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Molecular Biology and Genetics |
Department of Surgery, University of Texas Health Science Center, San Antonio, Texas 78229-3900
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Lack of sensitivity to TGF-ß can provide tumor cells with a selective growth advantage and accelerate malignant progression. Several studies indicate that a variety of carcinoma cells lose their growth inhibitory response to TGF-ß because of a loss of TGF-ß receptors (13, 14, 15, 16) . The heteromeric complex of RI and RII is essential for TGF-ß signaling, and loss of these receptors can cause a loss of sensitivity to TGF-ß. Expression of RII cDNA restored the growth inhibitory effect of TGF-ß in a variety of human cancer cell lines that do not express RII (15 , 17, 18, 19) . These studies indicate that RII is a tumor suppressor gene (16 , 20) . Other studies demonstrated that mutations in simple repeats of the RII gene that are targeted by microsatellite instability plays a critical role in altering responsiveness to TGF-ß in cancer cells, especially in HNPCC (21, 22, 23) . Additional studies using the dominant negative mutant of RII also demonstrated the premise that the RII gene is a tumor suppressor (24 , 25) . Loss of growth-inhibitory response to TGF-ß was reported in pancreatic acinar cells from transgenic mice expressing a dominant negative mutant of RII (24) . Similarly expression of the dominant negative mutant of RII in transgenic mice caused an increase in the incidence of carcinoma (25) .
Another reason for the lack of TGF-ß signaling is mutations of the DPC4 (Smad4) gene. DPC4, a candidate tumor suppressor gene, is an important down-stream target of TGF-ß signaling (26, 27, 28)
. The DPC4 gene is reported to be homozygously deleted in 
30% of pancreatic cancers and inactivated in another 20% of these cancers (29)
. A recent study indicates that loss of TGF-ß signaling defects occur in >80% of pancreatic cancers because of genetic inactivation of one of the TGF-ß pathway components, including TGF-ß receptors and DPC4 (30)
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Although mutations in the RII gene were found to be responsible for loss of TGF-ß signaling in HNPCC, we found the occurrence of such mutations in the RII gene to be low (4%) in pancreatic adenocarcinoma (31) . Similar observations indicating a low frequency of mutations in RII were reported by others for pancreatic cancer (30 , 32) . We also recently demonstrated that down-regulation of RII mRNA expression was common in pancreatic tumor tissues and human pancreatic cancer cell lines (33) . Taken together, these findings suggest that the lack of RII expression in pancreatic cancer cells could be attributable to epigenetic mechanisms. One possible epigenetic mechanism is aberrant DNA methylation of CpG islands of the promoter regions. The promoter of the RII gene has been cloned and characterized (34 , 35) . The RII promoter lacks a TATA box or CAAT box. The promoter of the RII gene contains binding sites for Sp1 and AP1 transcription factors (34 , 35) , a novel ets-related transcription factor (36) , along with as-yet unidentified positive regulatory elements.
Previously, we showed that the pancreatic carcinoma cell line, MIA PaCa-2, is resistant to TGF-ß-induced growth inhibition because of a lack of expression of the RII gene (37) . Of the three TGF-ß receptors, MIA PaCa-2 cells express RI and RIII (33) . These cells do not have a homozygous deletion mutation of DPC4 (26) and express DPC4 mRNA (33 , 38) . Restoration of RII expression in MIA PaCa-2 cells by transfecting the cells with a RII cDNA reduced their tumorigenic phenotype and rendered these cells sensitive to radiation-induced growth inhibition (39) . Hence, the only component of TGF-ß signaling that is lacking in MIA PaCa-2 cells is RII expression and, thus, provides the basis for the insensitivity of MIA PaCa-2 cells to TGF-ß-induced growth inhibition.
In this study we analyzed the mechanism responsible for the repression of the RII gene in MIA PaCa-2 cells. We show that MIA PaCa-2 cells possess a low level of the transcription factor Sp1 because of a de novo transcriptional repression. The transcriptional repression of Sp1 was reversed by treating the cells with 5-aza-2'-dc, a methyl transferase inhibitor (40 , 41) . Treatment of cells with 5-aza-2'-dc caused an increase in the rate of Sp1 transcription and restoration of RII expression. Expression of Sp1 cDNA in MIA PaCa-2 cells also resulted in the induction of RII mRNA expression and restoration of TGF-ß sensitivity, demonstrating the direct role of Sp1 to transactivate the RII gene. When RII-expressing cells (BxPC-3 and MIA PaCa-2 cells transfected with Sp1 cDNA) were treated with mithramycin, an inhibitor of Sp1 binding (42 , 43) , it caused a reduction of RII expression and RII promoter-CAT activity, demonstrating the Sp1 requirement for RII transcription. The findings of this study indicate that a deficit of Sp1 protein may be responsible for the transcriptional repression of the RII gene in pancreatic cancer cell line MIA PaCa-2.
