Induction of Transforming Growth Factor-β Receptor Type II Expression in Estrogen Receptor-positive Breast Cancer Cells through SP1 Activation by 5-Aza-2′-deoxycytidine*

Previous studies suggest that estrogen receptor-positive (ER+) breast cancer cells acquire resistance to transforming growth factor-β (TGF-β) because of reduced expression levels of TGF-β receptor type II (RII). We now report that treatment of ER+ breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-2′-dC) leads to accumulation of RII transcript and protein in three different cell lines. RII induction restored TGF-β response in MCF-7L breast cancer cells as indicated by the enhanced activity of a TGF-β responsive promoter-reporter construct (p3TP-Lux). A transiently transfected RII promoter-reporter element (RII-chloramphenicol acetyltransferase) showed an increase in activity in the 5-aza-2′-dC-treated MCF-7L cells compared with untreated cells, suggesting the activation of a transactivator of RII transcription. Using electrophoretic mobility shift assays, the enhanced binding of proteins from 5-aza-2′-dC-treated MCF-7L nuclear extracts to radiolabeled Sp1 oligonucleotides was demonstrated. An RII promoter-chloramphenicol acetyltransferase construct containing a mutation in the Sp1 site was not expressed in the 5-aza-2′-dC-treated MCF-7L cells, further demonstrating that induction of Sp1 activity by 5-aza-2′-dC in the MCF-7L cells was critical to RII expression. Northern analysis indicated that 5-aza-2′-dC treatment did not affect the Sp1 transcript levels. Western blot analysis revealed an increase of Sp1 protein in the 5-aza-2′-dC-treated MCF-7L cells, but there was no change in the c-Jun levels. Studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5-aza-2′-dC-treated MCF-7L extracts compared with untreated control extracts. These results indicate that the transcriptional repression of RII in the ER+ breast cancer cells is caused by suboptimal activity of Sp1, whereas treatment with 5-aza-2′-dC stabilizes the protein thus increasing steady-state Sp1 levels and thereby leads to enhanced RII transcription and subsequent restoration of TGF-β sensitivity.

family that includes activins, inhibins, bone morphogenetic proteins, and mü llerian-inhibiting substances (1,2). TGF-␤ plays an important role in cellular proliferation, differentiation, and synthesis of extracellular matrix proteins (1,2). Three major TGF-␤-binding proteins have been identified. They are referred to as type I (RI), type II (RII), and type III (RIII). RI and RII are glycoproteins of 53 and 75 kDa, respectively, whereas RIII is a 280 -330-kDa proteoglycan (3). RI and RII are serine/threonine kinases and form a hetero-oligomeric complex that is required for the TGF-␤-mediated signaling cascade (4 -7). RIII lacks a signaling motif, and its role appears to be limited to presenting TGF-␤ to the signaling receptors (8).
One of the important effects of TGF-␤ is the inhibition of growth of epithelial cells as well as some cancer cells. Because RI and RII are both required for TGF-␤-mediated growth suppression, loss of either receptor may contribute to TGF-␤ resistance and subsequent malignant progression. TGF-␤ resistance caused by defects in RII expression has been reported in various cell lines (9 -11). Previous work has indicated an association between defective RII expression and malignant progression of several cell types (9,10,(12)(13)(14) including breast carcinoma cells (15). RII replacement in breast and colon carcinoma cells restored TGF-␤ response and reduced malignant behavior (12,15). It has also been demonstrated that exogenous RI expression in an RI-defective colon carcinoma cell line reversed malignancy (16). These studies underline the importance of both RI and RII as tumor suppressors. Loss of RII expression was observed in gastric cancer cells as well as a subset of colon cancer cells in association with deletions or gene mutations (10,17). RII repression caused by decreased binding of nuclear proteins to the positive regulatory elements of the RII promoter has been shown to cause TGF-␤ resistance of adenovirus E1A-transformed mouse keratinocytes (18).
