract from DMBA-treated cells, in which the maximum binding was observed with 25 µM of DMBA treatment (Figure 6A, lanes 1–4). These results suggest that there is a factor(s) whose level is increased in the nuclear extract of MCF10A cells treated with DMBA. Next, we determined which one of the known GC-box binding proteins could be involved in the DNA–protein complexes in MCF10A cells and whether its level is changed after DMBA treatment. There are eight major Sp-family proteins binding to the GC-box region; among them are the most studied proteins, Sp1 and Sp3 (reviewed in ref. 29). In our studies we determined the levels of Sp1, Sp3 and Sp4 by western blot analysis. We found an increased level of Sp1 and Sp3 but not Sp4 in the nuclear extracts of MCF10A cells treated with DMBA (Figure 6B, lanes 1–5). The maximum induction was seen at 25 µM of DMBA treatment, which corresponds to the increased level of DNA–protein complex observed in EMSA (Figure 6A, compare lane 1 with 3). From these results it appears that Sp1 and Sp3 might be binding to the GC-box site of the pAPCP promoter and enhancing its activity in response to DMBA treatment.


Figure 6
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  		Fig. 6. DMBA treatment increases the levels of Sp proteins. (A) EMSA of 32P-labeled GC-box oligonucleotide with nuclear extracts of MCF10A cells treated with different concentrations of DMBA for 48 h. The free 32P-labeled probe is not shown in the figure. (B) Protein levels of Sp1, Sp3 and Sp4 in nuclear extracts of DMBA-treated MCF10A cells were determined by western blot analysis. The {alpha}-tubulin protein levels were used as loading controls. Data are representative of three separate experiments. (C) RT–PCR of the Sp1 and Sp3 mRNA levels. Cells were treated with different concentrations of DMBA for 48 h. Total RNA was isolated and processed for the RT–PCR using primers for the amplification of Sp1, Sp3 and ß-actin mRNA levels. A representative photograph of an agarose gel of three different experiments is shown in this figure. Amplification of the ß-actin mRNA levels served as an internal control for these experiments.

 
We further determined whether the increased Sp1 and Sp3 protein levels in MCF10A cells treated with DMBA were due to increased mRNA. We isolated total RNA from the MCF10A cells treated with 25 µM of DMBA for 48 h and determined the Sp1 and Sp3 mRNA levels by RT–PCR. To determine the RT–PCR accuracy and the quantitative analysis of the Sp1 and Sp3 mRNA levels, ß-actin mRNA levels were simultaneously amplified. Results showed no difference in the Sp1 or Sp3 mRNA levels in MCF10A cells after DMBA treatment (Figure 6C). From these results, it is concluded that the increase in the Sp1 and Sp3 protein levels after DMBA treatment might be a post-translational effect instead of a transcriptional effect.

To identify which Sp protein(s) might be binding to the GC-box oligonucleotide of the pAPCP promoter, we performed a super-shift EMSA by using Sp antibodies. In these experiments we used two types of GC-box oligonucleotides. One is a well-characterized consensus GC-box oligonucleotide (Con-GC-box; 5'-ATTCGATCGGGGCGGGGCGAGC-3') (30) and the other is from the pAPCP promoter (GC-box; 5'-GCGGTTGGGCGGGGCCCTGT-3'). The Con-GC-box oligonucleotide was used as a control for the verification of the super-shift results. We found a super-shift of the DNA–protein complexes I and II with anti-Sp3 antibody but not with anti-Sp1 or anti-Sp4 antibodies (Figure 7A, lanes 3 and 7). To further confirm whether the binding of Sp3 with the APC promoter was specific only to MCF10A cell line or applicable to other cell lines, we included MCF7 cells in our studies. A similar banding pattern of DNA–protein complexes were found with MCF7 cell nuclear extract, except that the shifted band III was a major band as compared with MCF10A cells in which the shifted band I was the major band. Also, there was a difference in the migration of shifted band IV in these two cells lines (Figure 7B). The difference in the intensity of the DNA–protein complexes and migration patterns in MCF10A and MCF7 cell lines could be due to differences in the expression levels of Sp-family proteins. Anti-Sp3 but not anti-Sp1 antibody shifted the DNA–protein complexes I and II of the MCF7 cells with both 32P-labeled Con-GC and APC promoter's GC-box oligonucleotides (Figure 7B, lanes 3 and 6). These results suggest that the Sp3 is a genuine protein binding to the GC-box site of the pAPCP promoter.