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MATERIALS AND METHODS |
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mRNA Expression of RII.
RNA was extracted using TRI ZOL reagent (Life Technologies, Inc., Gaithersburg, MD). One µg of total RNA was reverse transcribed into cDNA using a commercially available kit (Perkin-Elmer Corp., Norwalk, CT) according to the manufacturer’s instructions. Specific primers were used to determine the mRNA expression of the RII and ß-actin genes, as described previously (33)
. Expression of ß-actin gene levels were used as an internal control to normalize the expression levels. To determine the effect of 5-aza-2'-dc or mithramycin, cells were treated as detailed above in the cell culture section. RNA was extracted from untreated or 5-aza-2'-dc-treated MIA PaCa-2 cells and reverse transcribed into cDNA. Similarly RII mRNA expression was analyzed from MIA PaCa-2 (Neo) or MIA PaCa-2 (Sp1) cells. Wherever appropriate, RNA extracted from BxPC-3 cells was used as a control for RII mRNA expression. The PCR products were run in 2% agarose gels, stained with ethidium bromide, and photographed.
DNA Sequencing of RII cDNA.
RT-PCR was performed as described above to amplify the complete cDNA of RII as two overlapping fragments using two sets of primers. The primers were designed for the RII gene based on the published sequence (GenBank accession no. M85079). The first set of primers are: sense primer RII.297U, 5'-CGC TGG GGG CTC GGT CTA TG-3'; and antisense primer-RII.1139D, 5'-ACT GCC ACT GTC TCA AAC TGC TCT-3'. The second set of primers are: sense primer-RII.1029U, 5'-GCC AAC AAC ATC AAC CAC AAC ACA-3'; and antisense primer-RII.2061D, 5'-GGG GCA GCC TCT TTG GAC AT-3'. The PCR resulted in 866-bp and 1052-bp fragments, respectively. The yield of PCR product in MIA PaCa-2 cells was always undetectable in agarose gels stained with ethidium bromide. Hence, these PCR products were purified by using QiaQuick columns (Qiagen, Valencia, CA), and subjected to a second round PCR using the same set of primers. This procedure yielded sufficient quantities of PCR products to perform DNA sequencing. The purified PCR products were sequenced in both directions by using an automated Applied Biosystems 373A DNA sequencer and dye terminator cycle sequencing with DNA Taq polymerase fluorescent sequencing.
Nuclear Run-off Transcription Assays.
Nuclear run-off transcription assays were performed as described previously (44)
, with some modifications to determine the rate of transcription of RII and Sp1 genes in MIA PaCa-2 cells. BxPC-3 cells were used as a control to determine the rate of transcription of RII. Briefly, blots were prepared by using plasmid DNA immobilized on positively charged nylon membranes (Boehringer Mannheim, Mannheim, Germany) using a Schleicher and Schuell slot blot system (Schleicher & Schuell, Keene, NH). The amount of RII or Sp1 cDNA loaded were 2.0 and 4.0 µg/slot. An equal amount of empty vector was also loaded to check the background hybridization. As a loading control, GAPDH cDNA was used. The amount of GAPDH cDNA loaded was 0.5 and 1.0 µg/slot. Slots were loaded with the appropriate quantities of alkali-denatured plasmids, as indicated, and washed with 2 M ammonium acetate (pH 7.0). After drying the blots at room temperature, the plasmid DNA was UV-cross-linked in a Stratalinker UV-cross-linker (Stratagene, La Jolla, CA). Nuclei were prepared from exponentially growing cells. After trypsinization, cells were suspended in NP40 lysis buffer [10 mM TrisCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% NP40] and homogenized in a dounce homogenizer. Nuclei were pelleted after a brief spin at 2500 rpm for 1 min at 4°C. The isolated nuclei were stored in storage buffer [0.02 M Tris (pH 8.0), 20% glycerol, 0.14 M KCl, 0.01 M MgCl2, and 14 mM ß-ME] at -80°C until used for transcription reactions. An equal number of nuclei were suspended in 100 ml of transcription reaction buffer [0.02 M Tris (pH 8.0); 10% glycerol; 0.14 M KCl; 0.01 M MgCl2; 14 mM ß-ME; 1 mM each of ATP, CTP, GTP; and 100 µCi of (
-32P)-UTP [(3000 Ci/mmol); Amersham Pharmacia Biotech Inc., Piscataway, NJ], and the reaction was incubated at 30°C for 30 min. The 32P-labeled nascent RNA was extracted using TRI ZOL reagent (Life Technologies, Inc.) and precipitated in the presence of glycogen as a carrier. Equal amounts of radiolabeled RNA were hybridized to blots in ULTRAhyb hybridization buffer (Ambion, Inc., Austin, TX). Hybridization was performed at 45°C for 48 h. Blots were sequentially washed twice in 2x SSC for 10 min and in 0.2x SSC- 0.1% SDS at 65°C for 1 h. Routine autoradiographic techniques were performed to develop the blots.