Breast cancer cell lines that express estrogen receptor (ER ϩ ) are refractory to TGF-␤ effects, whereas estrogen receptornegative (ER Ϫ ) cells are often TGF-␤-sensitive (19). Loss or undetectable expression of RII has been reported to contribute to TGF-␤ resistance in ER ϩ breast cancer cells (11,15). Several different ER ϩ MCF-7 strains have been reported in the literature. Comparison of ER ϩ MCF-7 early (MCF-7E) and MCF-7 late (MCF-7L) passage cells from our laboratory has shown that MCF-7E cells express RII and are TGF-␤-responsive, but MCF-7L cells lack RII and are TGF-␤-resistant, suggesting possible defects at the transcriptional or post-transcriptional level (20). A transiently transfected RII promoter element exhibited markedly decreased activity in the MCF-7L cells com-pared with MCF-7E cells, pointing toward a possible defect in transcription.
Gene inactivation caused by methylation of CpG sites in the vicinity of promoter regions has long been associated with tissue-specific and developmentally regulated genes (21). However, recent studies have cited gene methylation as a mode of inactivation of several genes including some that are involved in cell cycle control (14). DNA methyltransferase inhibitors 5-azacytidine and 5-aza-2Ј-deoxycytidine (5-aza-2Ј-dC) are the agents used most frequently to reverse methylation and reconstitute the expression of these genes (22).
To delineate the mechanism of RII repression in the MCF-7L and other ER ϩ breast cancer cells, we have carried out studies using 5-aza-2Ј-dC and now provide evidence that RII expression is low or undetectable because of suboptimal activity of Sp1 transcripton factor. Treatment with 5-aza-2Ј-dC leads to increased Sp1 steady-state levels as a result of increased protein stability and, consequently, concomitant induction of RII expression. RII expression resulted in restoration of TGF-␤ sensitivity. These results shed light on a novel mechanism by which epithelial cells escape negative regulatory effects of TGF-␤ leading to uncontrolled growth and hence tumor formation and progression.

EXPERIMENTAL PROCEDURES
Cell Culture-All of the breast cancer cell lines used were obtained from American Type Culture Collection (ATCC). The BT20 strain in our laboratory has a constitutively active mutated estrogen receptor, hence we refer to it as ER ϩ . Cells were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum (Sigma), amino acids, antibiotics, pyruvate, and vitamins (Life Technologies, Inc.). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO 2 . For experiments in which 5-aza-2Ј-dC was used, cells were seeded at a density of 4 ϫ 10 5 cells/10-cm culture dish (day 0). 5-Aza-2Ј-dC (Sigma) was added to the growth medium (1 or 2 g/ml as indicated) in two 24-h pulses on days 2 and 5. Cells were used on day 7 for RNA determinations, transfections, or isolation of nuclear extracts for electrophoretic mobility shift assays (EMSAs) and Sp1 Western immunoblots.
RNA Analysis-Total RNA from the breast cancer cells was extracted by guanidine thiocyanate homogenization and ultracentrifugation through a cesium gradient as described previously (23). RI, RII, and actin riboprobes were described previously (15). RNase protection assays were performed as described previously (24). Briefly, radioactive riboprobes were allowed to hybridize overnight with the RI and RII mRNA in 40 g of total RNA. After RNase A and T 1 treatment, the protected double-stranded RNA fragments were analyzed by urea-PAGE and visualized by autoradiography. Actin was used to normalize sample loading.
Receptor Cross-linking-Simian TGF-␤1 was purified as described (25) and iodinated by the chloramine-T method (26). Cells were seeded at a density of 6 ϫ 10 4 /well in a six-well plate (day 0). Wherever indicated, 1 or 2 g of 5-aza-2Ј-dC was added in two 24-h pulses on days 2 and 5, and receptor binding studies were carried out on day 7 using 200 pM 125 I-TGF-␤ as described previously (27). Labeled cells were solubilized in 200 l of 1% Triton X-100 with 1 mM phenylmethylsulfonyl fluoride. Equal amounts of cell lysate protein were separated by 4 -10% gradient SDS-PAGE under reducing conditions and exposed for autoradiography.
