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Endocrinology, doi:10.1210/en.2004-0686
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Endocrinology Vol. 145, No. 12 5774-5785
Copyright © 2004 by The Endocrine Society

Peroxisome Proliferator-Activated Receptor {gamma}-Dependent Activation of p21 in Panc-28 Pancreatic Cancer Cells Involves Sp1 and Sp4 Proteins

Jun Hong, Ismael Samudio, Shengxi Liu, Maen Abdelrahim and Stephen Safe

Institute of Biosciences and Technology, Texas A&M University System Health Science Center (J.H., I.S., S.L., S.S.), Houston, Texas 77030; and Department of Veterinary Physiology and Pharmacology, Texas A&M University (I.S., M.A., S.S.), College Station, Texas 77843

Address all correspondence and requests for reprints to: Dr. Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, Veterinary Research Building 409, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (DIM-C-pPhCF3) and troglitazone activate peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) in Panc-28 pancreatic cancer cells and also inhibit cell proliferation. DIM-C-pPhCF3 was more active than troglitazone and was used as a model to investigate the mechanism of PPAR{gamma}-dependent inhibition of Panc-28 cell growth. DIM-C-pPhCF3 significantly inhibited G0/G1->S phase progression, as determined by FACS analysis, and this was associated with decreased retinoblastoma protein phosphorylation and increased p21 protein and mRNA expression, but no change in p27 or cyclin D1. PPAR{gamma} antagonists blocked DIM-C-pPhCF3-induced growth inhibition and induction of p21 protein, and similar inhibitory effects were observed in Panc-28 cells transfected with a construct (pWWP) containing a –2325 to +8 p21 promoter insert. Deletion analysis of the p21 promoter indicated that PPAR{gamma}-dependent activation of p21 promoter constructs by DIM-C-pPhCF3 required GC-rich sites 3 and 4 in the proximal region (–124 to –60) of the p21 promoter. The results of RNA interference and protein expression/DNA binding assays suggest that DIM-C-pPhCF3 induced p21 expression through a novel mechanism that involves PPAR{gamma} interactions with both Sp1 and Sp4 proteins bound to the proximal GC-rich region of the p21 promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PANCREATIC DUCTAL adenocarcinoma (PDAC) is a major cause of cancer-related deaths in developed countries, and it is estimated that in 2003, more than 30,000 new cases will be diagnosed in the United States (1). PDAC is a highly aggressive disease that invariably evades early diagnosis (2, 3, 4, 5). The mean survival time for patients with metastatic disease is only 3–6 months, and the 1-yr survival time for all pancreatic cancers cases is approximately 20–30% (1). Several factors are associated with increased risk for pancreatic cancer, and these include chronic pancreatitis, prior gastric surgery, smoking, diabetes, exposure to certain classes of organic solvents, and radiation (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21). Heritable germline mutations in several genes, including Peutz-Jeghers, hereditary pancreatitis, familial atypical multiple melanoma, familial breast cancer 2, and hereditary nonpolyposis colorectal cancer syndromes, also increase the risk for pancreatic cancer (4, 5, 11, 22, 23, 24). Several acquired gene mutations have been identified in sporadic pancreatic tumors; these include K-ras, p16, p53, and smad4, which are mutated in more than 50% in pancreatic tumors (22).

Pancreatic cancers are frequently detected at an advanced stage, and treatment regimens for this disease have provided very limited improvements in tumor regression and overall survival times after diagnosis (1, 2, 3, 4, 25, 26). 5-Fluorouracil alone and in combination with other drugs has been used to treat pancreatic cancer, and gemcitabine, a deoxycytidine analog, has partially replaced 5-fluorouracil due to increased clinical benefits in terms of response rate, time to progression, and median survival. Several other classes of drugs, including antimetabolites, taxanes, topoisomerase I inhibitors, K-ras inhibitors, various combinations of these drugs, and other novel mechanism-based agents are currently being investigated for the treatment of pancreatic cancer (25, 26, 27, 28).

Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) is a member of the nuclear receptor family of ligand-induced transcription factors, and PPAR{gamma} agonists such as 15-deoxy-{Delta}12,14-prostaglandin J2 and synthetic thiazolidinediones (TZDs) induce fat cell differentiation and are used for treatment of type II diabetes (29, 30, 31, 32, 33). PPAR{gamma} agonists also exhibit antitumorigenic activity, and their inhibition of cancer cell growth is associated with a G0/G1->S phase block, decreased cyclin D1, and/or increased p21 or p27 expression and induction of apoptosis. The growth inhibitory effects induced by PPAR{gamma} agonists are variable and dependent on both ligand structure and cell context. For example, troglitazone induced p21 expression and several differentiation markers in BxPC-3 pancreatic cancer cells (34), whereas p27, but not p21, expression was induced by troglitazone in Panc-1 cells (35), and similar results were observed in PK-1 pancreatic cancer cells (36). Recent studies in this laboratory (37) have identified a series of 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes [C-substituted diindolylmethanes (DIMs)] that induce PPAR{gamma}-dependent transactivation and inhibit the growth of multiple cancer cell lines. The most active C-substituted DIMs contained p-trifluoromethylphenyl (DIM-C-pPhCF3) (1), p-t-butyl (DIM-C-pPhtBu) (4), and p-phenyl (DIM-C-pPhC6H5) (9) substituents, and in MCF-7 cells, these compounds induce proteasome-dependent down-regulation of cyclin D1 and apoptosis. In the present study we investigated the mechanisms of DIM-C-pPhCF3-induced inhibition of Panc-1 cell growth, and the results show that DIM-C-pPhCF3 induced p21 expression through a unique PPAR{gamma}/Sp1-Sp4 complex targeted to the proximal GC-rich sites in the p21 promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
No experimental animals were used in this study.

Cell lines, plasmids, and reagents
The Panc-28 cell line was a gift from Dr. Paul Chiao (University of Texas M. D. Anderson Cancer Center, Houston, TX). DMEM/Ham’s F-12 with and without phenol red, 100x antibiotic/antimycotic solution, and propidium iodide were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Troglitazone was purchased from IKT Laboratories (St. Paul, MN). Fetal bovine serum was purchased from Intergen (Purchase, NY). Antibodies for p21, phospho-retinoblastoma (phospho-Rb), cyclin D1, PPAR{gamma}, Sp1, Sp3, Sp4, and ß-tubulin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The PPRE-luc construct contains three tandem PPAR{gamma} response elements (PPREs) with a minimal TATA sequence in pGL2. p21 promoter reporter constructs pWWP, pWWP124, and pWWP101 were provided by Dr. Toshiyuki Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan). pWWP60 was generated by digesting pWWP with SmaI, followed by religation of the purified vector. Mutant p21 reporter constructs mut5/6, mut4, mut2, and mut1 were provided by Dr. Dimitris Kardassis (University of Crete Medical School, Heraklion, Greece). The luciferase reporter Sp13-luc construct contains three consensus Sp1-binding sites as described previously (38). Lysis buffer, luciferase reagent, and ribonuclease were obtained from Promega Corp. (Madison, WI). All other chemicals and biochemicals were of the highest quality available from commercial sources. DIM-C-pPhCF3 was synthesized in our laboratory (37), and a solution of 5 x 10–2 M DIM-C-pPhCF3 was prepared in dimethylsulfoxide (DMSO). The PPAR{gamma} agonists GW9662 (2-chloro-5-nitro-N-phenylbenzamide) and T007 [N-(4'-aminopyridyl)-3-chloro-2-nitrobenzamide] (39, 40) were synthesized by condensation of the corresponding acid chlorides and aromatic amines and purified by thin layer chromatography, and structures were confirmed by gas chromatography-mass spectrometry. PPAR{gamma} and GL2 (luciferase) inhibitory RNAs (iRNAs) were prepared by IDT (Coralville, IA) and targeted to the coding region of PPAR{gamma} and GL2, respectively. Sp1, Sp3, and Sp4 iRNAs were synthesized using the Silencer small inhibitory RNA (siRNA) construction kit from Ambion, Inc. (Austin, TX) according to the manufacturer’s instructions. The iRNA duplexes used in this study are summarized in Table 1Go.