Protein-DNA Binding Studies.
EMSA, competitive analyses, and antibody supershifts were carried out using specific wild-type and mutant oligonucleotides representing the two Sp1-binding regions, as described previously (45)
. These two oligonucleotides were referred to as Sp1 (-37/-8) and Sp1 (-155/-125), based on the nucleotide positions relative to transcriptional initiation site (35)
. To determine the effect of mithramycin on Sp1 binding to the RII promoter, oligonucleotide probes were preincubated for 1 h at 4°C in the presence of mithramycin (0.1 µM) before adding to the nuclear extracts, as described previously (43)
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Western Immunoblots.
Western immunoblots were prepared to analyze the protein levels of Sp1, MAPK, IGF-1R, RII, and human ß-actin. MIA PaCa-2 and BxPC-3 cells were harvested at indicated time points after treatment with 5-aza-2'-dc, and total cellular proteins were extracted by using Laemmli buffer. Fifty µg of total cellular proteins from cells were electrophoresed on a 7.5% SDS-polyacrylamide gel and then transferred to Hybond-P, PVDF membrane (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The blots were probed with specific antibodies for the above-mentioned proteins (Sp1, RII, and IGF-1Rß; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; MAPK; New England Biolabs, Beverly, MA; and human ß-actin; Sigma Chemical Co., St. Louis, MO). Bound antibodies were detected by an enhanced chemiluminescence detection system (NEN Life Science Products, Boston, MA).
Determination of Sp1 Protein Stability.
To determine the stability of Sp1 protein, MIA PaCa-2 cells, either untreated or treated with 5-aza-2'-dc, were treated with cycloheximide (10 µg/ml) for 0, 3, 6, 9, and 12 h. Cells were harvested at indicated time points, and protein extractions were analyzed for Sp1 as described in the Western immunoblot section.
CAT Assays.
RII promoter-CAT constructs were as described previously (35)
. Two different RII promoter-CAT constructs (-274/+50 and -47/+50; Fig. 1B
) were used to assess basal RII promoter-CAT activity in MIA PaCa-2 cells. The RII promoter-CAT construct (-47/+50) contains one Sp1 binding site and the other construct, -274/+50, contains the core promoter region with two Sp1 binding sites and other positive/negative regulatory elements. As a control, MIA PaCa-2 cells were transfected with a positive control vector (pCAT-control; Promega, Madison, WI) to monitor the general transcriptional activity. Transfections were performed by using commercially available FuGENE6 transfection reagent (Boehringer Mannheim). To assess the overexpression of Sp1 on RII promoter activity, cells were cotransfected with Sp1 cDNA and RII promoter-CAT construct (-274/+50). Cells were harvested after 48 h of transfection and normalized by the ß-gal activity, and standard CAT-assays were performed using TLC plates. However, for cells stably transfected with Sp1 cDNA, normalization was based on the total protein level as measured by using the Bradford-Lowrey reaction (Bio-Rad, Hercules, CA). This procedure was adopted because, in pilot experiments, cotransfection with pSV-ß-galactosidase control vector (Promega) showed higher ß-galactosidase activity in cells overexpressing Sp1, probably because of the presence of several Sp1 binding sites in the promoter of pSV-ß-galactosidase vector. Hence, for the cells transfected with Sp1, total protein levels were measured for normalization.
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Luciferase Assay to Determine TGF-ß Responsiveness.
Responsiveness of MIA PaCa-2 cells to exogenously added TGF-ß was determined as described previously (6)
. Briefly, cells were transiently transfected with a TGF-ß responsive reporter construct, p3TP-Lux. Cells were grown in 6-cm culture dishes for 24 h after transfection and treated with 10 ng/ml of human recombinant TGF-ß1 (R&D Systems, Minneapolis, MN) for 24 h. Cells were harvested in 300 µl of reporter lysis buffer (Promega) after 48 h of transfection. Luciferase activity was measured with 20 µl of lysate using a luciferase assay kit (Promega), as per the manufacturer’s suggestion, in a luminometer (TROPIX, Inc., Bedford, MA) and expressed as relative units after being normalized to protein levels.
MTT and Soft Agar Assays.