Luciferase Assay-The TGF-␤-responsive plasminogen activator inhibitor promoter-luciferase reporter construct (p3TP-Lux) was used to determine TGF-␤ sensitivity as described previously (5). The p21 promoter in which the TGF-␤-responsive element was deleted (p21/WAF1/ CIP1/smaI⌬) was used as a control (28). 5-Aza-2Ј-dC-treated and untreated MCF-7L cells were transiently transfected with 30 g of p3TP-Lux and 5 g of ␤-galactosidase plasmid by electroporation with a Bio-Rad gene pulser at 250 mV and 960 microfarads. The electroporated cells were plated into six-well tissue culture plates. Cells were grown for 24 h and then treated with 5 or 10 ng/ml TGF-␤1 for 24 h. Cells were harvested in 200 l of lysis buffer (luciferase assay system, Promega) 48 h after transfection. In the first 10 s after the addition of substrate, luciferase activity was measured using a luminometer (Berthold lumat LB 9501) and was expressed as relative units after normalization to ␤-galactosidase activity.
Chloramphenicol Acetyltransferase (CAT) Assay-RII and insulinlike growth factor II (IGF-II) promoter-CAT constructs were described previously (29,30). The RII construct (Ϫ274/ϩ50) containing the core promoter with two Sp1 sites and two enhancer regions (PRE1 and PRE2), a Ϫ47 RII-CAT (wild type Sp1 site), a Ϫ47 Spm RII-CAT (mutated Sp1 site), and IGF-II constructs (Ϫ58/ϩ124) containing a distinct TATA box in combination with either two wild type Sp1 (Ϫ58 IGF-II-CAT) or two mutated Sp1 sites (Ϫ58 Spm IGF-II-CAT) sites were used in this study. MCF-7L cells that were untreated or treated with 1 g/ml 5-aza-2Ј-dC, respectively, were transiently transfected with 30 g of Ϫ274/ϩ50 RII promoter-CAT construct or Ϫ58/ϩ124 IGF-II promoter-CAT construct by electroporation with a Bio-Rad gene pulser at 250 mV and 960 microfarads. For normalization of transfection efficiency, 5 g of Rous sarcoma virus ␤-galactosidase was cotransfected into the cells. Cells were plated into 10-cm Petri dishes, and 48 h later cells were harvested to carry out the standard ␤-galactosidase (31) and CAT assays (32). ␤-Galactosidase was analyzed using a molecular dynamics microtiter plate reader and the Softmax software package. Results from CAT assays were analyzed by TLC, and the TLC plate was quantitated directly using an Ambis system as well as by autoradiography.
EMSA-Double-stranded oligonucleotides representing the two Sp1 sites (Ϫ25 bp and Ϫ143 bp relative to start site), the two positive regulatory regions (PRE1 and PRE2), and a mutant Sp1 oligonucleotide (18,29) were custom designed and obtained from Genosys. The oligonucleotides were end labeled using [␥-32 P]ATP (50 Ci at 3,000 Ci/ mmol) and T 4 polynucleotide kinase. The labeled oligonucleotides were purified using probe Quant TM G-50 microcolumns (Amersham Pharmacia Biotech). Binding reactions were performed using 3 g of nuclear extracts, buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol), 2 g of poly(dI-dC), and 10,000 cpm of 32 P-labeled oligonucleotide in a volume of 20 l. Reactions were incubated at room temperature for 15 min. Competition reactions were performed by adding an unlabeled double-stranded oligonucleotide to the reaction mixture. Samples were analyzed by nondenaturing 4% polyacrylamide gel at 150 V for 1.5 h in 100 mM Tris borate-EDTA buffer. Gels were vacuum dried and analyzed by autoradiography. For the supershift assay, the 32 P-labeled oligonucleotide plus nuclear extract was incubated further at room temperature for 15 min with 2 g of Sp1 antibody (anti-rabbit, Santa Cruz) before electrophoresis and autoradiography.