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  		TABLE 1. Summary of siRNA sequences

 
Cell proliferation experiments
Panc-28 cells were maintained in DMEM supplemented with 10% fetal calf serum at 37 C in a humidified atmosphere of 5% CO2 in air. For the cell proliferation experiments, Panc-28 cells were seeded at a density of 1–2 x 105 cells in six-well plates. Twenty-four hours after the seeding, cells were treated with various concentrations of DIM-C-pPhCF3 for the indicated periods of time. Adherent cells were collected by trypsinization, and cell numbers were determined using a Z1 Dual Coulter Counter (Beckman Coulter, Palatine, IL). In experiments using GW9662, Panc-28 cells were pretreated with 5 µM GW9662 for 1 h and then treated with various concentrations of DIM-C-pPhCF3. All treatments were replicated at least three times, and results were confirmed in at least two separate experiments.

Transient transfection assay
Cells were cultured in 12-well plates in DMEM/Ham’s F-12 medium supplemented with 10% fetal bovine serum. After the cells reached 50–60% confluence, reporter gene constructs and/or iRNA duplexes were transfected using TransFast Transfection Reagent (Promega Corp., Madison, WI). Cells were treated with compounds 30 min after transfection. For cotreatment of Panc-28 cells with GW9662, Panc-28 cells were pretreated with GW9662 for 1 h and then treated with various concentrations of compounds. Cotreatment of Panc-28 cells with 5 µM T007 was performed without pretreatment of the antagonist, and 0.2 µg reporter constructs and/or 0.03 µg iRNA duplex were transfected in each well. Cells were harvested 32–42 h after transfection by manual scraping in 1x lysis buffer (Promega Corp.). Lysates were assayed for luciferase activity using luciferase assay reagent (Promega Corp.), and luciferase values were normalized to the amount of the protein in cell lysates. All conditions were examined in triplicate, and results were confirmed at least three times.

Protein isolation and Western blot analysis
Panc-28 cells (1.5 x 105) were seeded in six-well plates and treated with compounds for the indicated time periods. Cells were washed twice with cold PBS, and collected in 200 µl sodium dodecyl sulfate sample buffer [50 mM Tris-HCl (pH 6.8), 10% glycerol, 1% sodium dodecyl sulfate, 5% 2-mercaptoethanol, and 0.025% bromophenol blue]. The protein extract was boiled for 3 min and loaded onto 15% (for p21 detection) or 10% (for pRB, cyclin D1, and PPAR{gamma} detection) polyacrylamide gel, electrophoresed, and transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). Proteins were detected by incubation with polyclonal primary antibodies p21 (sc-397), p-Rb (sc-7986-R), cyclin D1 (sc-718), PPAR{gamma} (sc-7196), Sp1 (sc-59), Sp3 (sc-644), Sp4 (sc-645), and ß-tubulin (sc-9104; all at 1:1000 dilution), followed by blotting with horseradish peroxidase-conjugated secondary antirabbit antibody (1:5000 dilution). Immune complexes were visualized using chemiluminescent substrate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and luminescent signal was recorded on Kodak X-OMAT AR autoradiography film (Eastman Kodak, Rochester, NY). Band intensities were determined by Image software (Scion Corp., Frederick, MD).

FACS analysis
After treatment for the indicated time periods, cells were removed from culture dishes by trypsinization, and approximately 2 x 106 cells were resuspended in 1 ml staining solution containing 50 µg/ml propidium iodide, 4 mM sodium citrate, 30 U/ml ribonuclease, and 0.1% Triton X-100, pH 7.8. Cells were then incubated at 37 C for 10 min, and before FACS analysis, sodium chloride was added to give a final concentration of 0.15 M. Cells were analyzed on a FACSCalibur flow cytometer (BD Pharmingen, San Diego, CA) using CellQuest (BD Pharmingen) acquisition software. Propidium iodide (PI) fluorescence was collected through a 585/42-nm bandpass filter, and list mode data were acquired on a minimum of 12,000 single cells defined by a dot plot of PI width vs. PI area. Data analysis was performed in ModFit LT (Verity Software House, Topsham, ME) using PI width vs. PI area to exclude cell aggregates. FlowJo (Treestar, Inc., Palo Alto, CA) was used to generate the plots shown in the figures.

RNA isolation and Northern blot analysis by RT-PCR
One hundred and fifty thousand cells were seeded in six-well tissue culture plates and treated with vehicle and DIM-C-pPhCF3 for 16 h. RNA was harvested using the SV Total RNA Isolation System (Promega Corp.) according to the manufacturer’s instructions. Ten percent of the total RNA was used for cDNA synthesis using the RT system (Promega Corp.), and amplification of p21 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs was carried out in multiplex reactions in the presence of 1 M betaine and 3% DMSO. The primer sequences used are: GAPDH: forward, 5'-GGT CTC CTC TGA CTT CAA CAG CG-3'; and reverse, 5'-GGT ACT TTA TTG ATG GTA CAT GAC-3'; and p21: forward, 5'-CTG CCG CCG CCT CTT C-3'; and reverse, 5'-GCC TCC TCC CAA CTC A-3'. PCR products were run on 2% agarose gels prestained with ethidium bromide and visualized in a UV transluminator; digital images of PCR products were captured using the Kodak EDAS 290 Electrophoresis Documentation and Analysis System (Eastman Kodak Co., New Haven, CT).

EMSA
A consensus GC-rich oligonucleotide was synthesized and annealed, and 5-pmol aliquots were 5'-end-labeled using T4 kinase and [{gamma}-32P]ATP. A 30-µl EMSA reaction mixture contained approximately 100 mM KCl, 3 µg crude nuclear protein, 1 µg poly(dI-dC), with or without unlabeled competitor oligonucleotide, and 10 fmol radiolabeled probe. After incubation for 20 min on ice, antibodies against Sp1, Sp3, and/or Sp4 proteins were added and incubated for another 20 min on ice. Protein-DNA complexes were resolved by 5% PAGE as previously described (38). Specific DNA-protein and antibody-supershifted complexes were observed as retarded bands in the gel. The GC box oligonucleotide used for gel-shift experiments is given below and contains the consensus Sp1/Sp3 binding site present in the six GC boxes in the p21 promoter that also bind both proteins (41): 5'-AGC TTA TTC GAT CGG GGC GGG GCG AGC G-3' (GC box oligonucleotide).

Immunofluorescent staining
Panc-28 cells were washed three times in PBS, fixed on coverslips with 2.5% paraformaldehyde (Sigma-Aldrich Corp.) for 10 min at room temperature, rinsed twice with PBS, and treated with 0.5% Triton X-100 (Roche, Indianapolis, IN) for 10 min at room temperature. Cells were then blocked with 100 µl 3% BSA for 1 h, followed by 100 µl p21 antibody diluted 1:500 in 3% BSA overnight at 4 C, and then washed with PBS (three times, 5 min each time). The fluorescein isothiocyanate-conjugated goat antirabbit antibody (Molecular Probes, Eugene, OR) was diluted 1:500, 100 µl of the antibody solution were placed on each coverslip for 1 h at room temperature, and cells were washed with PBS (three times, 5 min each time). Nuclei were stained with 0.1 µg/ml PI. The coverslips were mounted face down on microscope slides with mounting medium (Vector Laboratories, Inc., Burlingame, CA) to be viewed on a Zeiss 410 confocal microscope (Carl Zeiss, Oberkochen, Germany). The slides were stored in a light-proof black box.

