p21 Expression Is Induced by Activation of Nuclear Nerve Growth Factor–Induced Bα (Nur77) in Pancreatic Cancer Cells

The nuclear receptor family of transcription factors includes the steroid and thyroid hormone, vitamin D, retinoid and ecdysone receptors, adopted orphan receptors, and orphan receptors with no known ligands (1, 2). Nuclear receptors influence diverse aspects of normal physiology in multiple tissues; however, despite their functional diversity, this family of proteins exhibits several common structural features and molecular mechanisms of gene activation. Nerve growth factor (NGF)–inducible gene B (NGFI-B) is part of a subfamily of orphan nuclear receptors that were initially identified after treatment of PC12 pheochromocytoma cells with NGF (3). Members of this subfamily include Nur77 (NGFI-Bα, TR₃), Nurr1 (NGFI-Bβ), and Nor1 (NGFI-Bγ). Nur77 is expressed in multiple tissues; Nurr1 has been detected in thymus osteoblasts, liver, and pituitary gland; and Nor1 is highly expressed in the pituitary gland with low expression in other tissues (4-7). The physiologic roles for NGFI-B proteins are not fully understood; however, gene targeting knockout experiments show several important functions of these proteins that correlate, in part, with other in vitro and in vivo studies (7-11). For example, Nurr1 knockout mice have severe impairments in midbrain neuronal development and dopamine expression, and these animals die soon after birth (7, 8).

NGFI-B proteins bind to specific promoter DNA response elements as monomers, homodimers, or heterodimers complexed with RXR (12-14). Ligands that bind or activate NGFI-B proteins through AF-2 domains have not been identified; however, 6-mercaptorpurine activates both Nurr1 and Nor1 through their respective NH₂-terminal AF-1 domains (15, 16). 6-Mercaptorpurine also activates Nur77-dependent genes, including hypoxia-inducible factor 1α, in HepG2 liver cancer cells. Nur77 plays an important role in thymocyte-negative selection and in T-cell receptor–mediated apoptosis in thymocytes, and overexpression of Nur77 in transgenic mice results in high levels of apoptosis in thymocytes (17, 18). Several studies suggest that Nur77 plays a role in cell death pathways activated by apoptosis-inducing agents (19-28). The mechanisms associated with these responses are drug and cell context dependent; however, these responses are primarily due to nuclear export of Nur77. In some cases, Nur77 binds bcl-2 to form a proapoptotic complex (20).

Studies in this laboratory have identified a series of 1,1-bis(3′-indolyl)-1-(p-substituted phenyl)methanes in which the p-trifluoromethyl (DIM-C-pPhCF₃), p-methoxy (DIM-C-pPhOCH₃), and unsubstituted (DIM-C-pPh) analogues activate Nur77 in colon and pancreatic cancer cells (29, 30). A second compound containing a p-hydroxyl substituent (DIM-C-pPhOH) antagonizes activation of Nur77 by the Nur77-active compounds. DIM-C-pPhOCH₃ has been extensively used as a prototype compound, which induces apoptosis and inhibits colon and pancreatic cell and tumor growth, and in cell culture studies, the proapoptotic responses are dependent on nuclear Nur77 (29, 30). DIM-C-pPhOCH₃ induces G₀-G₁–phase to S-phase arrest in Panc1 cells, and this is accompanied by Nur77-dependent induction of the cyclin-dependent kinase inhibitor p21. Induction of p21 is Krüppel-like factor 4 (KLF4) independent but is accompanied by recruitment of Nur77 to the GC-rich proximal region of the p21 promoter and interactions of Nur77 with Sp1 and Sp4.