The growth rate of MIA PaCa-2 (Neo) and two different clones of MIA PaCa-2 (Sp1) cells was determined by MTT assays. Cells (1 x 103) were plated in 96-well plates and MTT assays were performed at days 1, 2, 4, and 5. On the day of assay, the growth media was replaced with serum-free medium containing 0.5 mg/ml MTT (Sigma Chemical Co.) and incubated at 37°C for 2 h. At the end of the incubation period, cells were solubilized in 200 µl of DMSO, and colorimetric determination was performed at 570 nm absorbance with a plate reader. The data were represented as the mean values from four independent experiments. The tumorigenic potential of cells was determined in soft agar assays as described previously (33)
. Briefly, MIA PaCa-2 (Neo) or MIA PaCa-2 (Sp1) cells were suspended in 0.4% NOBLE agar in DMEM medium containing 10% fetal bovine serum and plated on top of 1 ml of 1% agar in the same medium in six-well tissue culture plates. Wherever necessary, 10 ng/ml of TGF-ß1 (R&D Systems) was added to the medium. The cells were then incubated for 14 days at 37°C in a CO2 humidified incubator. Colonies were visualized by staining with 0.5 mg/ml MTT and photographed.
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RESULTS |
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Transcriptional Rate of RII Is Low in MIA PaCa-2 Cells.
Because there were no mutations found in the coding region of the RII gene, we determined whether a dysfunction of transcriptional activation might account for the lower level of RII expression in MIA PaCa-2 cells. Nuclear run-off assays were used to assess whether there was any impairment of the endogenous RII promoter activity. Nuclear run-off assays showed that the rate of transcription of RII gene is low in MIA PaCa-2 cells compared with the control cell line BxPC-3 (Fig. 1A)
. Bx PC-3 cells that express higher levels of RII mRNA showed a higher rate of RII transcription (Fig. 1A)
.
One possibility for the lower rate of RII transcription in MIA PaCa-2 cells is that there is a defect in the endogenous RII promoter. This defect could be attributable to a mutation within the promoter region or to the silencing of gene expression by aberrant methylation or be caused by a lack of appropriate transcriptional activation. Previous studies showed the presence of two Sp1 binding sites in the RII promoter and absence of TATA or CAAT boxes (Fig. 1B
; 34, 35). To ascertain the possibility of any defects of the RII promoter, we initially performed CAT assays using two wild-type RII promoter-CAT constructs. The first construct (-274/+50) contains two Sp1 binding sites along with positive regulatory elements, and the second construct (-47/+50) has one Sp1 binding site. When MIA PaCa-2 cells were transfected with either of these two different wild-type RII promoter-CAT constructs, a negligible level of RII promoter-CAT activity was observed in these cells irrespective of the constructs used (Fig. 1C)
, suggesting a lack of appropriate transcriptional activation. To rule out the possibility of any artifacts in transfections, MIA PaCa-2 cells were transfected with a positive-control CAT vector that contains SV40 promoter to monitor the general transcriptional activity and transfection. The positive control showed a significant CAT activity indicating that MIA PaCa-2 cells failed to show RII promoter-CAT activity, irrespective of the constructs used.
Sp1 Protein Levels Are Low in MIA PaCa-2 Cells and Can Be Restored by Treatment with 5-aza-2'-dc.
Because the CAT assays with wild-type RII promoter-CAT constructs showed very low levels of CAT activity, we further assessed the possibility of the absence of the transcriptional activator Sp1 in MIA PaCa-2 cells. Western immunoblot analysis of MIA PaCa-2 cells showed very low levels of Sp1 protein expression (Fig. 2A)
. The control pancreatic cancer cell line BxPC-3 that expresses higher levels of RII mRNA (33)
also showed higher levels of Sp1 protein (Fig. 2A)
. Thus, a deficiency in Sp1 expression might account for the lack of RII expression in MIA PaCa-2 cells.
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. We next determined whether treatment of MIA PaCa-2 cells with 5-aza-2'-dc caused a general increase of the protein levels. Western immunoblot analysis showed that the levels of MAPK, IGF-1R, and another general transcription factor, AP-1 (c-jun), were not dramatically altered by this treatment (Fig. 2B)
. This suggests that 5-aza-2'-dc did not result in a global increase in the levels of various proteins.
5-aza-2'-dc Treatment Caused Increased Binding of Sp1 Protein to the RII Promoter in MIA PaCa-2 Cells.