Chromatin immunoprecipitation (ChIP) assay
Panc28 cells (2 x 107 cells) were treated with DMSO (time zero) or 20 µM DIM-C-pPhCF3 for varying times. Cells were then fixed with 1.5% formaldehyde, and the cross-linking reaction was stopped by the addition of 0.125 M glycine. After washing twice with PBS, cells were scraped and pelleted. Cells were then hypotonically lysed, and nuclei were collected and sonicated to the desired chromatin length (500–1000 bp). The chromatin was precleared by addition of protein A-conjugated beads (Upstate Biotechnology, Inc., Lake Placid, NY) and incubated at 4 C for 1 h with gentle agitation. The beads were pelleted, and the precleared chromatin supernatant was immunoprecipitated with antibodies to Sp1, Sp3, Sp4, and PPAR{gamma} (Santa Cruz Biotechnology, Inc.) at 4 C overnight. The protein-antibody complexes were collected by addition of protein A-conjugated beads at room temperature for 1 h. The beads were extensively washed, and protein-DNA cross-links were reversed. DNA was purified by phenol/chloroform extraction and ethanol precipitation. PCR was performed with the following primers: p21: forward, 5'-GCT GGC CTG CTG GAA CTC-3'; and reverse, 5'-GGC AGC TGC TCA CAC CTC-3' (amplified a 193-bp region of the human p21 promoter) CNAP1: forward, 5'-ATG GTT GCC ACT GGG GAT CT-3'; and reverse, 5'-TGC CAA AGC CTA GGG GAA GA-3' [amplified a 174-bp region between the GAPDH gene and the chromosome condensation-related structural maintenance of chromosome-associated protein gene (CNAP1)]; GAPDH: forward, 5'-TAC TAG CGG TTT TAC GGG CG-3'; and reverse, 5'-TCG AAC AGG AGG AGC AGA GAG CGA-3' [amplified a 167-bp region of human GAPDH promoter that binds transcription factor IIB (TFIIB)]. The corresponding region of the CNAP1 gene does not bind TFIIB and serves as a negative control (Active Motif, Carlsbad, CA). PCR products were resolved on a 2% agarose gel in the presence of 0.5 µg/ml ethidium bromide.

Statistical analysis
Statistical significance was determined by ANOVA, and the levels of probability are noted. The results are expressed as the mean ± SD for at least three separate (replicate) experiments for each treatment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of PPAR{gamma} in Panc-28 cells
Ligand-dependent activation of PPAR{gamma} in Panc-28 cells was determined in cells transfected with PPRE-luc and treated with DIM-C-pPhCF3 and troglitazone alone and in combination with the PPAR{gamma} antagonist GW9622 (Fig. 1AGo). The results show that 15 µM troglitazone and DIM-C-pPhCF3 induced 3- and 26-fold increases in luciferase activity, whereas 5 µM GW9622 alone did not enhance luciferase activity. In Panc-28 cells cotreated with the PPAR{gamma} agonist GW9622 plus troglitazone or DIM-C-pPhCF3, there was a significant decrease in the induced activity. Confirmation that DIM-C-pPhCF3 activated PPRE-luc was further investigated in transactivation experiments in Panc-28 cells treated with DMSO or 15 µM DIM-C-pPhCF3 and cotransfected with small inhibitory RNA for PPAR{gamma} (iPPAR{gamma}) and nonspecific scrambled (iScr) RNA. iPPAR{gamma} significantly inhibited transactivation by DIM-C-pPhCF3, whereas only minimal effects on transactivation were observed with iScr. Moreover, iPPAR{gamma} significantly decreased PPAR{gamma} protein in whole cell lysates (Fig. 1CGo). These results confirm that DIM-C-pPhCF3 activates PPAR{gamma} in Panc-28 cells, and this is consistent with the results of comparable transactivation studies in MCF-7 cells (37).



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  		FIG. 1. Ligand-dependent activation of PPAR{gamma} in Panc-28 cells. A, Inhibition by GW9662. Panc-28 cells were transfected with PPRE-luc, treated with DMSO and various PPAR{gamma} agonists and antagonists, and luciferase activity was determined as described in Materials and Methods. B, Inhibition by RNA interference. Panc-28 cells were treated with DMSO or 15 µM DIM-C-pPhCF3, transfected with PPRE-luc and iPPAR{gamma} or iScr, and luciferase activity was determined as described in Materials and Methods. iScr is a nonspecific RNA used as a control in this experiment. Results in A and B are the mean ± SD for at least three replicates for each treatment. A significant (P < 0.05) induction (*) by PPAR{gamma} agonists alone or inhibition (**) after cotreatment of agonists plus GW9662 is indicated. C, iPPAR{gamma} decreases PPAR{gamma} protein. Panc-28 cells were treated essentially as described in B, and PPAR{gamma} protein in whole cell lysates was determined by Western blot analysis as described in Materials and Methods. PPAR{gamma} protein was significantly decreased (*, P < 0.05) by iPPAR{gamma}.

 
Inhibition of cell proliferation and cell cycle progression
PPAR{gamma} agonists typically inhibit the proliferation of pancreatic and other cancer cell lines, and the results in Fig. 2Go, A and B, show that both troglitazone and DIM-C-pPhCF3 inhibit the growth of Panc-28 cells. Growth inhibition by troglitazone was observed at concentrations of 10, 20, and 30 µM, and the 50% inhibitory concentration was between 20 and 30 µM. One to 15 µM DIM-C-pPhCF3 also inhibited the proliferation of Panc-28 cells; the 50% inhibitory concentration was approximately 5 µM, showing that DIM-C-pPhCF3 was also more potent than troglitazone in growth inhibition and transactivation studies (Fig. 1Go). The role of PPAR{gamma} in mediating the growth inhibitory effects of DIM-C-pPhCF3 was confirmed in Panc-28 cells treated for 48 h with 10 µM DIM-C-pPhCF3 alone or in combination with GW9622. DIM-C-pPhCF3 alone inhibited the growth of Panc-28 cells, and this response was partially reversed (Fig. 2CGo) after cotreatment with GW9622, suggesting that PPAR{gamma} plays a role in mediating inhibition of Panc-28 cell growth by DIM-C-pPhCF3.



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  		FIG. 2. PPAR{gamma} agonists inhibit Panc-28 cell growth. Inhibition by triglitazone (A) or DIM-C-pPhCF3 (B) is shown. Cells were treated for 144 h with 10–30 µM troglitazone or 1–15 µM DIM-C-pPhCF3, and cell numbers were determined as described in Materials and Methods. Significant inhibition was observed at all concentrations after treatment of the cells with PPAR{gamma} agonists for 144 h. C, Effects of GW9662. Cells were treated as indicated for 144 h, and significant (P < 0.05) inhibition of cell proliferation (*) was observed for DIM-C-pPhCF3. This response was partially reversed (**) after cotreatment with 5 µM GW9662. All experiments are the mean ± SD for at least three different determinations for each treatment group.

 
The effects of 15 µM DIM-C-pPhCF3 on distribution of cells in G0/G1, S, and G2/M phases of the cell cycle was investigated in Panc-28 cells after treatment for 12 or 24 h. Cells were initially cultured in serum-free medium for 48 h; the medium was then replaced with 2.5% charcoal-stripped serum and treated with DMSO or 15 µM DIM-C-pPhCF3 (Fig. 3Go). Treatment with DMSO for 12 and 24 h resulted in a time-dependent decrease in the percentage of cells in G0/G1 and an increase of cells in S and G2/M phase. In contrast, DIM-C-pPhCF3 inhibited G0/G1->S phase progression; after treatment with DIM-C-pPhCF3 for 24 h, there was a more than 27% increase and a more than 20% decrease in Panc-28 cells in G0/G1 and S phases, respectively, compared with cells treated with DMSO. Similar results were observed in duplicate studies. These data suggest that the growth inhibitory effects of DIM-C-pPhCF3 may be due in part to modulation of cell cycle genes/proteins associated with G0/G1->S phase progression, and these were further investigated in Panc-28 cells.



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  		FIG. 3. Effects of DIM-C-pPhCF3 on cell cycle distribution. Growth-arrested Panc-28 cells were treated with 15 µM DIM-C-pPhCF3 for 12 h (A) or 24 h (B) in 2.5% charcoal-stripped serum, and the percent distribution of the cells in G0/G1, S, and G2/M phases were determined by FACS analysis as described in Materials and Methods. The percentages are indicated, and similar results were observed in duplicate determinations.