DIM-C-pPhOCH₃ activates Nur77 in pancreatic and colon cancer cells (29, 30) and induces proapoptotic responses. However, in preliminary microarray studies, DIM-C-pPhOCH₃ also induced p21 expression after treatment for 6 hours (data not shown). Using Panc1 cells as a model, Fig. 1A shows that 5 or 10 μmol/L of DIM-C-pPhOCH₃ induced p21 protein levels and Fig. 1B shows the time course induction of p21 mRNA by DIM-C-pPhOCH₃. p21 was significantly induced 3 to 6 hours after treatment and was maximally induced after 18 to 24 hours. In addition, luciferase activity was also induced in Panc1 cells transfected with pWWP, a construct containing the p21 promoter (−2325 to +8) linked to a luciferase reporter gene. Because p21 is important for cell proliferation and cell cycle progression, the effects of DIM-C-pPhOCH₃ on Panc1 cell survival (Fig. 1C) and the percentage distribution of cells in G₀-G₁, S, and G₂-M were determined by cell counting and fluorescence-activated cell sorting (FACS) analysis, respectively (Fig. 1D). Treatment with 5 to 15 μmol/L of DIM-C-pPhOCH₃ for 24, 48, 72, and 96 hours decreased cell survival in a time- and concentration-dependent manner, and a significant decrease in cell survival was observed at all concentrations after treatment for 96 hours (Fig. 1C). Treatment of Panc1 cells with 10 μmol/L DIM-C-pPhOCH₃ for 24 hours also significantly increased the percentage of cells in G₀-G₁ and decreased the percentage of cells in S phase (Fig. 1D). These results are consistent with the effects of DIM-C-pPhOCH₃ on p21 expression in Panc1 cells.

Previous studies showed that Nur77-active C-DIMs activated apoptosis in colon and pancreatic cancer cells and Nur77 was detected primarily in the nuclear fraction, whereas studies in other cell lines reported that apoptosis-inducing agents such as the phorbol esters and the retinoid CD437 induced export of nuclear Nur77 (19-21). Results in Fig. 2 illustrate the effects of DMSO (solvent control), 10 μmol/L DIM-C-pPhOCH₃, 2 μmol/L CD437, and 200 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA) on subcellular localization of Nur77 in Panc1 cells. 4′,6-Diamidino-2-phenylindole (DAPI) staining was used to identify the cell nuclei and the overlay shows colocalization of Nur77 and DAPI staining. In DMSO-treated cells, Nur77 staining was primarily in the nucleus and treatment of Panc1 cells with 10 μmol/L DIM-C-pPhOCH₃ did not change the Nur77 staining pattern. In contrast, both CD437 and TPA induced nuclear export of Nur77 out of the nucleus in Panc1 within 3 hours after treatment and this was consistent with previous reports for these compounds in other cancer cell lines (19-21). Thus, induction of p21 by Nur77-active DIM-C-pPhOCH₃ is not accompanied by nuclear export of Nur77.

The role of Nur77 in mediating the induction of p21 by DIM-C-pPhOCH₃ was further investigated in Panc1 cells treated with DIM-C-pPhOCH₃ alone or in combination with DIM-C-pPhOH, a compound that inhibits activation of Nur77 (29). DIM-C-pPhOH alone decreased basal p21 protein expression and also blocked induction of p21 by DIM-C-pPhOCH₃ (Fig. 3A). In addition, induction of p21 by DIM-C-pPhOCH₃ was not affected by cotreatment with leptomycin B (LMB; a nuclear export inhibitor), confirming that this response was due to activation of nuclear Nur77 (Fig. 3B). A small inhibitory RNA (siRNA) for Nur77 (siNur77) was also used to confirm the role of the receptor in mediating induction of p21. In Panc1 cells transfected with siNur77, there was a significant decrease in Nur77 protein and this was accompanied by a significant decrease in the induction of p21 by DIM-C-pPhOCH₃ (Fig. 3C). siNur77 alone decreased basal expression of p21, suggesting that this cyclin-dependent kinase inhibitor may also be regulated by endogenous Nur77. siNur77 also decreased Nur77 mRNA levels, and Nur77 knockdown inhibited induction of p21 mRNA by DIM-C-pPhOCH₃ in Panc1 cells (Fig. 3D).

We also examined the effects of the Nur77-active DIM-C-pPhOCH₃ and DIM-C-Ph on induction of p21 mRNA levels in L3.6pl pancreatic cancer cells, and both compounds were active in this cell line (Fig. 4A). In addition, siNur77 decreased Nur77 in L3.6pl cells and Nur77 knockdown also decreased induction of p21 by DIM-C-pPhOCH₃ compared with L3.6pl cells transfected with nonspecific siScr (Fig. 4B). Thus, induction of p21 by DIM-C-pPhOCH₃ was Nur77 dependent in both Panc1 and L3.6pl cells. Recent studies show that 5,5′-dibromoDIM, a ring-substituted DIM, also induces p21 in colon cancer cells and this is KLF4 dependent (31). Nur77-active DIM-C-pPhOCH₃ and DIM-C-Ph induced KLF4 mRNA levels in Panc1 cells; however, RNA interference studies with siNur77 show that induction of KLF4 was Nur77 independent and KLF4 knockdown did not affect p21 induction by DIM-C-pPhOCH₃ (Supplementary Fig. S1). The differences between DIM derivatives and their induction of p21/KLF4 are currently being investigated.