EMSAs were performed to analyze the nuclear protein complexes that bind to the Sp1 consensus elements in the promoter of the RII gene. The two oligonucleotides (Sp1 -37/-8 and Sp1 -155/-125) used in this study were described previously (45)
. Nuclear extracts from untreated and 5-aza-2'-dc-treated MIA PaCa-2 cells were subjected to gel-shift analysis. Nuclear proteins from 5-aza-2'-dc-treated MIA PaCa-2 cells contain greater amounts of DNA-protein complexes than the nuclear proteins from untreated MIA PaCa-2 cells (Fig. 2C)
. The increase in the binding of DNA- protein complexes was observed for both the oligonucleotides representing the two Sp1 consensus elements of RII promoter. The protein-DNA complexes represent the specific binding of proteins to Sp1 consensus elements, because addition of excess unlabeled wild-type oligonucleotides showed a decrease in the binding complexes that was not seen with a mutant oligonucleotide (Fig. 2C)
. To assess whether these DNA-protein complexes contain Sp1 protein, supershift assays were carried out by incubating the oligonucleotide DNA-protein complexes with an antibody to Sp1. The addition of Sp1 antibody caused a shift for human recombinant Sp1 protein (Fig. 2D)
. However, for nuclear protein complexes from MIA PaCa-2 cells, the shifted complex was low (Fig. 2D
, arrow), but a large depletion of bound protein complexes to the oligonucleotides was observed (Fig. 2D
, arrowheads). This depletion of bound complexes with Sp1 antibody suggests that the bound complexes contain Sp1 protein.
Because 5-aza-2'-dc treatment caused an increase in Sp1 binding as determined by EMSA, we next tested whether these cells also show an increase in RII mRNA expression. RT-PCR analysis revealed that 5-aza-2'-dc treatment caused an increase in RII mRNA expression in MIA PaCa-2 cells (Fig. 2E)
.
5-aza-2'-dc Treatment Increases the Rate of Transcription of Sp1 Gene, but not the Stability of Sp1 Protein in MIA PaCa-2 Cells.
To determine the mechanism by which 5-aza-2'-dc increases the expression of Sp1, MIA PaCa-2 cells were treated with cycloheximide (10 µg/ml), and proteins were extracted at 0, 3, 6, 9 and 12 h after treatment. Western immunoblots were performed to detect Sp1 protein levels. Treatment with 5-aza-2'-dc did not lead to any change in the half-life of Sp1 protein when compared with untreated cells (Fig. 3A)
. However, treatment of MIA PaCa-2 cells with 5-aza-2'-dc had led to an increase in the rate of transcription of the Sp1 gene as determined by nuclear run-off assays (Fig. 3B)
.
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. MIA PaCa-2 cells cotransfected with the Sp1 cDNA and RII promoter-CAT construct showed an increase in RII promoter-CAT activity, indicating the transcriptional up-regulation of RII in cells that express the Sp1 cDNA (Fig. 4B)
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. Among the numerous MIA PaCa-2 (Sp1) clones established, two individual clones (clone 7 and 8) were used for additional analysis to rule out the possibility of any clonal variation. These two clones showed an increase in Sp1 protein levels and a concurrent increase of RII protein levels when compared with MIA PaCa-2 (Neo) cells (Fig. 4D)
. These two clones also showed an increase in RII promoter-CAT activity similar to that observed in MIA PaCa-2 cells transiently transfected with Sp1 cDNA (data not shown) and were assessed for growth, clonogenicity, and responsiveness to TGF-ß as determined by the p3TP-Lux-reporter assay.
Ectopic Expression of Sp1 cDNA in MIA PaCa-2 Cells Restores Responsiveness to TGF-ß and Causes a Decrease in Growth and Clonogenicity.
The responsiveness to TGF-ß was analyzed by the p3TP-Lux-reporter assay. Expression of Sp1 increased 3TP-lux activity in MIA PaCa-2 (Sp1) cells (Fig. 5A)
. The level of responsiveness was increased further by the addition of exogenous TGF-ß (Fig. 5A)
. However, parental MIA PaCa-2 cells or MIA PaCa-2 (Neo) cells did not show considerable p3TP-Lux activity even after the addition of exogenous TGF-ß (Fig. 5A)
. The increased basal levels of p3TP-Lux-reporter activity in MIA PaCa-2 (Sp1) cells suggests restoration of autocrine TGF-ß activity after an increase in the expression of the RII receptor. Similar restoration of autocrine TGF-ß signaling attributable to an increase of RII was also observed in other cell systems (47
, 48)
.
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. The clonogenic potential of MIA PaCa-2 (Sp1) cells was assessed by soft agar assays. MIA PaCa-2 (Sp1) cells showed a dramatic reduction in clonogenicity as compared with MIA PaCa-2 (Neo) cells (Fig. 5C)
. Exogenous TGF-ß did not add to a further-pronounced reduction of growth in soft agar. This suggests that restoration of autocrine TGF-ß signaling by Sp1 expression was sufficient for the reduction of growth in soft agar. Because the observations on MTT and soft agar assays were similar for both MIA PaCa-2 (Sp1) clones 7 and 8, the data are shown only for MIA PaCa-2 (Sp1) clone 8 in Fig. 5
.