Figure 7
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  		Fig. 7. Characterization of the binding of Sp proteins to the GC-box site of the pAPCP promoter. An EMSA was performed with MCF10A or MCF7 cell nuclear extracts and 32P-labeled GC-box oligonucleotide with or without antibodies to Sp1, Sp3 or Sp4. The DNA–protein complexes were resolved on a 4% non-denaturing gel electrophoresis. The super-shifted band is shown with an arrow.

 
ChIP to confirm the interaction of Sp3 with pAPCP promoter in vivo
To determine the binding of the Sp-family proteins to the pAPCP promoter in vivo in response to DMBA treatment, we performed a ChIP assay. The MCF10A cells were treated with 25 µM of DMBA for different periods. The ChIP assay on the fixed cells was performed by using a ChIP assay kit (Upstate Biotechnology). We observed an amplification of the GC-box binding site DNA immunoprecipitated by anti-Sp3 antibody, which increased in a time-dependent manner in the DMBA-treated cells unlike in the untreated cells (Figure 8, compare lane 10 with 11–13). No DNA amplification was seen with samples that were immunoprecipitated with anti-Sp1 antibody (Figure 8, compare lane 6 with 7–9). From these results, along with the gel-shift analysis results, it is clear that Sp3 binds to the GC-box site of the pAPCP promoter and responds to the DMBA treatment.


Figure 8
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  		Fig. 8. ChIP analysis of Sp1- and Sp3-binding proteins with pAPCP promoter. MCF10A cells were treated with 25 µM of DMBA for 0, 24, 36 and 48 h and processed for the ChIP analysis as described in Materials and methods. The expected 147 bp of the pAPCP promoter DNA after the ChIP assay is shown with an arrow. Lane 1, molecular marker; lane 2, the sample was run simultaneously without antibody to serve as a background control; lane 3, IgG control; lanes 4 and 5 were the positive controls showing the expected signal from the input or the pAPCP DNA, respectively. The ChIP analysis was performed with Sp–DNA complex of the MCF10A cells treated with 25 µM of DMBA for different periods. An increased signal of the Sp3-bound (compare lane 10 with 11–13), and not the Sp1-bound DNA (compare lane 6 with 7–8) from the DMBA-treated cells is shown in the figure.

 
RNA-interference assay to determine the role of Sp3 in DMBA-induced transcriptional regulation of pAPCP promoter
These experiments were set up to examine whether the interaction of Sp3 is critical for DMBA-induced transcriptional upregulation of the pAPCP promoter. We tested this hypothesis by knocking down the Sp3 protein levels by the RNA-interference technique using a plasmid-based Sp3 SiRNA expression system as described in Materials and methods. The plasmid harboring the insert, named pSiRNA-Sp3, was transfected into MCF10A and HeLa cells using FuGENE-6 or Lipofectamine 2000 reagents as described by Abdelrahim et al. (31). The use of HeLa cells in these experiments served as a control since the Sp3-knockdown conditions have been standardized in this cell line. The Sp3 protein levels and pAPCP promoter activity were determined by western blot analysis and CAT-reporter assay, respectively, as previously described (19,22). The results showed ~80% knockdown of the Sp3 protein level in both HeLa and MCF10A cell lines after transfection with pSiRNA-Sp3 plasmid (Figure 9A, compare lane 1 with 2–4 and lane 5 with 6–8). Since the maximum downregulation of Sp3 protein level was achieved with 2 µg pSiRNA-Sp3 plasmid, we used the same concentration of the plasmid in the rest of the experiments. We found that the cells transfected with pSiRNA-Sp3 plasmid showed no induction in the pAPCP promoter activity after treatment with DMBA (Figure 9B, compare lane 1 and 2 with 3 and 4 for HeLa cells and lanes 9 and 10 with 11 and 12 for MCF10A cells). Here, the pAPCP(GC-mut) promoter served as a control in which the DMBA response to the pAPCP promoter is lost due to mutations at the GC-binding element (Figure 9B, compare lanes 5 and 6 with 7 and 8 for HeLa cells and lanes 13 and 14 with 15 and 16 for MCF10A cells). As a control, a non-specific pSiRNA plasmid was used in these studies. The results showed an increased pAPCP promoter activity in MCF10A or HeLa cells transfected with non-specific pSiRNA plasmid after treatment with DMBA (data not shown). From these results we conclude that the increased pAPCP promoter activity in HeLa and MCF10A cell lines in response to DMBA treatment is mediated by Sp3. In preliminary studies, we have found that DMBA (Jaiswal,A.S. and Narayan,S., unpublished data) and cigarette smoke condensate (CSC) (32) can induce transformation of MCF10A cells in vitro. Whether Sp3-mediated expression of the APC gene is involved in the transformation of MCF10A cells in response to DMBA or CSC treatment is currently under investigation.