The mechanism of Nur77-dependent induction of p21 was further investigated in Panc1 cells transfected the pWWP construct that contains the −2325 to +8 p21 promoter sequence linked to a luciferase reporter gene and various deletion mutants (Fig. 5A). DIM-C-pPhOCH₃ significantly induced luciferase activity in cells transfected with pWWP; however, this promoter sequence does not contain Nur77 binding sites. Comparable induction response was also observed in cells transfected with the pWWP124 deletion mutant, which contains six well-characterized GC-rich sites in the proximal region of the p21 promoter but does not contain the distal p53 binding site. DIM-C-pPhOCH₃ induced luciferase activity in Panc1 cells transfected with pWWP101 (containing GC sites 1-4) but not pWWP60 (containing GC sites 1 and 2), indicating that sites 3 and 4 are necessary for the induction response. Panc1 cells were also transfected with pWWP101 and the mutant constructs pWWP101-mt3 and pWWP101-mt4 and treatment with DIM-C-pPhOCH₃ induced activity in cells transfected with pWWP101, whereas there was a significant decrease in both basal and inducible activity in cells transfected with the mutant constructs (Fig. 5B). These results imply that Sp proteins, such as Sp1, which bind GC-rich motifs and Nur77 may be important for DIM-C-pPhOCH₃–dependent activation of pWWP101, and this was confirmed in RNA interference studies, which showed that both siSp1 and siNur77 inhibited induction of luciferase activity by DIM-C-pPhOCH₃ (Fig. 5B). These results imply that Nur77-dependent induction of p21 may involve Sp proteins that bind GC-rich sites. Previous studies show that Sp1, Sp3, and Sp4 are the major Sp proteins expressed in pancreatic and other cancer cell lines (32). In Panc1 cells transfected with siRNAs for Sp1 (siSp1), Sp3 (siSp3), and Sp4 (siSp4), there was a specific decrease in these proteins as previously reported (32). Moreover, siSp1 and siSp4 but not siSp3 decreased induction of p21 by DIM-C-pPhOCH₃ (Fig. 5C). Chromatin immunoprecipitation (ChIP) analysis of interactions of Sp proteins and Nur77 with the proximal GC-rich region of the p21 promoter showed that treatment with DIM-C-pPhOCH₃ increased recruitment of Nur77 to the promoter (Fig. 5D). In addition, there was a time-dependent increase in Sp1 and Sp4 interactions with the p21 promoter, whereas the constitutive binding of Sp3 was relatively unchanged. In a control experiment, we observed binding of TF_IIB to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter but not to exon I of the CNAP1 gene (data not shown). These results show that induction of p21 in Panc1 cells is Nur77 dependent and involved novel Nur77 interactions with Sp proteins in the proximal region of the p21 promoter. However, these results do not exclude a role for direct interactions of Nur77 with promoter DNA in more distal regions of the p21 promoter. The interactions of Nur77 with Sp1 and Sp4 were investigated in coimmunoprecipitation studies in Panc1 cells (Fig. 6A) and in cells transfected with Flag-Nur77 followed by coimmunoprecipitation with Flag antibodies (Fig. 6B). Results of both studies show coimmunoprecipitation of Nur77 with both Sp1 and Sp4. In addition, we also transfected Panc1 cells with EYFP-Nur77 and these cells were also stained with Sp1 and Sp4 antibodies, and analysis by immunofluorescence microscopy showed that Nur77 colocalized in the nucleus with both Sp1 and Sp4 (Fig. 6C). Both Nur77 and Sp1 are exclusively localized in nuclear extracts of Panc1 cells; however, Sp4 protein is detected in both the nuclear and cytosolic fraction (Fig. 6B). Thus, the role of Nur77, Sp1, and Sp4 in activation of p21 by DIM-C-pPhOCH₃ is accompanied by association of these proteins in ChIP, coimmunoprecipitation, and colocalization assays.