Northern Analysis-Total RNA from the control and 5-aza-2Ј-dCtreated MCF-7L cells was extracted by guanidine thiocyanate homogenization and ultracentrifugation through a cesium gradient as described previously (23). Total RNA (10 g) was fractionated on 1.2% agarose gel containing formaldehyde and transferred to nitrocellulose membrane. Prehybridization and hybridization were performed at 65°C using RIPA buffer (Amersham Pharmacia Biotech). The cDNA probe for Sp1 was labeled with [ 32 P]dCTP (Ͼ3,000 Ci/mmol; Amersham Pharmacia Biotech) using a random primed DNA labeling kit (Boehringer Mannheim).
Western Immunoblot Analysis of Sp1-Nuclear extracts (4 g) from 5-aza-2Ј-dC-treated and untreated MCF-7L cells were resolved using 7.5% SDS-PAGE and transferred to nitrocellulose membranes by wet electrophoretic transfer (Bio-Rad). Nonspecific binding was blocked with 5% non-fat milk in TTBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) overnight at 4°C. The membrane was probed with a rabbit anti-human Sp1 polyclonal antibody (Santa Cruz) in the same buffer for 1 h at room temperature and washed three times with TTBS for 10 min each. Bound antibodies were detected with an anti-rabbit peroxidase-conjugated IgG and an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The membrane was reprobed with rabbit anti-human c-Jun polyclonal antibody (Santa Cruz). The MCF-7L cells were treated with 10 g/ml cyclohexamide and harvested at the indicated time points for the Sp1 protein stability studies (33).

Expression of TGF-␤ Receptors-Previous studies indicate
that ER ϩ breast cancer cells express low RII levels and hence are TGF-␤-resistant (11,15). However, several different strains of various ER ϩ breast cancer cell lines have been reported which exhibited differential sensitivities to TGF-␤. Hence we have screened for RI and RII transcripts in TGF-␤-resistant ER ϩ breast cancer cell lines (MCF-7L, BT20, ZR75, T47D) using an RNase protection assay. The TGF-␤-sensitive ER Ϫ

5-Aza-2Ј-dC Induction of TGF-␤ RII
cell line MDA MB 231 was used as a positive control. All of the ER ϩ cell lines expressed RI mRNA, but RII mRNA was undetectable ( Fig. 1). The ER Ϫ MDA MB 231 cell line has both RI and RII transcripts.
Because RI and RII are interdependent for TGF-␤ binding and signaling, we carried out receptor binding studies with 125 I-TGF-␤1 to determine the expression of cell surface receptors RI and RII. Cell surface receptors were not detected in the ER ϩ cell lines but were present in the ER Ϫ cell line and the mink lung epithelial cell line CCL64, which were used as positive controls (Fig. 2). The specificity of binding was demonstrated by competing with a 50-fold excess of cold TGF-␤1.
Effect of 5-Aza-2Ј-dC on the Induction of RII-RNase protection assays were performed to determine whether treatment with the DNA methyltransferase inhibitor 5-aza-2Ј-dC leads to expression of RII transcript. Accumulation of RII transcript was observed in all of the ER ϩ cell lines (Fig. 3). To examine whether RII expression permitted TGF-␤ binding to RI, cell surface receptor binding studies with 125 I-TGF-␤1 were carried out. The data show that expression of RII resulted in the cell surface binding of TGF-␤ to both RII and RI (Fig. 4). Increased binding for RIII was also noted. This phenomenon has also been observed after RII transfection in previous studies (12,15). Binding specificity was demonstrated by competing with a 50-fold excess of TGF-␤1.