Mithramycin Decreases Binding of Sp1 to the RII Promoter and Inhibits RII Promoter-CAT Activity and RII mRNA Expression.
To ascertain the dependency of RII promoter on Sp1, additional experiments were performed by treating cells with mithramycin, an inhibitor of Sp1 binding (42
, 43)
. Initially, EMSA assays were performed to determine the effect of mithramycin treatment on the binding of Sp1 protein to the RII promoter that contains the Sp1 consensus element. Nuclear extracts from MIA PaCa-2 (Neo) cells and MIA PaCa-2 (Sp1) cells were used for the study. The nuclear extracts from MIA PaCa-2 (Sp1) cells showed more binding of protein-DNA complexes than those from the nuclear extracts from MIA PaCa-2 (Neo) cells. The increase in the bound complexes were found for both the oligonucleotides (Sp1 -37/-8 and Sp1-155/-125). When oligonucleotides were preincubated with mithramycin, a considerable reduction of the bound protein-DNA complexes were observed (Fig. 6A)
. This suggests that mithramycin reduced the binding of Sp1 to the oligonucleotide probes that contain the Sp1 consensus elements of the RII promoter. MIA PaCa-2 (Sp1) cells also showed an inhibition in the RII promoter-CAT activity upon treatment with mithramycin (Fig. 6B)
. When Bx PC-3 or MIA PaCa-2 (Sp1) cells that show RII expression were treated with mithramycin, a reduction in the RII mRNA expression was observed (Fig. 6C)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transcriptional repression of a gene may be attributable to several mechanisms, including mutations within the promoter region, absence of transcriptional regulation, or aberrant DNA methylation of CpG islands. Recent studies indicate that the pathways of inactivating genes that led to the development of carcinogenesis could be attributable to the silencing of regulatory genes, especially tumor suppressor genes (49, 50, 51, 52) . The RII promoter lacks TATA and CAAT boxes, but does contain binding sites for Sp1, AP1, and (34 , 35) , ets-related transcription factor (36) , as well as other unidentified positive regulatory elements (35) .
The importance of Sp1 for basal transcriptional activity was suggested for promoters that lack TATA or CAAT boxes (53 , 54) . We found by Western blot analysis that MIA PaCa-2 cells express a lower level of Sp1 protein than BxPC-3 cells that express RII. Treatment of MIA PaCa-2 cells with 5-aza-2'-dc caused an increase in the expression of Sp1 protein. However, treatment with 5-aza-2'-dc did not result in a generalized increase of other protein levels such as MAPK, a constitutively expressed protein, IGF-1R, a growth factor signaling protein, and another general transcription factor, AP-1 (c-Jun). A recent study (45) indicated that RII expression was increased in MCF-7 L cells treated with 5-aza-2'-dc because of an increase in Sp1 protein stability (45) . In contrast to the previous study on MCF-7 L cells, treatment of MIA PaCa-2 cells with 5-aza-2'-dc did not result in a prolonged stability of Sp1 protein, but resulted in an increase in the rate of Sp1 transcription. This indicates that a deficit of Sp1 protein occurs because of separate mechanisms in different cancer cell types. However, at this point it is not clear how 5-aza-2'-dc treatment causes a transcriptional up-regulation of the Sp1 message in MIA PaCa-2 cells. It is possible that the low levels of Sp1 may be attributable to the methylation of the Sp1 promoter or of the promoter of a transactivator that regulates the expression of Sp1. In this study, however, we have not determined these possibilities, because a cloned Sp1 promoter is not available.
Furthermore, we demonstrated that RII expression is specifically mediated by Sp1 by transfecting MIA PaCa-2 cells with a vector expressing the Sp1 cDNA. Transient transfection of MIA PaCa-2 cells with the Sp1 cDNA led to an increase in Sp1 protein levels and RII promoter-CAT activity. MIA PaCa-2 (Sp1) cells stably transfected with the Sp1 cDNA showed an increase in RII mRNA and protein expression. The biological significance of the expression of Sp1 in MIA PaCa-2 (Sp1) cells was also assessed by growth characteristics. MTT assays showed a reduction in the rate of growth of MIA PaCa-2 (Sp1) cells as compared with MIA PaCa-2 (Neo) cells. The clonogenicity of MIA PaCa-2 (Sp1) cells was analyzed by soft agar assays. One of the characteristic features of MIA PaCa-2 cells is the colony formation in soft agar assays (33 , 37) . Also, these cells demonstrate an anchorage-independent growth in the presence of TGF-ß. Expression of Sp1 cDNA caused a dramatic reduction of clonogenicity in MIA PaCa-2 (Sp1) cells even in the absence of exogenously supplied TGF-ß, indicating a reduction of their tumorigenic phenotype. The increase in responsiveness to TGF-ß was also observed in p3TP-lux assays. The reduced rate of both growth and clonogenicity that is associated with an increase in the basal levels of 3TP-lux reporter activity in MIA PaCa-2 (Sp1) cells are probably the result of restoration of autocrine TGF-ß activity after the restoration of the RII receptor (47 , 48) .