 
Modulation of cell cycle proteins in Panc-28 cells treated with DIM-C-pPhCF3
Panc-28 cells were maintained in serum-free medium for 48 h; 2.5% stripped serum was then added, and cells were treated with DMSO or 15 µM DIM-C-pPhCF3 for up to 48 h. Whole cell lysates were obtained at different time points and analyzed for phospho-Rb, cyclin D1, and p21 protein by Western blot analysis. In cells treated with DMSO, there was a time-dependent increase in phospho-Rb and cyclin D1, and there was also an increase in p21 protein levels after 48 h (Fig. 4AGo). In contrast, DIM-C-pPhCF3 induced a time-dependent decrease in phospho-Rb protein levels, whereas cyclin D1 protein levels were relatively unchanged throughout the experiment (Fig. 4BGo). Decreased phosphorylation of Rb is consistent with the inhibition of G0/G1 to S phase progression induced by DIM-C-pPhCF3. Thus, DIM-C-pPhCF3 inhibited the time-dependent increase in cyclin D1 protein in Panc-28 cells after treatment with DMSO (Fig. 4BGo). DIM-C-pPhCF3 induced levels of p21 protein within 3 h after treatment, and levels of this protein were enhanced up to 48 h. The cyclin-dependent kinase inhibitor p27 was expressed at low levels in Panc-28 cells, and treatment with DMSO or DIM-C-pPhCF3 had minimal effects on p27 expression (data not shown). Figure 4CGo shows the concentration-dependent induction of p21 after treatment of Panc-28 cells with 5, 10, or 15 µM DIM-C-pPhCF3, and a more than 2.5-fold increase in immunostained p21 protein was observed. In addition, RT-PCR analysis showed a significant 2.0-fold induction of p21 mRNA levels in Panc-28 cells treated with 15 µM DIM-C-pPhCF3 for 16 h (Fig. 4DGo). DIM-C-pPhCF3 induced higher levels of p21 protein at different time points than observed for p21 mRNA levels (2-fold induction) after treatment for 16 h. The differences in fold induction of p21 protein and mRNA levels may be due to posttranscriptional processing and/or less than optimal time points for induced p21 mRNA determination. The role of PPAR{gamma} in mediating induction of p21 protein by DIM-C-pPhCF3 was investigated by treating cells with the agonist alone and in combination with 2.5 or 7.5 µM of the PPAR{gamma} antagonist GW9622. The results (Fig. 4EGo) show that induction of p21 protein expression by DIM-C-pPhCF3 is blocked by 7.5 µM GW9622, confirming that PPAR{gamma} mediates the expression of p21 in Panc-28 cells.



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  		FIG. 4. Effects of DIM-C-pPhCF3 on cell cycle genes. A and B, Time-course study. Panc-28 cells were treated with DMSO (A) or 15 µM DIM-C-pPhCF3 (B) for up to 48 h, and protein levels in whole cell lysates were determined by Western blot analysis as described in Materials and Methods. C, Quantitative induction of p21 protein. Panc-28 cells were treated for 24 h with 5–15 µM DIM-C-pPhCF3 (in triplicate), and p21 protein levels (relative to a nonspecific control) were determined as indicated in A. Significant (P < 0.05) induction of p21 protein was observed (*). D, Induced p21 mRNA levels. Panc-28 cells were treated for 16 h with 15 µM DIM-C-pPhCF3, and p21 mRNA levels, relative to a standard control, were determined (in triplicate) by RT-PCR as described in Materials and Methods. Significant (P < 0.05) induction of p21 mRNA levels was observed (*). E, Effects of GW9662. Cells in the various treatment groups were analyzed for p21 protein as described in A. DIM-C-pPhCF3-induced p21 protein levels were inhibited after cotreatment with GW9662 in multiple (more than three) separate experiments.

 
Mechanism of PPAR{gamma}-dependent activation of p21 by DIM-C-pPhCF3
Initial studies of the mechanism of PPAR{gamma}-dependent induction of p21 focused on ligand-dependent activation of pWWP, which contains the –2325 to +8 region of the p21 promoter linked to a luciferase reporter gene. The results summarized in Fig. 5AGo show that 15 µM DIM-C-pPhCF3 induced reporter gene activity in cells transfected with pWWP. The PPAR{gamma} antagonist GW9622 alone did not affect transactivation, but in combined treatment with DIM-C-pPhCF3, GW9622 inhibited induced transactivation. Additional confirmation of the role of PPAR{gamma} in mediating transactivation in cells transfected with pWWP was determined in Panc-28 cells cotransfected with siRNAs, iPPAR{gamma}, or iScr (Fig. 5BGo). The results showed that iPPAR{gamma} significantly inhibited DIM-C-pPhCF3-induced transactivation, confirming the role of this receptor in mediating activation of p21. Figure 5CGo summarizes the induction of luciferase activity by DIM-C-pPhCF3 in Panc-28 cells transfected with pWWP and three constructs with p21 promoter inserts containing 6 (pWWP124, –124 to +8), 4 (pWWP101, –101 to +8), and 2 (pWWP60, –60 to +8) of the well characterized proximal GC-rich sites in the p21 promoter (41, 42, 43, 44, 45, 46, 47, 48). The results show that DIM-C-pPhCF3 induced transactivation in cells transfected with pWWP, pWWP124, and pWWP101, but not pWWP60, and induction by DIM-C-pPhCF3 was inhibited by the PPAR{gamma} antagonist T007. Because pWWP101, but not pWWP60, is activated by DIM-C-pPhCF3, GC-rich sites 3 and 4 are sufficient for PPAR{gamma}-dependent transactivation.



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  		FIG. 5. PPAR{gamma}-dependent activation of p21 promoter constructs. A, Inhibition by GW9662. Panc-28 cells were transfected with pWWP and treated with DMSO or PPAR{gamma} agonists/antagonists, and luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) induction (*) or inhibition (**) is indicated. B, Inhibition by iPPAR{gamma}. Panc-28 cells were treated with DMSO or 15 µM DIM-C-pPhCF3 transfected with pWWP and iPPAR{gamma} or iScr, and luciferase activity was determined as indicated in A. Significant (P < 0.05) induction (*) or inhibition (**) is indicated. C, Deletion analysis. The p21-derived promoter constructs were transfected into Panc-28 cells treated with DMSO and 15 µM DIM-C-pPhCF3 alone or in combination with 5 µM T007 (a PPAR{gamma} antagonist), and luciferase activity was determined as described in A. Significant (P < 0.05) induction (*) or inhibition (**) is indicated. Results are presented as the mean ± SD for at least three replicate determinations for each treatment group. The pWWP, pWWP124, pWWP101, and pWWP60 constructs contain –2325 to +8, –124 to +8, –101 to +8, and –60 to +8 p21 gene promoter inserts, respectively.

 
Several studies have previously reported the critical role of one or more of the proximal GC-rich sites in activation of p21 expression by multiple stimuli (41, 42, 43, 44, 45, 46, 47, 48), and these have been linked to cellular expression of Sp1 and Sp3. Recent studies in this laboratory have identified Sp4 protein in several cancer cell lines, and Western blot analysis confirms that Sp4 along with Sp1 and Sp3 is also expressed in Panc-28 cells (Fig. 6AGo). Gel mobility shift analysis of nuclear extracts from Panc-28 cells treated with DMSO (lane 2), 10 or 15 µM DIM-C-pPhCF3 (lanes 3 and 4, respectively), and the radiolabeled GC-rich oligonucleotide containing the same consensus GC-rich sequences observed in the p21 promoter show a familiar pattern of retarded bands that has previously been characterized in MCF-7 breast cancer cells (lanes 12 and 13) (49). Coincubation of the radiolabeled GC-rich oligonucleotide ([32P]Sp1) and nuclear extracts with antibodies to Sp1 ({alpha}Sp1; lane 5), Sp3 ({alpha}Sp3; lane 6), and Sp4 ({alpha}Sp4; lane 7) gave supershifted bands. Coincubation with a 100-fold excess of unlabeled GC-rich oligonucleotide decreased retarded band intensities (lanes 8 and 9), whereas incubation with a 100-fold excess of unlabeled PPRE oligonucleotide or PPAR{gamma} antibody (lanes 10 and 11, respectively) did not affect retarded band intensities. These results confirm that Sp1, Sp3, and Sp4 interact with the consensus GC-rich sequence, and this band was not directly associated with PPAR{gamma} under the conditions used for the gel mobility shift assay. Previous studies of activation of genes through nuclear receptor-Sp protein interactions with GC-rich sites have also not detected receptor-Sp1-DNA ternary complexes (50, 51, 52, 53). Direct interactions of nuclear receptors with Sp1 protein have been reported, and a recent study (51) showed that PPAR{gamma} interacts with both Sp1 and Sp3 proteins.



