Cancer Research Cancer Epigenetics Genetics and Biology of Brain Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qin, C.
Right arrow Articles by Safe, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qin, C.
Right arrow Articles by Safe, S.
[Cancer Research 63, 958-964, March 1, 2003]
© 2003 American Association for Cancer Research

Endocrinology

Peroxisome Proliferator-activated Receptor {gamma} Agonists Induce Proteasome-dependent Degradation of Cyclin D1 and Estrogen Receptor {alpha} in MCF-7 Breast Cancer Cells1

Chunhua Qin, Robert Burghardt, Roger Smith, Mark Wormke, Jessica Stewart and Stephen Safe2

Departments of Veterinary Physiology and Pharmacology [C. Q., M. W., J. S., S. S.], Veterinary Anatomy and Public Health [R. B.], and Veterinary Pathobiology [R. S.], Texas A&M University, College Station, Texas, and Institute of Biosciences and Technology, The Texas A&M University System Health Science Center, Houston, Texas 77843-4466 [S. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) {gamma} agonists ciglitazone or 15-deoxy-{Delta}12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) {alpha} proteins, and this was accompanied by decreased cell proliferation and G1-G0->S-phase progression. Down-regulation of cyclin D1 and ER{alpha} by PPAR{gamma} agonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-{Delta}12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ER{alpha} antibodies, there was enhanced formation of ubiquitinated cyclin D1 and ER{alpha} bands. Thus, PPAR{gamma}-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER{alpha}), and this pathway may be important for other cancer cell lines.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferators were initially characterized from a large group of synthetic industrial and pharmaceutical chemicals that induced hepatic hypertrophy and hyperplasia in rodents (1, 2, 3) . The effects of these compounds were accompanied by increased numbers and size of liver peroxisomes and induction of enzymes required for oxidative metabolism of fatty acids and members of the cytochrome P4504A (CYP4A) family. The intracellular receptor for peroxisome proliferator-induced hepatic responses was first reported in 1990 as PPAR{alpha}3 (4) , and subsequent studies in several laboratories have also characterized PPARß (or PPAR{delta}), PPAR{gamma}, and several isoforms that arise from alternative splicing and promoter use (5, 6, 7, 8, 9) . PPARs are differentially expressed in various tissues and tumors and play a critical role in fatty acid metabolism and energy homeostasis (reviewed in Refs. 1, 2, 3 ). PPARs are ligand-activated transcription factors and members of the nuclear receptor superfamily (10 , 11) . Activation of PPARs is a multistep process that involves ligand binding and heterodimerization with the retinoic X receptor, interaction with sequence-specific gene promoter elements, and recruitment of coactivators and other nuclear coregulatory proteins. PGJ2 is the most potent eicosanoid activator of PPAR{gamma} (12 , 13) ; thiazolidinediones such as ciglitazone are synthetic PPAR{gamma} agonists used extensively for their antidiabetic properties and treatment of insulin-resistant type II diabetes (14, 15, 16, 17) .

PPAR{gamma} is widely expressed in multiple tumors and cell lines, and this receptor has also become a target for developing new anticancer drugs that will take advantage of the antiproliferative effects mediated through PPAR{gamma}. For example, a recent study investigated PPAR{gamma} expression in 339 clinical tumor samples from colon, breast, lung, prostate, osteosarcomas, glioblastomas, acute myelogenous leukemia, adult T-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell non-Hodgkin’s lymphoma, and myelodisplastic syndrome (18) . Wild-type PPAR{gamma} mRNA was expressed in all tumor specimens, and receptor mutants were not detected in any of these samples. The growth-inhibitory effects of endogenous and synthetic PPAR{gamma} agonists have been investigated in several tumors and cancer cell lines (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) , and a number of these studies show that ligands for this receptor induce apoptosis and/or decrease G0-G1->S-phase cell cycle progression, which is accompanied by a decrease in cyclin D1 or modulation of cdk inhibitors and other factors involved in cell growth.