Conversely, we reasoned that inhibition of Sp1 causes a decrease in RII expression. Mithramycin has been shown to prevent the binding of Sp1 to its consensus sequences in promoters and to inhibit Sp1-dependent gene expression in several cell types (42 , 43 , 55 , 56) . EMSAs showed that treatment of mithramycin caused a reduction of Sp1 binding to its consensus sequence of the RII promoter. When MIA PaCa-2 (Sp1) cells that show RII mRNA expression were treated with mithramycin, a reduction in RII promoter-CAT activity and RII mRNA expression was observed. These studies further support the notion that transcriptional activation by Sp1 is critical for RII gene expression. We suggest that the RII gene may represent a unique subpopulation of genes that require threshold levels of Sp1 for transcriptional activation. It is probable that other transcription factors or cofactors, or activators that interact with Sp1, may efficiently drive the transcription of many genes that depend on Sp1 in cells such as MIA PaCa-2, which have low levels of Sp1. The essential role of Sp1 for RII transcription in other pancreatic cancer cell lines is being investigated currently in this laboratory.
In summary, this study indicates that RII expression in MIA PaCa-2 cells is attributable to suboptimal levels of the transcriptional factor Sp1 that is essential for RII transcription. The increase of expression of Sp1 by treatment of MIA PaCa-2 cells with a methyl transferase inhibitor (5-aza-2'-dc) or by ectopically overexpressing Sp1 increased the transcription of the RII gene. Conversely, RII expression was reduced in pancreatic cancer cells that express RII after treatment with mithramycin, a drug that blocks the binding of Sp1 to its consensus binding sequence. Thus, this study suggests that a threshold level of Sp1 is required for the expression of the RII gene. Clearly, in the case of MIA PaCa-2, 5-aza-2'-dc treatment increased Sp1 expression by up-regulating the transcription rate of the Sp1 gene. Reversion of transcriptional repression of Sp1 causes an increase in RII expression and responsiveness to TGF-ß in MIA PaCa-2 cells. Modulation of Sp1 expression may prove a useful target in restoration of RII expression and TGF-ß signaling in cells that otherwise possess an intact TGF-ß signaling pathway.
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FOOTNOTES |
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1 Supported by National Institutes of Health Grant CA 69122 and the Lustgarten Foundation for Pancreatic Cancer Research (to J. W. F.). 
2 To whom requests for reprints should be addressed, at Department of Surgery, University of Texas Health Sciences Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. Phone: (210) 567-5298; Fax: (210) 567-4664; E-mail: freemanjw{at}uthscsa.edu
3 The abbreviations used are: TGF-ß, transforming growth factor ß; RI, RII, and RIII, TGF-ß receptor types I, II, and III, respectively; RT-PCR, reverse transcription-PCR; HNPCC, hereditary non-polyposis colon cancer; DPC4, deleted in pancreatic cancer; 5-aza-2'-dc, 5-aza-2'deoxycytidine; CAT, chloramphenicol acetyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LOH, loss of heterozygosity; EMSA, electrophoretic mobility shift assay; PVDF, polyvinylidene difluoride; MAPK, mitogen activated protein kinase; IGF-1R, insulin-like growth factor-1 receptor; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-thiazolyl blue; ets, epithelial specific; p3TP-Lux, TGF-ß-responsive plasminogen activator inhibitor promoter-luciferase reporter construct.
Received 11/28/00. Accepted 6/ 8/01.