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  		FIG. 6. Role of Sp proteins in PPAR{gamma}-dependent activation of p21. A, Western blot analysis. Whole cell lysates from Panc-28 cells were analyzed by Western blot analysis for Sp1, Sp3, and Sp4 proteins as described in Materials and Methods. Sp4 was identified in Panc 28 cells, and in ongoing studies, Sp4 has also been identified in five other pancreatic cancer cell lines. B, Gel mobility shift assays. Nuclear extracts from Panc-28 or MCF-7 cells treated with DMSO (D) or 10 µM (lane 3) or 15 µM (lanes 4 and 9–11) DIM-C-pPhCF3 were isolated and analyzed by gel mobility shift assays as indicated in Materials and Methods. FP, Incubation with free probe alone (no nuclear extracts). The retarded bands associated with Sp1, Sp3, and Sp4 and the supershifted (SS) bands are indicated. The less intense supershifted bands obtained with Sp4 antibodies are more intense using guanosine and thymidine oligonucleotides (data not shown). C, iRNA for Sp proteins modulate p21 promoter-dependent transactivation. Panc-28 cells were cotransfected with pWWP-101-luc and siRNAs and treated with 15 µM DIM-C-pPhCF3, and luciferase activity was determined as described in Materials and Methods. Significant (P < 0.05) induction (*) or inhibition (**) of activity is indicated, and results are expressed as the mean ± SD for three separate determinations for each treatment group. D, siRNA effects on p21 protein. Panc-28 cells were treated with DMSO or DIM-C-pPhCF3 and transfected with siRNA, and protein levels in whole cell lysates were analyzed by Western blot analysis as described in Materials and Methods. The untreated (UT) group in this study and in E was not transfected with siRNAs. E, siRNA effects on Sp proteins. Panc-28 cells were treated essentially as described in C, and Sp proteins were analyzed by Western blot analysis as described in Materials and Methods. iSp1 specifically decreased Sp1 protein expression (data not shown), as previously described in other cell lines (49 54 ). +, Treatment with 15 µM DIM-C-pPhCF3. F, ChIP analysis of the p21 promoter. Interaction of Sp proteins with the p21 promoter was determined by ChIP assays 0, 15, and 60 min after treatment with DIM-C-pPhCF3, and ligand-induced interactions of PPAR{gamma} were determined at 0 and 30 min after treatment as described in Materials and Methods. Similar results were obtained in replicate studies. Controls for this experiment determined interactions of TFIIB with the GAPDH and CNAP1 genomic sequences in which TFIIB interaction was only observed with the former promoter.

 
RNA interference has previously been used in this laboratory for decreasing Sp protein expression and Sp-dependent transactivation (49, 54). The results presented in Fig. 6CGo show that siRNAs for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) differentially affect PPAR{gamma}-dependent activation of pWWP. iSp1 and iSp4 decrease DIM-C-pPhCF3-induced transactivation, whereas iSp3 did not have any significant effect. Cotransfection with iSp1 and iSp4 further decreased PPAR{gamma}-dependent transactivation induced by DIM-C-pPhCF3. Thus, RNA interference with iPPAR{gamma}, iSp1, and iSp4, but not iSp3 or iScr, decreased activation of pWWP by DIM-C-pPhCF3, suggesting that both PPAR{gamma}/Sp1 and PPAR{gamma}/Sp4 cooperatively activate p21 in Panc-28 cells. Additional confirmation of PPAR{gamma}/Sp-dependent activation of p21 is observed in whole cell lysates from Panc-28 cells treated with DMSO or 15 µM DIM-C-pPhCF3 in the presence or absence of cotransfected iSp1-iSp4, iSp3, or nonspecific iScr oligonucleotides (Fig. 6DGo). Transfection of iSp1-iSp4 markedly decreased DIM-C-pPhCF3-induced p21 protein expression, as determined by Western blot analysis of whole cell lysates compared with induction observed in the iSp3 and iScr treatment groups. The specificity of the siRNAs for Sp proteins was also confirmed for Sp4 and Sp3 (Fig. 6EGo). Similar results were obtained using iSp1 (data not shown) as previously described in other cell lines (49, 54). We also examined interactions of Sp proteins and PPAR{gamma} with the proximal region of the p21 promoter by ChIP assays. Sp proteins were constitutively bound to the promoter, and treatment with DIM-C-pPhCF3 enhanced binding of Sp1, Sp4, and PPAR{gamma}, but not Sp3 (Fig. 6FGo). Figure 7Go summarizes p21 immunostaining of Panc-28 cells after treatment with DMSO (A), 15 µM DIM-C-pPhCF3 (B), DMSO/iSp3 (C), DIM-C-pPhCF3/iSp3 (D), DMSO/iSp1-iSp4 (E), DIM-C-pPhCF3/iSp1-iSp4 (F), DMSO/iScr (G), and DIM-C-pPhCF3/iScr (H). The results confirm that DIM-C-pPhCF3 induces nuclear p21 protein (F, D, and H), which is decreased after cotransfection with iSp1-iSp4 (F), but not iSp3 (D). These data demonstrate that PPAR{gamma}-dependent induction of p21, which is observed in several cancer cell lines, is mediated through PPAR{gamma} interactions with Sp1 and Sp4 bound to proximal GC-rich sites in the p21 promoter.



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  		FIG. 7. Immunostaining for p21 protein. Panc-28 cells were treated with DMSO (A), 15 µM DIM-C-pPhCF3 (B), DMSO/iSp3 (C), DIM-C-pPhCF3/iSp3 (D), DMSO/iSp1-iSp4 (E), DIM-C-pPhCF3/iSp1-iSp4 (F), DMSO/iScr (G), and DIM-C-pPhCF3/iScr (H) and immunostained for p21 protein as described in Materials and Methods. p21 Immunostaining was predominantly nuclear.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Structurally diverse PPAR{gamma} agonists inhibit the growth of multiple cancer cell lines (55, 56, 57, 58, 59, 60, 61, 62, 63, 64), including pancreatic cancer cells; however, the mechanisms of growth inhibition are dependent on ligand structure and cell context. For example, several colon cancer cells were differentially responsive to the growth inhibitory effects of rosiglitazone (64). The growth of HT-29, but not HCT-15, colon cancer cells was inhibited by rosiglitazone, and nonresponsiveness in the latter cell line was associated with a K422Q mutation in the ligand-binding domain of PPAR{gamma}. Recent studies in this laboratory have identified a series of PPAR{gamma}-active, C-substituted DIMs that induce PPAR{gamma}-mediated transactivation and inhibit the growth of breast (37) and other cancer (Qin, C., M. Abdelrahim, and J. Stewart, unpublished results) cell lines. DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5 were the most active compounds in terms of PPAR{gamma}-dependent transactivation and growth inhibition, and the former compound was used as a prototype for this research on Panc-28 cells. DIM-C-pPhCF3 induced PPAR{gamma}-dependent transactivation in Panc-28 cells and was more potent than troglitazone. This study primarily focused on the activity of DIM-C-pPhCF3 in Panc-28 cells; however, these responses were also observed for other PPAR{gamma}-active C-substituted DIMs (Safe, S., and J. Hong, unpublished results).

TZDs such as troglitazone inhibit the growth of several pancreatic cancer cells, including PK-1, PK-8, PK-9, MiaPaca2, Capan-1, AsPC-1, BxPC3, Panc-1, and KMP3 cells, and studies of PK-1, BxPC3, and Panc-1 cells show that growth inhibition is accompanied by inhibition of G0/G1->S phase progression (34, 35, 36). The results summarized in Figs. 2Go and 3Go show that troglitazone and DIM-C-pPhCF3 inhibit the growth of Panc-28 cells, and both compounds activate PPAR{gamma}-dependent transactivation. DIM-C-pPhCF3 also inhibited G0/G1->S phase progression. DIM-C-pPhCF3 was more potent than troglitazone in both transactivation and growth inhibition studies, and similar potency differences have been observed for PPAR{gamma}-active C-substituted DIMs and TZDs in breast cancer cells (37).