Studies in breast cancer cells show that PPAR{gamma} agonists inhibit growth of ER-positive and -negative cell lines. Treatment of ER-positive MCF-7 cells with PPAR{gamma} agonists inhibits activation of epidermal growth factor receptors through inhibition of tyrosine phosphorylation (42) and up-regulates PTEN expression in MCF-7 and other cancer cell lines (41) . PGJ2 also repressed cyclin D1 mRNA and protein in MCF-7 cells, and inhibition of transactivation was associated with enhanced recruitment of limiting cellular levels of p300 to PPAR{gamma} (39) . This study further investigates the mechanism of PPAR{gamma}-induced inhibition of cancer cell growth using MCF-7 human breast cancer cells as a model. The results show that both PGJ2 and ciglitazone (a thiazolidinedione) induce proteasome-dependent degradation of cyclin D1 and ER{alpha}, and this represents a novel pathway for PPAR{gamma}-mediated growth arrest in breast cancer cells and is consistent with their inhibition of G0-G1->S-phase progression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, Chemicals, Biochemicals, and Other Materials.
MCF-7 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in MEM with phenol red and supplemented with 0.22% sodium bicarbonate, 10% FBS, 0.011% sodium pyruvate, 0.1% glucose, 0.24% HEPES, 10-6% insulin, and 10 ml/liter antibiotic solution. Cells were grown in 150-cm2 culture plates in an air:carbon dioxide (95:5) atmosphere at 37°C and passaged every 6 days. Cells were seeded in DMEM:Ham’s F-12 with 5% FBS, and cell proliferation studies were determined using different concentrations of PGJ2 or ciglitazone; cell numbers were determined using a Coulter Z1 cell counter. DMSO, PBS, and 100x antibiotic solution were purchased from Sigma Chemical Co. (St. Louis, MO). PGJ2 (PG-050) and ciglitazone were purchased from Biomol Research Laboratories Inc. (Plymouth Meeting, PA). MG132, PSII, calpeptin, and CII were purchased from CalBiochem-Novabiochem Co. (San Diego, CA). FBS was obtained from Intergen (Purchase, NY). Horseradish peroxidase substrate for Western blot analysis was purchased from New England Nuclear Life Science Products (Boston, MA). Antibodies for cyclin D1 (sc-718 and sc-246), ER{alpha} (sc-544 and sc-8005 for Western blot and immunoprecipitation, respectively), PPAR{gamma} (sc-7196), Sp1 (sc-59 and sc-420), ubiquitin (sc-8017), cdk4 (sc-260), and preimmune IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoprecipitation antibody for cyclin D1 was obtained from Biosource International (Camarillo, CA). Restriction enzymes, T4-polynucleotide kinase, RNase ONE, and pGL2 luciferase reporter vector were purchased from Promega (Madison, WI) and Boehringer Mannheim (Indianapolis, IN). Reporter Lysis Buffer and Luciferase Reagent for luciferase studies were purchased from Promega. ß-Gal reagent was purchased from Tropix (Bedford, MA). SuperFect transfection and plasmid preparation kits were purchased from Qiagen (Santa Clarita, CA). All other chemicals and biochemicals were the highest quality available from commercial sources. Lab-Tek Chamber slides were purchased from Nalge Nunc International (Naperville, IL). InstantImage and Luminometer were purchased from Packard (Meriden, CT).

Cloning and Plasmid Preparation.
The PPRE3Luc luciferase reporter was constructed using the TpGL2 vector, which was constructed by inserting a minimal TATA sequence (GCT-GTA-GGG-TAT-ATA-ATG-GAT-CA) with linkers into the BglII and HindIII sites in the pGL2 basic vector (Promega). The triple consensus PPRE nucleotides were synthesized by Genosys/Sigma (The Woodlands, TX) as a tandem repeat inserted between the SacI and BglII sites. The Gal4 reporter (pGAL4Luc) containing five tandem GAL4 response elements was kindly provided by Dr. Marty Mayo (University of North Carolina, Chapel Hill, NC). Gal4DBD-PPAR{gamma} construct (gPPAR{gamma}) was a gift of Dr. Jennifer L. Oberfield (Glaxo Wellcome Research and Development, Research Triangle Park, NC), and PPAR{gamma} expression plasmid was a gift of Dr. Bruce M. Spiegelman (Harvard University, Boston, MA). All constructs were transformed into TOP10F' competent cells (Invitrogen, Carlsbad, CA). Plasmids were confirmed by restriction enzyme mapping and DNA sequencing. High quality plasmids for transfection were prepared using Qiagen Plasmid Megaprep Kit.