Figure 9
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  		Fig. 9. Sp3 is the functional protein which responds to DMBA treatment and induces pAPCP promoter activity in HeLa and MCF10A cell lines. (A) Sp3 protein levels. HeLa and MCF10A cell lines were transfected with pSiRNA-Sp3 plasmid. After 48 h of transfection, cells were harvested and processed for western blot analysis. The {alpha}-tubulin protein levels were used as loading controls. (B) Effect of Sp3-knockdown on the pAPCP promoter activity. Cells were co-transfected with pSiRNA-Sp3, pAPCP or pAPCP(GC-mut), and ß-gal plasmids. After 24 h post-transfection, cells were treated with 25 µM of DMBA for an additional 48 h. After the treatment, cells were harvested and CAT-reporter activity was determined. Data are the mean ± SE of three different experiments. Asterisk indicates significantly different from control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Despite the broad biological functions of APC protein in various cell types, the transcriptional regulation of the APC gene in normal and cancer cells is not well studied. In the present study we examined the mechanisms by which DMBA, which is present in cigarette smoke and is a well-known breast carcinogen, induces APC gene expression in the normal breast epithelial cell line, MCF10A. Our results suggest that the GC-box binding site and the proteins binding to this site are critical for DMBA-induced transcriptional upregulation of APC gene expression. The sequential deletion analysis of the cloned promoter region of the APC gene (pAPCP) described here indicates that the GC-box site is important for DMBA response. The site-directed mutagenesis of the GC-box site further allowed a precise analysis of the role of GC-box site in the pAPCP promoter function in response to DMBA treatment. The importance of the GC-box site in the DMBA-induced transcriptional regulation of the pAPCP promoter was also confirmed by using mithramycin A which binds to the GC-box DNA and inhibits GC-box-mediated regulation of gene expression (27,28,33).

In order to understand which of the Sp-family proteins might be involved in DMBA-induced transcriptional regulation of APC gene expression, we characterized proteins binding to the GC-box site of the pAPCP promoter. Among the Sp-family proteins, Sp1 and Sp3 are ubiquitously expressed and well studied for their transcriptional role in gene regulation (reviewed in ref. 29). Our gel-shift and ChIP assay results suggest that Sp3 binds directly to the GC-box site of the pAPCP promoter DNA and activates its transcriptional activity. The regulation of Sp3 transcriptional activity is very complex and has been described both as an activator and an inhibitor (34). While the role of Sp3 is shown in the transcriptional upregulation of the cytokine-induced SOCS3 gene expression (35), its increased binding to the GC-rich box has been found critical for the downregulation of the ß myosine heavy chain (ß-MyHC) gene expression (36). In previous studies, it has been suggested that Sp3 competes for the binding with Sp1 to the GC-box and hence blocks Sp1-mediated transcriptional activation of gene expression (37). To test this possibility, we performed an RNA interference experiment in which the Sp3 protein levels in MCF10A cells were post-translationally knocked down by pSiRNA-Sp3. The results showed that the Sp3-knock-down did not have a significant effect on the pAPCP promoter activity in MCF10A cells after DMBA treatment. These results suggest that Sp3 and not Sp1 is involved in the DMBA-mediated transcriptional upregulation of APC gene expression in MCF10A cells. Since it is known that Sp1 and Sp3 compete for binding at the same GC-box site (37), we expected that after the Sp3-knockdown Sp1 would replace the function of Sp3 and activate the pAPCP promoter activity in response to DMBA treatment. However, our results did not confirm this possibility suggesting the presence of some unknown interference of the Sp1 binding to the pAPCP promoter. In future studies, it would be interesting to determine the mechanism by which Sp1 and Sp3 binding to the pAPCP promoter are discriminated in MCF10A cells after DMBA treatment. Nonetheless, it is intriguing that Sp3 is highly specific for the transcriptional regulation of the APC gene expression in MCF10A cells. It is known that Sp1 performs an additive or synergistic role, while Sp3 weakly activates or performs a repressive role in the cases of multiple adjacent GC-box sites in the promoters (38–42). Since the pAPCP promoter contains a single GC-box, Sp3 showed only a 2-fold induction of the promoter activity after DMBA treatment which is in agreement with the above findings. Furthermore, in earlier studies it has been shown that Sp3 functions as an activator once it is acetylated, suggesting that the acetylation may serve as a control switch from repressor to activator function of Sp3 (43). Whether the similar mechanism operates for the Sp3-mediated APC gene expression in MCF10A cells after DMBA treatment is currently not known.