RII Promoter Activity-Southern analysis of the RII promoter region did not reveal any methylated sites (data not shown). We have compared the RII promoter activity in control and 5-aza-2Ј-dC-treated MCF-7L cells using an RII promoter-CAT construct (Fig. 6A) to examine whether the induction of RII after 5-aza-2Ј-dC treatment is caused by the activation of a transactivator. This promoter element contains two Sp1 binding sites (at Ϫ25 bp and Ϫ143 bp relative to the start site) and two positive regulatory elements (PRE1, Ϫ219 bp/Ϫ172 bp; PRE2, ϩ1 bp/ϩ50 bp relative to the start site). 5-Aza-2Ј-dCtreated MCF-7L cells showed higher promoter activity than control cells, thus suggesting the activation of nuclear proteins that bind to the RII promoter and enhance its activity (Fig. 6B).
EMSAs-To narrow the identity of the proteins that are

FIG. 3. Expression of RII mRNA after 5-aza-2-dC treatment.
Total RNA was isolated from control and 5-aza-2Ј-dC-treated breast cancer cells as described under "Experimental Procedures." RII riboprobe was incubated with 40 g of total RNA, and the hybridized RNA was analyzed by RNase protection assay as described under "Experimental Procedures." Actin RNA levels were used for normalization.

5-Aza-2Ј-dC Induction of TGF-␤ RII
enhancing the RII promoter activity in the 5-aza-2Ј-dC-treated MCF-7L cells, EMSAs were performed using nuclear extracts and the 32 P-labeled oligonucleotides corresponding to the two Sp1 binding sites as well as the two positive regulatory elements (PRE1 and PRE2), which have been recognized previously (18). Oligonucleotides corresponding to the wild type RII promoter PRE1, PRE2, and the two Sp1 sites as well as a mutated Sp1 oligonucleotide were analyzed for binding to nuclear proteins (Figs. 7A and 8A). Gel shift analysis of nuclear extracts from 5-aza-2Ј-dC-treated and control MCF-7L cells do not reveal any differences in the proteins that bind to PRE1 and PRE2 (Fig. 7B). Cold oligonucleotides competed with 32 Plabeled PRE oligonucleotides for binding to the protein-DNA complexes (Fig. 7B). However, there was enhanced binding of nuclear proteins from the 5-aza-2Ј-dC-treated MCF-7L cells to both of the 32 P-labeled Sp1 oligonucleotides compared with untreated control cells (Fig. 8B). The low mobility complexes that were present in the 5-aza-2Ј-dC-treated extracts were absent in the control nuclear extracts. The mobilities of these complexes were similar to that of recombinant human Sp1 (Promega)-bound 32 P-labeled Sp1 oligonucleotide complex, which was used as a positive control. Wild type Sp1 oligonucleotides competed with 32 P-labeled Sp1 oligonucleotides for binding to the protein complexes, whereas the mutant Sp1 oligonucleotide could not, thus indicating the specificity of the shifts. To confirm further that the enhanced protein-DNA complex contains Sp1, supershift assays were carried out by incubating the protein-DNA complexes with 2 g of Sp1 antibody (described under "Experimental Procedures"). Sp1 antibody recognized the Sp1 in the protein-DNA complexes, resulting in a clear shift of the mobility of the protein bound to the 32 Plabeled Sp1 oligonucleotides (Fig. 9).
Effect of Sp1 Mutation on RII Promoter Activity-To confirm the role of Sp1 in the enhanced RII promoter activity, the Ϫ47 RII promoter-CAT construct containing either the wild type or mutant Sp1 site was transiently transfected into control and 5-aza-2Ј-dC-treated MCF-7 cells (Fig. 10A). 5-Aza-2Ј-dCtreated MCF-7L cells expressed higher wild type RII promoter activity compared with control untreated MCF-7L cells. The Sp1 mutant RII promoter-CAT construct was not expressed (Fig. 10B).
IGF-II Promoter Activity-To determine if 5-aza-2Ј-dC-mediated Sp1 activity leads to enhanced expression of other Sp1dependent promoters, we transiently expressed either the wild type or Sp1 site-mutated IGF-II promoter-CAT constructs (Fig.  11A) in 5-aza-2Ј-dC-treated or untreated MCF-7L cells. The IGF-II promoter contains a distinct TATA box and two Sp1 sites (30). 5-Aza-2Ј-dC-treated MCF-7L cells exhibited a significantly higher IGF-II promoter activity compared with untreated control cells. The 5-aza-2Ј-dC-induced IGF-II promoter activity disappeared but retained the basal promoter activity when the Sp1 sites were mutated (Ϫ58 Spm IGF-II-CAT). These data further confirm the activation of Sp1 by 5-aza-2Ј-dC in the MCF-7L cells (Fig. 11B).