Transient Transfection and Luciferase Activity Assay.
MCF-7 cells were seeded in 5% FBS DMEM:Ham’s F-12 in 12-well plates 1 day before transfection using the calcium phosphate-DNA coprecipitation method or SuperFect transfection kit. PPRE3Luc reporter plasmid (1.1 µg), PPAR{gamma} expression plasmid (0.4 µg), and ß-Gal DNA (0.1 µg) were used; alternatively, 1.5 µg of GAL4Luc reporter plasmid, 0.05 µg of gPPAR{gamma}, and 0.1 µg of ß-Gal DNA were used for transfection. After incubation for 16 h (with calcium phosphate) or 3 h (with SuperFect), cells were washed with PBS and treated with compounds as indicated for 16–20 h in fresh media. Cells were then lysed with 200 µl of 1x Reporter Lysis Buffer; 30 µl of cell extract were used for luciferase and ß-Gal assays. LumiCount was used to quantitate luciferase and ß-Gal activities, and the luciferase activities were normalized to ß-Gal activity.

Western Blot Analysis.
MCF-7 cells were seeded in 5% charcoal-stripped FBS and DMEM:Ham’s F-12 for 24 h and then treated with the indicated compounds. WCLs were obtained using 1x Western sampling buffer. Protein samples were heated at 100°C for 5 min, separated on 10% SDS-PAGE at 160 V for 3 h in 1x running buffer [25 mM Tris-base, 192 mM glycine, and 0.1% SDS (pH 8.3)], and transferred to polyvinylidene difluoride membrane (Amersham) at 100 V for 2 h at 4°C in 1x transfer buffer [48 mM Tris-HCl, 39 mM glycine, and 0.075% SDS]. The polyvinylidene difluoride membrane was blocked in 5% milk-TBS [10 mM Tris-HCl and 150 mM NaCl (pH 8.0)] with gentle shaking for 1 h and incubated in fresh 5% milk-TBS with 1:5000 (for cyclin D1), 1:1000 (for ER{alpha}, cdk4, and PPAR{gamma}), or 1:8000 (for Sp1) primary antibody (Santa Cruz Biotechnology) for 1 h with gentle shaking. After vigorous washing in 1x TBS for 30 min, secondary antibody (1:5000) in 5% milk-TBS was incubated with shaking for 1 h. The membrane was washed vigorously in TBS buffer for 30 min, incubated in 10 ml of chemiluminescent substrate (ECL; New England Nuclear Life Science Products, Inc.) for 1.0 min, and exposed to Kodak X-OMAT AR autoradiography film immediately. Band intensities were evaluated by scanning laser densitometry (Sharp Electronics Corp.) The same membrane was stripped and then used for Western blot analysis of specific proteins in the same treatment group. The aqueous stripping solution (1 liter) was prepared with Tris-HCl (9.85 g; pH 6.8), SDS (20 g) and ß-mercaptoethanol (7.8 ml).

Coimmunoprecipitation/Western Blot.
MCF-7 cells were seeded in 5% DMEM:Ham’s F-12 in 100-mm plates for 24 h and treated as indicated. Cells were rinsed with PBS at room temperature and harvested in 0.6 ml of R1PA buffer (1x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and 100 µg/ml phenylmethylsulfonyl fluoride). Cells were then transferred to a fresh tube using a syringe with a 21-gauge needle and incubated with 10 µl of 10 mg/ml phenylmethylsulfonyl fluoride for 30–60 min on ice. Supernatant was collected as WCL after microcentrifuging at 10,000 x g for 10 min at 4°C. WCL containing 1 mg of protein was aliquoted and made to 1 ml with RIPA buffer. Each aliquot of WCL was precleared by incubation with 20 µl of protein A-agarose beads at 4°C for 30 min with shaking. Beads were pelleted by centrifuging at 1,000 x g for 5 min at 4°C, and supernatant (cell lysate) was collected. To 1 mg of the precleared WCL, 2 µg of mouse CD1 or ER{alpha} antibody or normal mouse IgG were added. The reaction mixture was incubated at 4°C for 1 h, mixed with 30 µl of resuspended protein A-agarose beads, and incubated for 10 h on a rocker platform at 4°C. The pellet was collected by centrifugation at 1,000 x g for 5 min at 4°C and washed alternately with RIPA (2x) and PBS buffer (2x). After the final wash, the pellet was resuspended in 50 µl of 1x Western sampling buffer, and proteins were separated by 10% SDS-PAGE electrophoresis. Western blot analysis was performed as described above.