The effects of DIM-C-pPhCF3 on cell cycle proteins/genes associated with G0/G1->S phase progression suggested that up-regulation of p21 protein and mRNA was a critical response, whereas minimal changes in p27 or cyclin D1 protein expression were observed. Similar results were reported for BxPC3 cells (34) treated with TZDs, whereas in Panc-1 and PK-1 cells, p27 (and not p21) was induced by PPAR{gamma} agonists (35, 36). Previous studies in MCF-7 breast cancer cells showed that 15-deoxy-{Delta}12,14-prostaglandin J2, TZDs, and DIM-C-pPhCF3 induced proteasome-dependent degradation of cyclin D1, which was not inhibited by PPAR{gamma} antagonists (37, 62, 65, 66). In contrast, the PPAR{gamma} antagonist GW9622 partially reversed the inhibition of Panc-28 cell proliferation by 10 µM DIM-C-pPhCF3, suggesting that this response was in part PPAR{gamma} dependent. The rationale for differences in selective activation of genes/proteins associated with G0/G1->S phase progression in pancreatic and other cancer cell lines is unknown and is currently being investigated.

Activation of p21 by PPAR{gamma} agonists has been observed in several cancer cell lines; however, the mechanisms of this response have not been determined. p21 expression is induced by the p53 tumor suppressor gene through interactions with cognate response elements in the promoter (67); however, p21 is also activated by multiple differentiation-inducing agents, including PPAR{gamma} agonists (34, 68, 69, 70). The results in Fig. 5Go confirm that DIM-C-pPhCF3 induces transactivation in Panc-28 cells transfected with pWWP, a construct containing the –2325 to +8 region of the p21 promoter. Moreover, transactivation is inhibited by the PPAR{gamma} antagonists GW9622 and T007 and by cotransfection with siRNA for PPAR{gamma}. Deletion analysis of the p21 promoter shows that the distal region of the promoter, which contains a p53 response element, is not necessary for activation of p21, because pWWP124 is fully activated by DIM-C-pPhCF3. This region of the p21 promoter contains six well characterized, GC-rich sites that bind Sp1 and other Sp family proteins, and these cis-acting sequences are activated by multiple agents (34, 41, 42, 43, 44, 45, 46, 47, 48, 68, 69, 70). DIM-C-pPhCF3 also induced transactivation in Panc-28 cells transfected with pWWP101 and pWWP60, indicating that GC-rich sites 1–4 (–124 to –60) were associated with activation by PPAR{gamma}. Deletion of sites 1 and 2 (–24 to –101) did not result in a significant loss of transactivation (e.g. pWWP124 vs. pWWP101), whereas deletion of sites 3 and 4 (pWWP101 vs. pWWP60) resulted in a loss of DIM-C-pPhCF3-induced transactivation. These data suggest that sites 3 and 4 are necessary for PPAR{gamma}-dependent activation of p21. Single mutations of sites 3 and 4 did not result in a loss of PPAR{gamma} responsiveness (data not shown), suggesting that both sites 3 and 4 are required for DIM-C-pPhCF3-induced transactivation. It has also previously been reported that progesterone receptor and androgen receptor agonists induce p21 through receptor-Sp protein interactions with GC-rich sites 3 and 4 and site 3, respectively (71, 72).

The results of this study suggest that induction of p21 by DIM-C-pPhCF3 is dependent on PPAR{gamma} interactions with GC-rich sites 3 and 4 in the p21 promoter, and a previous study reported direct interactions of PPAR{gamma} and Sp1 proteins (51, 52). Ongoing studies in several pancreatic cancer cell lines indicate that in addition to Sp1 and Sp3, we detected expression of Sp4 protein; this was observed in Panc-28 cells by Western blot and gel mobility shift assays. Sp4 expression is associated with transactivation of genes/reporter genes, and results obtained from RNA interference and ChIP assays indicate that induction of p21 by DIM-C-pPhCF3 involves PPAR{gamma} interactions with both Sp1 and Sp4, but not Sp3, proteins. This study demonstrates that PPAR{gamma}, like other members of the nuclear hormone receptor family of transcription factors, preferentially activates Sp transcription factors bound to GC-rich sites that are also important for progesterone and androgen receptor-mediated transactivation of p21. Current studies are focused on regulation of p21, p27, and cyclin D1 by PPAR{gamma} agonists in other pancreatic cancer cells and on the mechanisms that selectively target specific cell cycle regulatory genes in different cell lines.


   Footnotes
 
This work was supported by National Institutes of Health Grants ES-09106 and CA-108178, M. D. Anderson Cancer Center Pancreatic Cancer Spore Grant P20-CA-10193, and the Texas Agricultural Experiment Station.

Abbreviations: ChIP, Chromatin immunoprecipitation; DIM, diindolylmethane; DIM-C-pPhCF3, 1,1-bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane; DIM-C-pPhC6H5, 1,1-bis(3'-indolyl)-1-(p-biphenyl)methane; DIM-C-pPhtBu, 1,1-bis(3'-indolyl)-1-(p-t-butylphenyl)methane; DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iPPAR{gamma}, siRNA for PPAR{gamma}; iRNA, inhibitory RNA; iScr, siRNA scrambled; PDAC, pancreatic ductal adenocarcinoma; phospho-Rb, phospho-retinoblastoma; PI, propidium iodide; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; PPRE, peroxisome proliferator-activated receptor {gamma} response element; siRNA, small inhibitory RNA; TFIIB, transcription factor IIB; TZD, thiazolidinedione.

Received May 28, 2004.