The Sp-family of proteins plays an important role in cell growth and differentiation, since, in many cases, the transcriptional regulation of growth-related genes have been implicated through the GC-box binding sites (reviewed in ref. 44). In a recent study, it has been shown that Sp1 and Sp3 transcription factors upregulate the human prostate-specific antigen gene expression through multiple Sp1/Sp3 binding sites (45). In a previous study, the binding of Sp3 led to the repression of quinine oxidoreductase 2 (NQO2) gene transcription by the promoter containing the 29 bp insertion polymorphism, suggesting that alterations in NQO2 activity might be an important factor in susceptibility to Parkinson's disease (46). The specific recognition of GC-box sequence by Sp1/Sp3 has been shown to increase resistin (RETN) promoter activity, leading to enhanced serum resistin levels, causing the induction of human type 2 diabetes mellitus (47). In some cases the genetic variation in the human cholesteryl ester transfer protein (CETP) promoter has been shown to be associated with high-density lipoprotein cholesterol (HDL-C) levels and cardiovascular disease, which are regulated by variation in the Sp1/Sp3 binding sites in the proximal promoter (48). These studies suggest that the expression of Sp1 and Sp3 may play an essential role during the transformation of normal cells into malignant cells as well as be associated with the development of many diseases. In fact, in a recent study, data indicate that overexpression of Sp1 plays a causal role in the malignant transformation of human fibroblasts (49). In preliminary studies, we have found that DMBA (Jaiswal,A.S. and Narayan,S., unpublished data) and CSC (32) can induce transformation of normal breast epithelial cell line MCF10A in vitro. Previously, we have reported an increase in APC gene expression upon exposure of colon cancer cells to DNA-damaging agents (20,22), suggesting the possibility of an interaction between APC and the DNA repair machinery. Our recent study suggests that in response to DNA damage, APC plays a role in the regulation of base excision repair by directly interacting with DNA polymerase ß (pol-ß) and blocking pol-ß-mediated strand-displacement synthesis of long-patch base excision repair (LP-BER) pathway. The blocked LP-BER then increases the sensitivity of colon cancer cells to DNA-methylating agent, methylmethane sulfonate (50). Whether the Sp3-mediated expression of the APC gene is also involved in DNA damage-mediated carcinogenesis of MCF10A cells in response to DMBA treatment is currently being investigated in our laboratory. The increased expression of APC in MCF10A cells after DMBA treatment appears to be transient and persists up to 48 h and then decreases. Since DNA damage is an early event in the cells and since the compromised DNA repair may cause persistence in the mutation, it seems likely that the DMBA-induced APC gene expression could block DNA repair in MCF10A cells before cell division. The doubling time for the MCF10A cells is ~20 h; thus, the APC-mediated block of DNA repair for 48 h may create a permanent mutation in the genome that may result in the transformation of the MCF10A cells. This hypothesis will be tested in our future studies.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We thank Dr Fazlul Sarkar from Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI, for the MCF10A cell line; Dr Daiqing Liao, Ms Mary Wall and Ms Shahnjayla Connors for critically reading the manuscript, and NCI/NIH for the financial support to SN (R01 CA-097031). We also thank Dr T. Thangasamy for her technical assistance in some CAT-reporter assays.

Conflict of Interest Statement: None declared.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
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Received May 2, 2005; revised August 9, 2005; accepted September 5, 2005.


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