Immunocytochemistry.
MCF-7 cells (5 x 104) were seeded in 5% FBS and DMEM:Ham’s F-12 on Lab-Tek Chamber slides (Nalge Nunc International) and treated with PGJ2, ciglitazone, or DMSO for various time intervals. Slides were then washed with PBS, fixed in -20°C methanol for 10 min, air dried, and washed in 0.3% Tween/PBS (use 0.3% Tween in 20 nM PBS for subsequent washing steps) for 5 min. Slides were blocked with 10% serum (the species used for secondary antibody) in 1x antibody dilution buffer (1% BSA in 0.3% Tween/PBS) for 1 h and incubated with 1:500 cyclin D1 or ER{alpha} antibody (Santa Cruz Biotechnology) overnight at 4°C in a humid chamber to prevent evaporation of the antibody solution. After washing in 0.3% Tween/PBS for 10 min (3x), slides were reprobed with secondary antibody for 2 h at room temperature in a dark humid chamber and then washed in 0.3% Tween/PBS for 10 min (4x) and rinsed in deionized water. Slides were stained with FITC fluorochrome, mounted with glycerol/phenylenediamine, and visualized. Samples without primary antibody were used as control.

Fluorescence-activated Cell-sorting Analysis.
MCF-7 cells were synchronized in serum-free media for 24 h and then treated with DMSO or different concentrations of the PPAR{gamma} agonists for 24 h. Trypsinized cells were then centrifuged and resuspended in staining solution containing 50 µg/ml PI, 4 mM sodium citrate, 30 units/ml RNase, and 0.1% Triton X-100 (pH 7.8). After incubation at 37°C for 10 min, sodium chloride was added to give a final concentration of 0.15 M, and cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA), using CellQuest (Becton Dickinson) acquisition software. 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 versus PI area. Data analysis was performed in ModFit LT (Verity Software House, Topsham, ME) using PI width versus PI area to exclude cell aggregates.

Statistical Analysis.
Statistical differences between different groups were determined by ANOVA and Scheffe’s test for significance. The data are presented as means ± SD for at least three separate determinations for each treatment.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth Inhibition and Transcription.
The growth-inhibitory effects of PPAR{gamma} agonists in MCF-7 cells were determined using PGJ2 and ciglitazone. The results showed that PGJ2 inhibited MCF-7 cell proliferation at a concentration as low as 5 µM (Fig. 1)Citation , whereas higher concentrations (30–40 µM) were required for ciglitazone (data not shown). Previous studies showed that PGJ2 decreased immunoreactive cyclin D1 mRNA and protein in MCF-7 cells, and this was accompanied by deceased phosphorylation of retinoblastoma protein and inhibition of G1 to S-phase cell cycle progression (39) . Our results also show that PGJ2 and ciglitazone inhibit G0-G1->S-phase progression in MCF-7 cells, and this was not accompanied by apoptosis (Table 1)Citation . The relative potencies of PGJ2 and ciglitazone in MCF-7 cells were also determined in transactivation studies in cells transfected with a consensus PPRE construct (pPPRE3-luc) or cotransfected with a GAL4-DNA binding domain-PPAR{gamma} fusion protein and a reporter construct containing five tandem GAL response elements linked to a luciferase reporter gene. Results of both assays gave comparable results (Fig. 1B)Citation showing that PGJ2 was a more potent ligand than ciglitazone in transactivation experiments, and this complements effects of PGJ2 and ciglitazone as inhibitors of MCF-7 cell proliferation.