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2(I) collagen to transforming growth factor-ß1. J. Biol. Chem., 272: 19738-19745, 1997. regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells. J. Biol. Chem., 273: 22128-22135, 1998.This article has been cited by other articles:
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  F. Crea, E. Giovannetti, F. Cortesi, V. Mey, S. Nannizzi, M. I. Gallegos Ruiz, S. Ricciardi, M. Del Tacca, G. J. Peters, and R. Danesi Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines Mol. Cancer Ther., July 1, 2009; 8(7): 1964 - 1973. [Abstract] [Full Text] [PDF]
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 M. J. Truty, G. Lomberk, M. E. Fernandez-Zapico, and R. Urrutia Silencing of the Transforming Growth Factor-{beta} (TGF{beta}) Receptor II by Kruppel-like Factor 14 Underscores the Importance of a Negative Feedback Mechanism in TGF{beta} Signaling J. Biol. Chem., March 6, 2009; 284(10): 6291 - 6300. [Abstract] [Full Text] [PDF]
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A. Thangasamy, J. Rogge, and S. Ammanamanchi Regulation of RON Tyrosine Kinase-mediated Invasion of Breast Cancer Cells J. Biol. Chem., February 29, 2008; 283(9): 5335 - 5343. [Abstract] [Full Text] [PDF]
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 W. Li, G. M. Venkataraman, and K. B. Ain Protein Synthesis Inhibitors, in Synergy with 5-Azacytidine, Restore Sodium/Iodide Symporter Gene Expression in Human Thyroid Adenoma Cell Line, KAK-1, Suggesting Trans-Active Transcriptional Repressor J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 1080 - 1087. [Abstract] [Full Text] [PDF]
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 M. Anbar, R. Bracha, Y. Nuchamowitz, Y. Li, A. Florentin, and D. Mirelman Involvement of a Short Interspersed Element in Epigenetic Transcriptional Silencing of the Amoebapore Gene in Entamoeba histolytica Eukaryot. Cell, November 1, 2005; 4(11): 1775 - 1784. [Abstract] [Full Text] [PDF]
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 K. Venkatasubbarao, A. Choudary, and J. W. Freeman Farnesyl Transferase Inhibitor (R115777)-Induced Inhibition of STAT3(Tyr705) Phosphorylation in Human Pancreatic Cancer Cell Lines Require Extracellular Signal-Regulated Kinases Cancer Res., April 1, 2005; 65(7): 2861 - 2871. [Abstract] [Full Text] [PDF]
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 R. L. Elliott and G. C. Blobe Role of Transforming Growth Factor Beta in Human Cancer J. Clin. Oncol., March 20, 2005; 23(9): 2078 - 2093. [Abstract] [Full Text] [PDF]
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 S.S. Prime, M. Davies, M. Pring, and I.C. Paterson THE ROLE OF TGF-{beta} IN EPITHELIAL MALIGNANCY AND ITS RELEVANCE TO THE PATHOGENESIS OF ORAL CANCER (PART II) Critical Reviews in Oral Biology & Medicine, November 1, 2004; 15(6): 337 - 347. [Abstract] [Full Text] [PDF]
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S. Ammanamanchi, J. W. Freeman, and M. G. Brattain Acetylated Sp3 Is a Transcriptional Activator J. Biol. Chem., September 12, 2003; 278(37): 35775 - 35780. [Abstract] [Full Text] [PDF]
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Y. Bontemps, B. Vuillermoz, F. Antonicelli, C. Perreau, J.-L. Danan, F.-X. Maquart, and Y. Wegrowski Specific Protein-1 Is a Universal Regulator of UDP-glucose Dehydrogenase Expression: ITS POSITIVE INVOLVEMENT IN TRANSFORMING GROWTH FACTOR-{beta} SIGNALING AND INHIBITION IN HYPOXIA J. Biol. Chem., June 6, 2003; 278(24): 21566 - 21575. [Abstract] [Full Text] [PDF]
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S. Zhao, K. Venkatasubbarao, S. Li, and J. W. Freeman Requirement of a Specific Sp1 Site for Histone Deacetylase-mediated Repression of Transforming Growth Factor {beta} Type II Receptor Expression in Human Pancreatic Cancer Cells Cancer Res., May 15, 2003; 63(10): 2624 - 2630. [Abstract] [Full Text] [PDF]
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R. Bracha, Y. Nuchamowitz, and D. Mirelman Transcriptional Silencing of an Amoebapore Gene in Entamoeba histolytica: Molecular Analysis and Effect on Pathogenicity Eukaryot. Cell, April 1, 2003; 2(2): 295 - 305. [Abstract] [Full Text] [PDF]
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K. B. Glaser, J. Li, M. E. Aakre, D. W. Morgan, G. Sheppard, K. D. Stewart, J. Pollock, P. Lee, C. Z. O'Connor, S. N. Anderson, et al. Transforming Growth Factor {beta} Mimetics: Discovery of 7-[4-(4-Cyanophenyl)phenoxy]-Heptanohydroxamic Acid, a Biaryl Hydroxamate Inhibitor of Histone Deacetylase Mol. Cancer Ther., August 1, 2002; 1(10): 759 - 768. [Abstract] [Full Text] [PDF]
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