Accepted for publication August 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. American Cancer Society 2003 Cancer facts and figures-2003. Atlanta: American Cancer Society; 4
  2. Evans DB, Abbruzzese JL, Willett CG 1997 Cancer of the pancreas. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer: principles and practice of oncology. Philadelphia: Lippincott, Williams & Wilkins; 1126–1161
  3. Anderson KE, Potter JD, Mack TM 1996 Pancreatic cancer. In: Schottenfeld D, Fraumeni Jr JF, eds. Cancer epidemiology and prevention. New York: Oxford University Press; 725–771
  4. Hruban RH 2001 Pancreatic cancer: from genes to patient care. J Gastrointest Surg 5:583–587[CrossRef][Medline]
  5. Bardeesy N, Depinho RA 2002 Pancreatic cancer biology and genetics. Nat Rev Cancer 2:897–909[CrossRef][Medline]
  6. Li D 2001 Molecular epidemiology of pancreatic cancer. Cancer J 7:259–265[Medline]
  7. Gold EB, Goldin SB 1998 Epidemiology of and risk factors for pancreatic cancer. Surg Oncol Clin North Am 7:67–91[Medline]
  8. Ahlgren JD 1996 Epidemiology and risk factors in pancreatic cancer. Semin Oncol 23:241–250[Medline]
  9. Weiderpass E, Partanen T, Kaaks R, Vainio H, Porta M, Kauppinen T, Ojajarvi A, Boffetta P, Malats N 1998 Occurrence, trends and environment etiology of pancreatic cancer. Scand J Work Environ Health 24:165–174[Medline]
  10. Lowenfels AB, Maisonneuve P, Cavallini G, Ammann RW, Lankisch PG, Andersen JR, Dimagno EP, Andren-Sandberg A, Domellof L 1993 Pancreatitis and the risk of pancreatic cancer. International Pancreatitis Study Group. N Engl J Med 328:1433–1437[Abstract/Free Full Text]
  11. Lowenfels AB, Maisonneuve P 1999 Pancreatic cancer: development of a unifying etiologic concept. Ann NY Acad Sci 880:191–200[CrossRef][Medline]
  12. Mack TM, Yu MC, Hanisch R, Henderson BE 1986 Pancreas cancer and smoking, beverage consumption, and past medical history. J Natl Cancer Inst 76:49–60
  13. Howe GR, Jain M, Burch JD, Miller AB 1991 Cigarette smoking and cancer of the pancreas: evidence from a population-based case-control study in Toronto, Canada. Int J Cancer 47:323–328[Medline]
  14. Silverman DT, Dunn JA, Hoover RN, Schiffman M, Lillemoe KD, Schoenberg JB, Brown LM, Greenberg RS, Hayes RB, Swanson GM 1994 Cigarette smoking and pancreas cancer: a case-control study based on direct interviews. J Natl Cancer Inst 86:1510–1516[Abstract/Free Full Text]
  15. Fuchs CS, Colditz GA, Stampfer MJ, Giovannucci EL, Hunter DJ, Rimm EB, Willett WC, Speizer FE 1996 A prospective study of cigarette smoking and the risk of pancreatic cancer. Arch Intern Med 156:2255–2260[Abstract/Free Full Text]
  16. Muscat JE, Stellman SD, Hoffmann D, Wynder EL 1997 Smoking and pancreatic cancer in men and women. Cancer Epidemiol Biomarkers Prev 6:15–19[Abstract/Free Full Text]
  17. Duell EJ, Holly EA, Bracci PM, Liu M, Wiencke JK, Kelsey KT 2002 A population-based, case-control study of polymorphisms in carcinogen-metabolizing genes, smoking, and pancreatic adenocarcinoma risk. J Natl Cancer Inst 94:297–306[Abstract/Free Full Text]
  18. Park RM, Mirer FE 1996 A survey of mortality at two automotive engine manufacturing plants. Am J Ind Med 30:664–673[CrossRef][Medline]
  19. Bardin JA, Eisen EA, Tolbert PE, Hallock MF, Hammond SK, Woskie SR, Smith TJ, Monson RR 1997 Mortality studies of machining fluid exposure in the automobile industry. V. A case-control study of pancreatic cancer. Am J Ind Med 32:240–247[CrossRef][Medline]
  20. Norell S, Ahlbom A, Olin R, Erwald R, Jacobson G, Lindberg-Navier I, Wiechel KL 1986 Occupational factors and pancreatic cancer. Br J Ind Med 43:775–778[Medline]
  21. Kauppinen T, Partanen T, Degerth R, Ojajarvi A 1995 Pancreatic cancer and occupational exposures. Epidemiology 6:498–502[Medline]
  22. Schmid RM 2002 Genetic basis of pancreatic cancer. Baillieres Best Pract Res Clin Gastroenterol 16:421–433[CrossRef]
  23. Jaffee EM, Hruban RH, Canto M, Kern SE 2002 Focus on pancreas cancer. Cancer Cell 2:25–28[CrossRef][Medline]
  24. Caldas C, Kern SE 1995 K-ras mutation and pancreatic adenocarcinoma. Int J Pancreatol 18:1–6[Medline]
  25. McKenna S, Eatock M 2003 The medical management of pancreatic cancer: a review. Oncologist 8:149–160[Abstract/Free Full Text]
  26. Cohen SJ, Ho L, Ranganathan S, Abbruzzese JL, Alpaugh RK, Beard M, Lewis NL, McLaughlin S, Rogatko A, Perez-Ruixo JJ, Thistle AM, Verhaeghe T, Wang H, Weiner LM, Wright JJ, Hudes GR, Meropol NJ 2003 Phase II and pharmacodynamic study of the farnesyltransferase inhibitor R115777 as initial therapy in patients with metastatic pancreatic adenocarcinoma. J Clin Oncol 21:1301–1306[Abstract/Free Full Text]
  27. Macdonald JS, Chansky K, Whitehead R, Wade J, Giguere J, Abbruzzese JL 2002 A phase II study of farnesyl transferase inhibitor R115777 in pancreatic cancer. A Southwest Oncology Group (SWOG) study. Proc Am Soc Clin Oncol 21:548 (Abstract)
  28. Moore M, Hamm J, Eisenberg P, Dagenais M, Hagan K, Fields A, Greenberg B, Schwartz B, Ottaway J, Zee B, Seymour L 2000 A comparison between gemcitabine and the matrix metalloproteinase inhibitor BAY12–9566 in patients with advanced pancreatic cancer. Proc Am Soc Clin Oncol 19:930 (Abstract)
  29. Murphy GJ, Holder JC 2000 PPAR-{gamma} agonists: therapeutic role in diabetes, inflammation and cancer. Trends Pharmacol Sci 21:469–474[CrossRef][Medline]
  30. Fajas L, Debril MB, Auwerx J 2001 Peroxisome proliferator-activated receptor-{gamma}: from adipogenesis to carcinogenesis. J Mol Endocrinol 27:1–9[Abstract]
  31. Desvergne B, Wahli W 1999 Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20:649–688[Abstract/Free Full Text]
  32. Escher P, Wahli W 2000 Peroxisome proliferator-activated receptors: insight into multiple cellular functions. Mutat Res 448:121–138[Medline]
  33. Willson RM, Lambert MH, Kliewer SA 2001 Peroxisome proliferator-activated receptor {gamma} and metabolic disease. Annu Rev Biochem 70:341–367[CrossRef][Medline]
  34. Elnemr A, Ohta T, Iwata K, Ninomia I, Fushida S, Nishimura G, Kitagawa H, Kayahara M, Yamamoto M, Terada T, Miwa K 2000 PPAR{gamma} ligand (thiazolidinedione) induces growth arrest and differentiation markers of human pancreatic cancer cells. Int J Oncol 17:1157–1164[Medline]
  35. Itami A, Watanabe G, Shimada Y, Hashimoto Y, Kawamura J, Kato M, Hosotani R, Imamura M 2001 Ligands for peroxisome proliferator-activated receptor {gamma} inhibit growth of pancreatic cancers both in vitro and in vivo. Int J Cancer 94:370–376[CrossRef][Medline]
  36. Motomura W, Okumura T, Takahashi N, Obara T, Kohgo Y 2000 Activation of peroxisome proliferator-activated receptor {gamma} by troglitazone inhibits cell growth through the increase of p27KiP1 in human pancreatic carcinoma cells. Cancer Res 60:5558–5564[Abstract/Free Full Text]
  37. Qin C, Morrow D, Stewart J, Spencer K, Porter W, Smith III R, Phillips T, Abdelrahim M, Samudio I, Safe S 2004 A new class of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists that inhibit growth of breast cancer cells: 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes. Mol Cancer Ther 3:247–259[Abstract/Free Full Text]
  38. Kim K, Nguyen T, Saville B, Safe S 2003 Domains of estrogen receptor {alpha} (ER{alpha}) required for ER{alpha}/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells. Mol Endocrinol 17:804–817[Abstract/Free Full Text]
  39. Leesnitzer LM, Parks DJ, Bledsoe RK, Cobb JE, Collins JL, Consler TG, Davis RG, Hull-Ryde EA, Lenhard JM, Patel L, Plunket KD, Shenk JL, Stimmel JB, Therapontos C, Willson TM, Blanchard SG 2002 Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662. Biochemistry 41:6640–6650[CrossRef][Medline]
  40. Lee G, Elwood F, McNally J, Weiszmann J, Lindstrom M, Amaral K, Nakamura M, Miao S, Cao P, Learned RM, Chen JL, Li Y 2002 T0070907, a selective ligand for peroxisome proliferator-activated receptor {gamma}, functions as an antagonist of biochemical and cellular activities. J Biol Chem 277:19649–19657[Abstract/Free Full Text]