View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. PGJ2 and ciglitazone activate PPAR{gamma} and inhibit growth of MCF-7 cells. A, growth inhibition. MCF-7 cells were treated with different concentrations of PGJ2 for 96 h, and the number of cells was determined as described in "Materials and Methods." All concentrations of PGJ2 significantly (P < 0.05) inhibited growth of MCF-7 cells, and results are expressed as means ± SD for three replicate determinations for each treatment. B, transactivation. MCF-7 cells were transfected with PPARE-luc or GAL4-PPAR{gamma}/pGAL45 and treated with different concentrations of PGJ2 or ciglitazone, and luciferase activity was determined as described in "Materials and Methods." Significant (P < 0.05) induction of luciferase activity is indicated with an asterisk. Results are expressed as means ± SD for three replicate determinations for each treatment group.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Effects of PPAR{gamma} agonists on cell cycle distribution of MCF-7 cellsa

 
PPAR{gamma} Agonists Induce Down-Regulation of Cyclin D1 and ER{alpha} in MCF-7 Cells.
Because PGJ2 and ciglitazone inhibit growth of MCF-7 cells and G1->S-phase progression, we also investigated the effects of both PPAR{gamma} agonists on cyclin D1 protein, which plays a critical role in progression through G1 to S-phase. The results in Fig. 2ACitation demonstrate that 30 or 80 µM PGJ2 or ciglitazone, respectively, induce a time-dependent decrease in cyclin D1 protein within 3–15 h after treatment, and similar results were also observed for ER{alpha} protein. The results in Fig. 2BCitation also illustrate a concentration-dependent decrease of both cyclin D1 and ER{alpha} in MCF-7 cells treated with ciglitazone (40–100 µM) or PGJ2 (10–40 µM) for 12 h. PGJ2 was the more potent PPAR{gamma} agonist and induced down-regulation of cyclin D1 and ER{alpha} at concentrations as low as 10 µM, and this was further investigated in a separate experiment using lower concentrations of PGJ2 (Fig. 2C)Citation . In this experiment, PGJ2 induced down-regulation of cyclin D1 and ER{alpha} at concentrations between 5 and 10 µM, whereas levels of PPAR{gamma} were unchanged at these concentrations but were slightly decreased after treatment with higher concentrations of PGJ2 (20 µM) for 24 h. In contrast, Sp1 protein was relatively unchanged during these experiments and is used as a loading control for quantitating relative protein levels.



View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. PPAR{gamma} agonists induce degradation of cyclin D1 (CD1) and ER{alpha} in MCF-7 cells. A, time course effects. MCF-7 cells were treated with 30 µM PGJ2 or 80 µM ciglitazone for 1, 3, 15, or 24 h, and WCLs were analyzed for immunoreactive cyclin D1, ER{alpha}, and Sp1 proteins by Western blot analysis as described in "Materials and Methods." B, concentration-dependent effects of PPAR{gamma} agonists. MCF-7 cells were treated with different concentrations of PGJ2 or ciglitazone, and ER{alpha}, cyclin D1, and Sp1 protein levels were determined as described in A. C, effects of low-dose PGJ2. MCF-7 cells were treated with 2.5, 5, 7.5, 10, 15, and 20 µM PGJ2 for 24 h, and immunoreactive Sp1, ER{alpha}, PPAR{gamma}, and cyclin D1 protein levels were determined as described in A.