Nur77 is an orphan receptor and a member of the nuclear receptor superfamily. The role of Nur77 in cancer has been extensively investigated, and the proapoptotic function of this nuclear receptor has paradoxically been associated with drug-induced apoptosis associated with extranuclear Nur77. Zhang and coworkers reported that various proapoptotic drugs, including the phorbol ester 12-O-tetradecanoyl-13-phorbol acetate and the adamantyl-derived retinoids 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene (AHPN or CD437) and 5-chloro-AHPN (chlorine substitution on the naphthalene group), induce apoptosis through translocation of Nur77 from the nucleus to the mitochondria in cancer cell lines (19, 20, 24-26). Moreover, results of drug-induced and Nur77 (wild-type/mutant) overexpression studies show that Nur77 specifically binds the antiapoptotic protein bcl-2 to form a proapoptotic bcl-2/Nur77 complex (20). Other studies have confirmed drug-induced nuclear export of Nur77 but they do not necessarily detect Nur77 association with mitochondria (21-23). For example, in colon and prostate cancer cells, proapoptotic agents such as 5-fluorodeoxyuridine, TPA, sulindac, and short chain fatty acids induce nuclear to cytosolic translocation of Nur77 and the subsequent enhancement of mitochondrial bax and release of cytochrome c (23).

Studies in this laboratory showed that DIM-C-pPh and DIM-C-pPhOCH₃ inhibit growth of colon and pancreatic cancer cells and also induce both Nur77-dependent and Nur77-independent apoptosis (29, 30). The Nur77-independent responses in colon cancer cells were primarily observed at higher concentrations of DIM-C-pPhOCH₃ and involved induction of ER stress, which is a general property of C-DIM compounds (33, 34). Microarray studies in several different cancer cell lines identified a large number of genes associated with growth and apoptosis, and RNA interference experiments in colon cancer cells confirmed that induction of p21 by DIM-C-pPhOCH₃ was Nur77 dependent (data not shown; ref. 30). p21 is a critical gene involved in regulating cell proliferation, cell cycle progression, and cell death pathways, and this study focused on the molecular mechanisms of Nur77-dependent induction of p21 by DIM-C-pPhOCH₃ in Panc1 cells.

Initial studies showed that DIM-C-pPhOCH₃ inhibited Panc1 cell survival (Fig. 1C), and FACS analysis of Panc1 cells in G₀-G₁, S, and G₂-M phases of the cell cycle showed that DIM-C-pPhOCH₃ induced a G₀-G₁–phase to S-phase arrest (Fig. 1D). These results are consistent with the induction of p21 mRNA, protein, and promoter activity (Fig. 1A and B). Results of RNA interference in Panc1 cells transfected with siNur77 confirm that induction of p21 protein and mRNA was Nur77 dependent (Fig. 3B and C). DIM-C-pPhOH has previously been characterized as a compound that acts as a Nur77 antagonist (29), and in this study, DIM-C-pPhOH also inhibited induction of p21 by DIM-C-pPhOCH₃ (Fig. 3A). We also observed that p21 was induced by DIM-C-pPhOCH₃ (and DIM-C-Ph) in L3.6pl pancreatic cancer cells, and this response was also inhibited after cotransfection with siNur77 (Fig. 4). Moreover, in Panc1 cells treated with DMSO or 10 μmol/L DIM-C-pPhOCH₃ for 3 hours, immunostaining showed that Nur77 remained in the nucleus, whereas treatment with TPA or CD437 for 3 hours resulted in transport of Nur77 out of the nucleus (Fig. 2) as previously reported for these apoptosis-inducing agents (19-21). The Nur77-dependent proapoptotic activity of DIM-C-pPhOCH₃ in pancreatic and colon cancer cells was dependent on nuclear Nur77 and was unaffected by LMB, an agent that inhibits nuclear protein export (29, 30). In this study, LMB also did not affect induction of p21 by DIM-C-pPhOCH₃ (Fig. 3B), confirming that this response was also dependent on nuclear Nur77.