  41. Sowa Y, Orita T, Minamikawa-Hiranabe S, Mizuno T, Nomura H, Sakai T 1999 Sp3, but not Sp1, mediates the transcriptional activation of the p21/WAF1/Cip1 gene promoter by histone deacetylase inhibitor. Cancer Res 59:4266–4270[Abstract/Free Full Text]
  42. Xiao H, Hasegawa T, Isobe K 1999 Both Sp1 and Sp3 are responsible for p21waf1 promoter activity induced by histone deacetylase inhibitor in NIH3T3 cells. J Cell Biochem 73:291–302[CrossRef][Medline]
  43. Xiao H, Hasegawa T, Isobe K-I 2000 p300 Collaborates with Sp1 and Sp3 in p21waf1/cip1 promoter activation induced by histone deacetylase inhibitor. J Biol Chem 275:1371–1376[Abstract/Free Full Text]
  44. Sowa Y, Orita T, Minamikawa S, Nakano K, Mizuno T, Nomura H, Sakai T 1997 Histone deacetylase inhibitor activates the WAF1/Cip1 gene promoter through the Sp1 sites. Biochem Biophys Res Commun 241:142–150[CrossRef][Medline]
  45. Nakano K, Mizuno T, Sowa Y, Orita T, Yoshino T, Okuyama Y, Fujita T, Ohtani-Fujita N, Matsukawa Y, Tokino T, Yamagishi H, Oka T, Nomura H, Sakai T 1997 Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line. J Biol Chem 272:22199–22206[Abstract/Free Full Text]
  46. Pagliuca A, Gallo P, Lania L 2000 Differential role for Sp1/Sp3 transcription factors in the regulation of the promoter activity of multiple cyclin-dependent kinase inhibitor genes. J Cell Biochem 76:360–367[CrossRef][Medline]
  47. Koutsodontis G, Tentes I, Papakosta P, Moustakas A, Kardassis D 2001 Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein. J Biol Chem 276:29116–29125[Abstract/Free Full Text]
  48. Koutsodontis G, Moustakas A, Kardassis D 2002 The role of Sp1 family members, the proximal GC-rich motifs, and the upstream enhancer region in the regulation of the human cell cycle inhibitor p21WAF-1/Cip1 gene promoter. Biochemistry 41:12771–12784[CrossRef][Medline]
  49. Abdelrahim M, Samudio I, Smith R, Burghardt R, Safe S 2002 Small inhibitory RNA duplexes for Sp1 mRNA block basal and estrogen-induced gene expression and cell cycle progression in MCF-7 breast cancer cells. J Biol Chem 277:28815–28822[Abstract/Free Full Text]
  50. Porter W, Saville B, Hoivik D, Safe S 1997 Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 11:1569–1580[Abstract/Free Full Text]
  51. Sassa Y, Hata Y, Aiello LP, Taniguchi Y, Kohno K, Ishibashi T 2004 Bifunctional properties of peroxisome proliferator-activated receptor {gamma}1 in KDR gene regulation mediated via interaction with both Sp1 and Sp3. Diabetes 53:1222–1229[Abstract/Free Full Text]
  52. Sugawara A, Uruno A, Kudo M, Ikeda Y, Sato K, Taniyama Y, Ito S, Takeuchi K 2002 Transcription suppression of thromboxane receptor gene by peroxisome proliferator-activated receptor-{gamma} via an interaction with Sp1 in vascular smooth muscle cells. J Biol Chem 277:9676–9683[Abstract/Free Full Text]
  53. Curtin D, Jenkins S, Farmer N, Anderson AC, Haisenleder DJ, Rissman E, Wilson EM, Shupnik MA 2001 Androgen suppression of GnRH-stimulated rat LHß gene transcription occurs through Sp1 sites in the distal GnRH-responsive promoter region. Mol Endocrinol 15:1906–1917[Abstract/Free Full Text]
  54. Stoner M, Wormke M, Saville B, Samudio I, Qin C, Abdelrahim M, Safe S 2004 Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor {alpha} and Sp proteins. Oncogene 23:1052–1063[CrossRef][Medline]
  55. Sato H, Ishihara S, Kawashima K, Moriyama N, Suetsugu H, Kazumori H, Okuyama T, Rumi MA, Fukuda R, Nagasue N, Kinoshita Y 2000 Expression of peroxisome proliferator-activated receptor (PPAR){gamma} in gastric cancer and inhibitory effects of PPAR{gamma} agonists. Br J Cancer 83:1394–1400[CrossRef][Medline]
  56. Takahashi N, Okumura T, Motomura W, Fujimoto Y, Kawabata I, Kohgo Y 1999 Activation of PPAR{gamma} inhibits cell growth and induces apoptosis in human gastric cancer cells. FEBS Lett 455:135–139[CrossRef][Medline]
  57. Inoue K, Kawahito Y, Tsubouchi Y, Kohno M, Yoshimura R, Yoshikawa T, Sano H 2001 Expression of peroxisome proliferator-activated receptor {gamma} in renal cell carcinoma and growth inhibition by its agonists. Biochem Biophys Res Commun 287:727–732[CrossRef][Medline]
  58. Kitamura S, Miyazaki Y, Hiraoka S, Nagasawa Y, Toyota M, Takakura R, Kiyohara T, Shinomura Y, Matsuzawa Y 2001 PPAR{gamma} agonists inhibit cell growth and suppress the expression of cyclin D1 and EGF-like growth factors in ras-transformed rat intestinal epithelial cells. Int J Cancer 94:335–342[CrossRef][Medline]
  59. Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S, Miyoshi I, Koeffler HP 1998 Ligand for peroxisome proliferator-activated receptor {gamma} (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 58:3344–3352[Abstract/Free Full Text]
  60. Suh N, Wang Y, Williams CR, Risingsong R, Gilmer T, Willson TM, Sporn MB 1999 A new ligand for the peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}), GW7845, inhibits rat mammary carcinogenesis. Cancer Res 59:5671–5673[Abstract/Free Full Text]
  61. Sarraf P, Mueller E, Jones D, King FJ, DeAngelo DJ, Partridge JB, Holden SA, Chen LB, Singer S, Fletcher C, Spiegelman BM 1998 Differentiation and reversal of malignant changes in colon cancer through PPAR{gamma}. Nat Med 4:1046–1052[CrossRef][Medline]
  62. Clay CE, Namen AM, Atsumi G, Willingham MC, High KP, Kute TE, Trimboli AJ, Fonteh AN, Dawson PA, Chilton FH 1999 Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells. Carcinogenesis 20:1905–1911[Abstract/Free Full Text]
  63. Qin C, Burghardt R, Smith R, Wormke M, Stewart J, Safe S 2003 Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists induce proteasome-dependent degradation of cyclin D1 and estrogen receptor {alpha} in MCF-7 breast cancer cells. Cancer Res 63:958–964[Abstract/Free Full Text]
  64. Gupta RA, Sarraf P, Mueller E, Brockman JA, Prusakiewicz JJ, Eng C, Willson TM, DuBois RN 2003 Peroxisome proliferator-activated receptor {gamma}-mediated differentiation: a mutation in colon cancer cells reveals divergent and cell type-specific mechanisms. J Biol Chem 278:22669–22677[Abstract/Free Full Text]
  65. Clay CE, Monjazeb A, Thorburn J, Chilton FH, High KP 2002 15-Deoxy-{Delta}12,14-prostaglandin J2-induced apoptosis does not require PPAR{gamma} in breast cancer cells. J Lipid Res 43:1818–1828[Abstract/Free Full Text]
  66. Campo PA, Das S, Hsiang CH, Bui T, Samuel CE, Straus DS 2002 Translational regulation of cyclin D1 by 15-deoxy-{Delta}12,14-prostaglandin J2. Cell Growth Differ 13:409–420[Abstract/Free Full Text]
  67. Vogelstein B, Lane D, Levine AJ 2000 Surfing the p53 network. Nature 408:307–310[CrossRef][Medline]
  68. Jiang H, Lin J, Su ZZ, Collart FR, Huberman E, Fisher PB 1994 Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21WAF1/CIP1 expression in the absence of p53. Oncogene 9:3397–3406[Medline]
  69. Liu M, Iavarone A, Freedman LP 1996 Transcriptional activation of the human p21(WAF1/CIP1) gene by retinoic acid receptor. Correlation with retinoid induction of U937 cell differentiation. J Biol Chem 271:31723–31728[Abstract/Free Full Text]
  70. Yan GZ, Ziff EB 1997 Nerve growth factor induces transcription of the p21 WAF1/CIP1 and cyclin D1 genes in PC12 cells by activating the Sp1 transcription factor. J Neurosci 17:6122–6132[Abstract/Free Full Text]
  71. Owen GI, Richer JK, Tung L, Takimoto G, Horwitz KB 1998 Progesterone regulates transcription of the p21WAF1 cyclin-dependent kinase inhibitor gene through Sp1 and CBP/p300. J Biol Chem 273:10696–10701[Abstract/Free Full Text]
  72. Lu S, Jenster G, Epner DE 2000 Androgen induction of cyclin-dependent kinase inhibitor p21 gene: role of androgen receptor and transcription factor Sp1 complex. Mol Endocrinol 14:753–760[Abstract/Free Full Text]



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