 
Effects of Proteasome and Protease Inhibitors on Degradation of Cyclin D1 and ER{alpha} by PPAR{gamma} Agonists.
Previous reports indicate that both antiestrogens and AhR agonists that inhibit hormone-induced growth of MCF-7 cells also induce proteasome-dependent degradation of ER{alpha} (43, 44, 45) . Retinoids also inhibit growth of MCF-7 and bronchial epithelial cells (46, 47, 48) and induce down-regulation of cyclin D1 through the proteasome pathway in the latter cells. Therefore, in initial studies, we investigated effects of the proteasome inhibitor MG132 (10 µM) and the protease inhibitor CII (10 µM) on PPAR{gamma} agonist-induced down-regulation of cyclin D1 and ER{alpha} in MCF-7 cells. In cells treated with solvent control (DMSO), MG132 slightly increased cyclin D1 but not ER{alpha} protein levels, whereas CII was inactive. Cotreatment with PPAR{gamma} agonists and MG132 or CII showed that MG132 but not CII blocked PGJ2- and ciglitazone-induced down-regulation of cyclin D1 and ER{alpha} proteins, suggesting that activation of proteasomes plays an important role in these responses. We further investigated the effects of proteasome and protease inhibitors by quantitatively determining ER{alpha} and cyclin D1 protein levels after treating MCF-7 cells with 30 µM PGJ2 for 8 h These experiments were carried out in triplicate, and results are expressed as means ± SD for levels of ER{alpha} and cyclin D1 proteins in each lane. The results illustrated in Fig. 3BCitation show that PGJ2-induced down-regulation of cyclin D1 and ER{alpha} protein levels was inhibited after cotreatment with 1–10 µM MG132 and PSII, another proteasome inhibitor. Levels of Sp1, PPAR{gamma}, and cdk4 were essentially unaffected by protease or proteasome inhibitors or PGJ2. Although PGJ2 decreased PPAR{gamma} levels after treatment for 24 h (Fig. 2C)Citation , levels of the protein were unchanged after 8 h (Fig. 3B)Citation , suggesting that the temporal patterns for degradation of PPAR{gamma} (8–24 h) are different from those observed for cyclin D1 and ER{alpha}. Parallel studies show that two kinase inhibitors, CII and calpeptin, did not affect PGJ2-induced degradation of cyclin D1 or ER{alpha} at inhibitor concentrations as high as 50 µM (Fig. 3C)Citation . These data (Figs. 2Citation and 3Citation ) demonstrate that PGJ2 activation of PPAR{gamma} results in coordinate proteasome-dependent degradations of ER{alpha} and cyclin D1 within 3–8 h after treatment, whereas PPAR{gamma} was only degraded after prolonged treatment. In contrast, Sp1 and cdk4 proteins are unaffected by PGJ2 or proteasome or protease inhibitors.



View larger version (62K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Effects of proteasome and protease inhibitors on PPAR{gamma} agonist-induced degradation of cyclin D and ER{alpha}. A, effects of proteasome and protease inhibitors. MCF-7 cells were treated with DMSO, 80 µM ciglitazone, or 30 µM PGJ2 in the presence of DMSO solvent, 10 µM MG132, or 10 µM CII for 9 h, and WCLs were analyzed by Western blot analysis for cyclin D1, ER{alpha}, or Sp1 protein as described in "Materials and Methods." Results illustrated in this figure were observed in at least two separate experiments. B, effects of different concentrations of proteasome inhibitors on PGJ2-induced responses. MCF-7 cells were treated with DMSO (Lane D) alone or 30 µM PGJ2 in the presence of solvent alone (Lane D) and 1–10 µM concentrations of MG132 or PSII for 8 h, and immunoreactive Sp1, ER{alpha}, PPAR{gamma}, cyclin D1, and cdk4 levels were determined by Western blot analysis as described in "Materials and Methods." C, effects of different concentrations of protease inhibitors on PGJ2-induced responses. MCF-7 cells were treated with 30 µM PGJ2 and different concentrations (2–50 µM) of CII and calpeptin protease inhibitors, and levels of immunoreactive protein in the different treatment groups were determined as described in B. Compared with the results obtained after treatment with PGJ2 alone, all concentrations of MG132 and PSII significantly (P < 0.05) inhibited down-regulation of ER{alpha} and cyclin D1 (C), whereas the protease inhibitors did not affect down-regulation of ER{alpha} and cyclin D1 by PGJ2 (C). These experiments were carried out in triplicate, and results are expressed as means ± SD.