Expression of Sp1 Protein-To determine whether 5-aza-2Ј-dC treatment stimulates the Sp1 protein expression, Western immunoblot was performed using 4 g each of nuclear extracts from 5-aza-2Ј-dC-treated and untreated MCF-7L cells. Western analysis showed two protein species of 95 and 105 kDa. The two species are the result of differential post-translational modification of the Sp1 polypeptide (34,35). Significant increases of both the species were observed in the nuclear extracts of 5-aza-2Ј-dC-treated MCF-7L cells, whereas there was no change in the c-Jun levels (Fig. 13). Sp1 protein stabil-ity studies after treatment with 10 g/ml cyclohexamide were performed as described previously for this protein (33) to determine if 5-aza-2Ј-dC stabilizes Sp1 protein indirectly. Sp1 protein from 5-aza-2Ј-dC-treated MCF-7L cells showed enhanced stability compared with untreated MCF-7L control cells (Fig. 14), thus indicating that the increased steady-state levels of Sp1 protein and oligonucleotide binding activity were the result of indirect actions of the demethylating agent on Sp1. DISCUSSION Breast cancer cells that express estrogen receptor (ER ϩ ) escape negative growth regulation by TGF-␤, leading to malig- nant behavior. Previous work from our laboratory (15) and Kalkhoven et al. (11) indicated that ER ϩ breast cancer cells acquire resistance to TGF-␤ because of a lack of or inadequate expression of RII. Replacement of RII in a TGF-␤ resistant ER ϩ MCF-7L cell line restored TGF-␤ response and reduced tumorigenicity in athymic nude mice (15). Reversal of malignancy of a human colon carcinoma cell line was also reported after RII expression (12). Hence, targeting re-expression of RII may offer potential novel approaches for treatment or chemoprevention of breast cancer. In this study, we have examined the ability of the DNA methyltransferase inhibitor 5-aza-2Ј-dC to restore endogenous RII expression in the ER ϩ breast cancer cells.
Treatment with 5-aza-2Ј-dC led to induction of RII expression in all three of the ER ϩ breast cancer cell lines examined (Figs. 3 and 4). Significantly, 5-aza-2Ј-dC-mediated RII induction resulted in restoration of TGF-␤ response in the MCF-7L cells (Fig. 5). However, the induction of RII expression after 5-aza-2Ј-dC treatment was not found to be a result of the direct demethylation of the RII gene. This raised the possibility of the involvement of increased activation of a transactivator as a cause for enhanced RII transcription.
RII repression resulting from decreased binding of nuclear proteins to the enhancer regions (PRE1 and PRE2) of the RII promoter has been reported in adenovirus E1A-transformed mouse keratinocytes (18). However, 5-aza-2Ј-dC-treated and untreated MCF-7L cells showed no differences in the nuclear proteins that bind to these enhancer regions (PRE1 and PRE2). The RII promoter lacks a distinct TATA box, and Sp1 has been reported to play an important role in the initiation of transcription from promoters lacking distinct TATA boxes (36). The human RII promoter contains two Sp1 sites at Ϫ25 and Ϫ143 bp relative to the start site (29). Enhanced RII promoter activity (Fig. 6) as well as the increased binding of nuclear proteins to the 32 P-labeled Sp1 oligonucleotides (Figs. 8B and 9) in the 5-aza-2Ј-dC-treated MCF-7L cells indicate that increased Sp1 activity was induced by 5-aza-2Ј-dC. An RII promoter-CAT construct with a mutated Sp1 site was not expressed in the 5-aza-2Ј-dC-treated MCF-7L cells, further demonstrating the specificity of enhanced Sp1 activity resulting from 5-aza-2Ј-dC treatment (Fig. 10B). Enhancement of Sp1 levels also led to the increased expression of the Sp1-dependent IGF-II promoter in 5-aza-2Ј-dC-treated MCF-7L cells (Fig. 11B). Consequently, the results presented in this study indicate that suboptimal activity of Sp1 results in transcriptional repression of RII in the ER ϩ breast cancer cells. This appears to have a role in uncontrolled growth and subsequent malignant progression as evidenced by studies showing that RII replacement reverses malignancy in MCF-7L cells (15).