p21 is induced through multiple p53-dependent and p53-independent pathways (35-37), and results in Fig. 5A show that DIM-C-pPhOCH₃ activates p21 promoter constructs containing the distal p53 response element (pWWP) or constructs containing only downstream GC-rich motifs (pWWP124 and pWWP101). Because the p21 promoter does not contain binding sites for Nur77 monomers or dimers, the promoter analysis studies suggest that Nur77-dependent activation of p21 by DIM-C-pPhOCH₃ is associated with the proximal GC-rich sites (6-3) in the p21 promoter. Transfection experiments using pWWP101 containing mutations in GC-rich sites 3 or 4 confirm the importance of these cis-elements for basal and inducible activation by DIM-C-pPhOCH₃ (Fig. 5B), and RNA interference confirms the importance of Nur77 and Sp1 for activation of pWWP101. Previous studies have shown that other nuclear receptors, including the androgen, progesterone, and retinoic acid receptors and peroxisome proliferator-activated receptor γ, induce p21 through interactions of the ligand-bound receptors with Sp1 or Sp1/Sp4 (for peroxisome proliferator-activated receptor γ) proteins bound to these sites (32, 38-40). Results of RNA interference and ChIP assays (Fig. 5C and D) show that treatment of Panc1 cells with DIM-C-pPhOCH₃ enhances Sp1, Sp4 (but not Sp3), and Nur77 binding to the GC-rich region of the p21 promoter, and knockdown of Sp1 or Sp4 but not Sp3 abrogates p21 induction. Moreover, coimmunoprecipitation and colocalization studies show the association of Nur77 with Sp1 and Sp4 (Fig. 6). Thus, activation of p21 by DIM-C-pPhOCH₃ involves Nur77-Sp1/Sp4 (protein-protein) association rather than direct binding of Nur77 with promoter DNA, and this pathway seems to be important not only for Nur77 but also for other nuclear receptors that induce p21 expression (32, 38-40). These results do not exclude the possibility that interactions of Nur77 with Sp1 or Sp4 may be indirect and involve other factors, and this is currently being investigated.

In summary, this study shows that DIM-C-pPhOCH₃ decreases Panc1 cell survival and inhibits G₀-G₁–phase to S-phase progression, and this is due, in part, to Nur77-dependent induction of p21. We show that p21 induction by DIM-C-pPhOCH₃ in Panc1 cells is KLF4 independent and involves association of Nur77 with Sp1 and Sp4 bound to proximal GC-rich sites in the p21 promoter. This represents a novel pathway for induction of p21 by activation of the orphan receptor Nur77 and shows for the first time that, like other nuclear receptors, Nur77 activation can result in transactivation through protein-protein (Nur77-Sp)-DNA association. Current studies are focused on the molecular mechanisms of Nur77-mediated induction of other genes and on development of Nur77-active C-DIMs for cancer chemotherapy.

Panc1 human pancreatic cancer cell lines were obtained from the American Type Culture Collection. The L3.6pl cell line was developed at the M. D. Anderson Cancer Center and kindly provided by Dr. I.J. Fidler (M. D. Anderson Cancer Center, Houston, TX). Panc1 cells were maintained in DMEM nutrient mixture with Ham's F-12 (DMEM/Ham's F-12; Sigma-Aldrich) supplemented with 0.22% sodium bicarbonate, 0.011% sodium pyruvate, 10% fetal bovine serum (FBS), and 10 mL/L 100× antibiotic antimycotic solution (Sigma-Aldrich). L3.6pl cells were maintained in RPMI 1640 supplemented with 10% FBS and 10 mL/L 100× antibiotic antimycotic solution. Cells were maintained at 37°C in the presence of 5% CO₂.