 
Studies illustrated in Figs. 2Citation and 3Citation were obtained by Western blot analyses of whole cell extracts, and we therefore examined the effects of PGJ2 on cyclin D1 (Fig. 4A)Citation and ER{alpha} (Fig. 4B)Citation by immunocytochemical analysis. Treatment with PGJ2 (30 µM) for 10 h decreased ER{alpha} and cyclin D1 protein staining, and this was blocked by cotreatment with MG132 but not CII. The inhibitors alone had minimal effects. These data complement results obtained for whole cell extracts and demonstrate PPAR{gamma}-induced intracellular degradation of both cyclin D1 and ER{alpha} proteins, which are primarily localized in the nucleus of MCF-7 cells.



View larger version (100K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. PGJ2-induced degradation of cyclin D1 (A) and ER{alpha} (B) proteins in the absence or presence of the proteasome inhibitor MG132 or the protease inhibitor CII. MCF-7 cells were seeded in 5% DMEM:Ham’s F-12 for 24 h and treated with 30 µM PGJ2 alone or in combination with 10 µM MG132 or 10 µM CII (protease inhibitor). Immunocytochemical analysis of cyclin D1 and ER{alpha} proteins was carried out as described in "Materials and Methods." C represents treatment with DMSO solvent.

 
PGJ2 Enhances Ubiquitination of Cyclin D1.
Proteasome-dependent degradation pathways involve multiple steps including initial ubiquitination of proteins targeted for proteolysis (49, 50, 51, 52) . We therefore investigated the effects of DMSO (solvent control) or 30 µM PGJ2 on formation of ubiquitinated cyclin D1 and ER{alpha} (Fig. 5)Citation . After treatment of MCF-7 cells for 4 h, whole cells extracts were immunoprecipitated with mouse cyclin D1 IgG2a antibodies or normal mouse IgG, and immunoprecipitated protein and input were analyzed by SDS gel electrophoresis and immunostaining with cyclin D1 or ubiquitin antibodies (Fig. 5A)Citation . Cellular protein immunoprecipitated with cyclin D1 antibodies gave a 3–4-fold increase in ubiquitinated bands after treatment with PGJ2, and lower intensity ubiquitinated bands were observed in the solvent (DMSO) control. Western blot analysis of the input band with cyclin D1 antibodies also shows decreased intensity of the cyclin D1 band after treatment with PGJ2. Analysis for ubiquitinated ER{alpha} was also determined using WCLs from cells treated with PGJ2 for 2 or 4 h. Western blot analysis of the input shows the ER{alpha} band that had not significantly decreased (Fig. 5B)Citation ; however, using ubiquitinated antibodies, there was approximately a 2-fold increase in intensity of the ubiquitinated bands in the 4-h treatment group. The increased ubiquitination of cyclin D1 and ER{alpha} was observed in replicate studies. Immunoprecipitation of WCLs with mouse IgG did not pull down ER{alpha}, cyclin D1, or their corresponding ubiquitinated bands, and in a separate study, we also showed that retinoic acid induced a similar pattern of ubiquitinated cyclin D1 bands (data not shown). A complex ladder of ubiquitinated bands was observed in whole cell extracts (input), and IgG heavy chain and nonspecific bands (obtained with beads alone) were observed as control bands in both immunoprecipitated extracts. These data suggest that PPAR{gamma}-induced degradation of cyclin D1 and ER{alpha} is accompanied by enhanced ubiquitination, which is consistent with activation of the proteasome pathway.