However, 5-aza-2Ј-dC studies on MCF-7L cells have raised some interesting questions. A similar drug (5-azacytidine) has been reported to increase Sp1 activity without altering protein expression leading to TGF-␣ transcription in melanoma cells (37). In our study, 5-aza-2Ј-dC-treated MCF-7L cells did not show any increase in the Sp1 transcript levels (Fig. 12). Thus, Sp1 induction as a result of the demethylation at the Sp1 gene locus was eliminated. However, 5-aza-2Ј-dC-treated MCF-7L cells exhibited enhanced Sp1 activity as well as increased Sp1 nuclear protein levels (Fig. 13). This may result from the effects of 5-aza-2Ј-dC at a different gene locus, whose product may be FIG. 9. Detection of protein-DNA complexes using Sp1 antibody. To confirm that protein-DNA complexes contained Sp1, supershift assays were carried out by incubating the nuclear extract plus 32 P-labeled Sp1 oligonucleotide complexes for 15 min at room temperature with 2 g of Sp1 antibody. A lower mobility complex resulting from the binding of Sp1 antibody to protein-DNA complexes was observed.
FIG. 10. Panel A, schematic of wild type/mutated Ϫ47 RII promoter-CAT construct. The construct contains one Sp1 binding site. Wild type and mutated sequences are indicated. Panel B, wild type and mutated RII promoter-CAT activity in 5-aza-2Ј-dC-treated MCF-7L cells. The Ϫ47 RII promoter-CAT construct with wild type or mutated Sp1 sites was transiently transfected into control or 5-aza-2Ј-dC-treated MCF-7L cells as described under "Experimental Procedures." 48 h after transfection, cells were harvested, normalized for ␤-galactosidase activity, and CAT assays were performed (see "Experimental Procedures"). required for Sp1 expression and activity. Sp1 undergoes posttranslational modifications such as phosphorylation and glycosylation (34,35). Glycosylation may aid in nuclear localization and DNA binding, and phosphorylation may assist in stabilizing the Sp1⅐DNA complex. It has been shown that glycosylation stabilizes Sp1, and hypoglycosylated Sp1 is susceptible to proteasome degradation (38). However, recent modeling studies of Sp1 indicated that the modification adversely affected proteinprotein interactions involving the transcription factor (39). Protein stability studies after cyclohexamide treatment suggested a significant increase in the Sp1 protein stability from 5-aza-2Ј-dC-treated MCF-7L nuclear extracts compared with control untreated MCF-7L nuclear extracts (Fig. 14). The mechanism for increased stability is not clear at this time. The available data indicate that the affected gene resulting in Sp1 protein stabilization is probably not affecting glycosylation. A transiently transfected RII promoter-CAT element exhibited enhanced promoter activity in MCF-7L cells when cotransfected with Sp1 cDNA, demonstrating further that low expression levels of Sp1 contribute to repression of RII expression in the MCF-7L cells. 2 In summary, the results of our present study demonstrated that the tumor suppressor gene RII is repressed in the ER ϩ breast cancer cells because of suboptimal activity of Sp1. 5-Aza-2Ј-dC treatment indirectly stabilizes and activates Sp1, thus leading to enhanced RII transcription and subsequent restoration of TGF-␤ response. These findings suggest a novel mechanism by which epithelial cells escape the negative growth regulatory effects of TGF-␤ leading to malignant behavior.