p21 promoter reporter constructs pWWP, pWWP124, pWWP101, pWWP101-mt3, and pWWP101-mt4 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. The Flag-tagged and YFP-tagged full-length Nur77 were constructed by inserting PCR-amplified full-length Nur77 fragments into the EcoRI/BamHI site of p3XFLAG-CMV-10 expression vector (Sigma-Aldrich) and pEYFP-C1 expression vector (BD Biosciences Clontech). The C-substituted DIMs were synthesized in this laboratory as previously described (29), and their identities and purity (>98%) were confirmed by gas chromatography-mass spectrometry. All other chemicals were obtained from Sigma Chemical unless otherwise indicated. Nur77, p21, and Sp1 antibodies were purchased from Imgenex, BD Pharmingen, and Upstate, respectively. All other antibodies, including Sp3 and Sp4, were purchased from Santa Cruz Biotechnology. Reporter lysis buffer and luciferase reagent were supplied by Promega. β-Galactosidase (β-Gal) reagent was obtained from Tropix, and Lipofectamine 2000 reagent was purchased from Invitrogen. For RNA interference assays, we used a nonspecific scrambled (siScr) oligonucleotide as described (30, 31). The siRNA for Nur77 was identical to the reported oligonucleotide (29, 30) and the siRNAs for Sp1, Sp3, and Sp4 were identical to the reported oligonucleotides (32). All the siRNAs were prepared by Dharmacon Research.

cDNA was prepared from the total RNA of cells using Reverse Transcription System (Promega). Each PCR was carried out in triplicate in a 20-μL volume using SYBR Green Mastermix (Applied Biosystems) for 15 min at 95°C for initial denaturing followed by 40 cycles of 95°C for 30 s and 60°C for 1 min in the Applied Biosystems 7900HT Fast Real-time PCR System. The ABI Dissociation Curves software was used following a brief thermal protocol (95°C for 15 s and 60°C for 15 s followed by a slow ramp to 95°C) to control for multiple species in each PCR amplification. Values for each gene were normalized to expression levels of TATA-binding protein. The sequences of the primers used for real-time PCR were as follows: p21, 5′-GGCAGACCAGCATGACAGATTTC-3′ (sense) and 5′-CGGATTAGGGCTTCCTCTTGG-3′ (antisense); TATA-binding protein, 5′-TGCACAGGAGCCAAGAGTGAA-3′ (sense) and 5′-CACATCACAGCTCCCCACCA-3′ (antisense). The PCR primers for Nur77 and KLF4 were purchased from Qiagen.

Cells (2 × 10⁵) were plated in six-well plates in DMEM/Ham's F-12 medium containing 2.5% charcoal-stripped FBS for 16 h and then treated with different concentrations of the compounds. Cellular lysates were prepared in a lysis buffer containing 50 mmol/L Tris-HCl (pH 7.5), 2 mmol/L EDTA, 150 mmol/L NaCl, 0.5% deoxycholate, 0.1% SDS, 1 mmol/L NaF, 1 mmol/L Na₃VO₄, 1 mmol/L phenylmethylsulfonyl fluoride, 5 μL/mL protease inhibitor cocktail (Sigma-Aldrich), and 1% NP40. The cells were disrupted and extracted at 4°C for 30 min. After centrifugation at 13,000 rpm for 15 min, the supernatant was obtained as the cell lysate. Nuclear extracts were obtained using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Inc.). Protein concentrations were measured using the Bio-Rad protein assay. Aliquots of cellular proteins were electrophoresed on 10% or 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was allowed to react with a specific antibody and detection of specific proteins was carried out by enhanced chemiluminescence (Perkin-Elmer). Loading differences were normalized using a polyclonal β-actin antibody. For immunoprecipitation, 1 mg of cell lysate was precleared with mouse IgG agarose (Sigma-Aldrich) for 2 h with agitation, and then the supernatant was incubated overnight with anti-Flag antibody-conjugated agarose beads (Sigma-Aldrich) with agitation. The immunoprecipitates were collected by centrifugation for 1 min at 8,000 × g and washed four times with TBS and then subjected to 8% SDS-PAGE. All immunoprecipitation steps were done at 2°C to 8°C.

Cells (1 × 10⁵ per well) were plated in 12-well plates and allowed to attach for 16 h. The medium was then changed to DMEM/Ham's F-12 medium containing 2.5% charcoal-stripped FBS, and either vehicle (DMSO) or different concentrations of the compound were added. Fresh medium and compounds were added every 48 h, and cells were then trypsinized and counted after 24, 48, 72, and 96 h using a Coulter Z1 cell counter (Beckman Coulter, Inc.).

Cells were treated with either the vehicle (DMSO) or the compound for 48 h. Cells were trypsinized, centrifuged, and resuspended in staining solution containing 50 μg/mL propidium iodide (PI), 4 mmol/L sodium citrate, and 30 units/mL RNase. After incubation at room temperature for 1 h, cells were analyzed on a FACSVantage SE DiVa made by Becton Dickinson using FACSDiva software V4.1.1. PI fluorescence was collected through a 610SP bandpass filter, and list mode data were acquired on a minimum of 50,000 single cells defined by a dot plot of PI width versus PI area. Data analysis was done in FACSDiva software V4.1.1 using PI width versus PI area to exclude cell aggregates.

Cells on coverslip were fixed in 1% formalin in PBS (pH 7.4) after washing with PBS and permeabilized by immersing the cells in 0.2% Triton X-100 solution in PBS for 10 min. Cells were then incubated with a specific antibody followed by anti-rabbit IgG conjugated with FITC or Texas red (Santa Cruz Biotechnology). For nuclear counterstaining, cells were mounted in mounting medium including DAPI (Vector Laboratories). Fluorescent images were collected and analyzed using a Zeiss Axioplan2 fluorescence microscope (Carl Zeiss).

Cells (1 × 10⁵ per well) were plated in 12-well plates in DMEM/Ham's F-12 medium supplemented with 5% charcoal-stripped FBS. After 16 h, various amounts of DNA [i.e., p21 promoter-luciferase reporter constructs (0.1 μg) and pCMV-β-Gal reporter plasmid (0.02 μg)] were transfected using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's protocol. After transfection for 6 h, the transfection mix was replaced with complete medium containing either vehicle (DMSO) or different concentrations of the compound for 18 h. Cells were then lysed with 150 μL of 1× reporter lysis buffer, and 30 μL of cell extract were used for luciferase and β-Gal assays. A multifunctional microplate reader (FLUOstar OPTIMA) was used to quantitate luciferase and β-Gal activities, and the luciferase activities were normalized to β-Gal activity.

Cells (1.5 × 10⁵ per well) were plated in six-well plates in DMEM/Ham's F-12 medium supplemented with 5% charcoal-stripped FBS. After 16 h, the cells were transfected with 100 nmol/L of each siRNA duplex for 7 h using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's protocol. The medium was then changed to DMEM/Ham's F-12 medium containing 5% charcoal-stripped FBS and incubated for 40 h. After incubation, the cells were treated with either vehicle (DMSO) or different concentrations of the compound and cells were collected for Western blot analysis and quantitative real-time PCR assay.

Panc1 cells (1 × 10⁷) were treated with DMSO and DIM-C-pPhOCH₃ (10 μmol/L) for 1, 2, or 6 h. Cells were then fixed with 1% formaldehyde, and the cross-linking reaction was stopped by addition of 0.125 mol/L glycine. After washing twice with PBS, cells were scraped and pelleted. Collected cells were hypotonically lysed, and nuclei were collected. Nuclei were then sonicated to desired chromatin length (∼500 bp). The chromatin was precleared by addition of protein A–conjugated beads (Pierce Biotechnology) and then incubation at 4°C for 1 h with gentle agitation. The beads were pelleted, and the precleared chromatin supernatant was immunoprecipitated with antibodies to IgG, Sp1, Sp3, Sp4, and Nur77 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 extract/ethanol precipitation followed by PCR amplification. The p21 primers were 5′-GCTGGCCTGCTGGAACTC-3′ (sense) and 5′-GGCAGCTGCTCACACCTC-3′ (antisense), and they amplified a 193-bp region of the human p21 promoter, which contains several GC-rich, Sp1 binding sites. The positive control primers were 5′-TACTAGCGGTTTTACGGGCG-3′ (sense) and 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′ (antisense), and they amplified a 167-bp region of human GAPDH gene. The negative control primers were 5′-ATGGTTGCCACTGGGGATCT-3′ (sense) and 5′-TGCCAAAGCCTAGGGGAAGA-3′ (antisense) and amplified a 174-bp region of genomic DNA between human GAPDH and CNAP1 genes. PCR products were resolved on a 2% agarose gel in the presence of CYBR gold (1:10,000).

The results are expressed as mean ± SE and differences between means for two groups were determined by unpaired Student's t test. The minimum significance level was set at P value of ≤0.05 for all analysis. All experiments were done at least thrice.

S. Safe is a consultant for Plantacor.