View larger version (66K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Ubiquitination of cyclin D1 and ER{alpha} after treatment with DMSO (C) and PGJ2 (PG) in MCF-7 cells. A, cyclin D1. Cells were treated with DMSO or 30 µM PGJ2 for 4 h; WCLs were immunoprecipitated with cyclin D1 antibodies and analyzed by immunoblot analysis with cyclin D1 (left panel) or ubiquitin (right panel) antibodies as described in "Materials and Methods." B, ER{alpha}. Cells were untreated (control, 0 h) or treated with 30 µM PGJ2 for 2 or 4 h; WCLs were immunoprecipitated with ER{alpha} antibodies and analyzed by immunoblot analysis with ER{alpha} (left panel) or ubiquitin antibodies (right panel) as described in "Materials and Methods." Input (1/20) was also used for the immunoblot experiments to show cyclin D1, ER{alpha}, and ubiquitin staining bands in the WCL. IgG heavy chain and nonspecific bands (observed with beads alone) are also indicated, and normal mouse IgG did not pull down ER{alpha}, cyclin D1, or their ubiquitinated complexes (data not shown).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PGJ2 and other PPAR{gamma} agonists inhibit growth of breast and other cancer cell lines, and in most cancer cells, these effects are linked to apoptosis or inhibition of G1->S-phase progression. Our results (Fig. 1)Citation confirm the growth-inhibitory effects of both PGJ2 and ciglitazone in MCF-7 cells, and this is accompanied by decreased G1->S-phase cell cycle progression (Table 1)Citation . Wang et al. (39) previously reported that PGJ2 also decreased cyclin D1 protein and mRNA levels in MCF-7 cells, and their studies indicated that transcriptional inhibition was due to competition between PPAR{gamma} and c-fos (bound to the cyclin D1 promoter) for limiting cellular levels of p300, an important coregulatory protein. In this study, we also observed that PGJ2 and ciglitazone decreased cyclin D1 protein in MCF-7 cells (Fig. 2)Citation , and this response was observed at concentrations of PGJ2 as low as 10 µM. Moreover, we also observed a similar pattern of ER{alpha} degradation induced by both PPAR{gamma} agonists, and 5–10 µM PGJ2 significantly decreased levels of ER{alpha} protein (Fig. 2C)Citation , and these responses were observed as early as 3 h after treatment. Thus, pharmacological concentrations (>=5 µM) of PGJ2 were required for these studies. We also observed some degradation of PPAR{gamma} at longer time points (Fig. 2C)Citation , whereas Sp1 protein is relatively unchanged by PPAR{gamma} agonists, as reported previously in other studies using AhR agonists and antiestrogens that also induce degradation of ER{alpha} (43 , 44) .

Cyclin D1 plays a critical role in G0/G1->S-phase cell cycle progression, and, not surprisingly, there are multiple cell-specific transcriptional and posttranscriptional mechanisms for regulation of cyclin D1 after mitogenic stimuli or after treatment with growth inhibitors (39 , 48 , 53 , 54) . As noted above, PPAR{gamma} agonists repress transcription of cyclin D1 by sequestering p300 (39) , and cyclin D1 transcription is also down-regulated in MCF-7 cells by flavopiridol, an inhibitor of cell cycle progression (53) . Serum starvation of NIH3T3 cells results in Ca2+-dependent calpain protease-induced degradation of cyclin D1, whereas in the same cells, cyclin B1 is degraded through the proteasome pathway (54) . In other cell lines, cyclin D1 is degraded by proteasomes, and retinoic acid induces proteasome-dependent degradation of cyclin D1 in immortalized human bronchial BEAS-2B epithelial cells (48) . Coordinate down-regulation of both cyclin D1 and ER{alpha} by PPAR{gamma} or other growth-inhibitory agents has not been reported previously; however, estrogens, some antiestrogens, and AhR agonists induce rapid proteasome-dependent degradation of ER{alpha} in breast cancer cells (43, 44, 45) . Therefore, based on the observation that both ciglitazone and PGJ2 induced coordinate degradation of ER{alpha} and cyclin D1 in MCF-7 cells (Fig. 2)Citation , we further investigated these responses in the presence of proteasome (MG132 and PSII) and protease (CII and calpeptin) inhibitors (Fig. 3)Citation . The results clearly demonstrate that over a range of concentrations, proteasome inhibitors block PPAR{gamma}-induced down-regulation of both cyclin D1 and ER{alpha}, whereas protease inhibitors did not block this response. Moreover, the responses determined by Western blot analysis of whole cell extracts were also observed directly by immunocytochemical analysis of cyclin D1 and ER{alpha} proteins after treatment with PGJ2 alone or in combination with MG132 or CII (Fig. 4)Citation . The proteasome inhibitor MG132 or the protease inhibitor CII has minimal effects on levels of cyclin D1 protein in MCF-7 cells; however, MG132 (but not CII) inhibited PGJ2-induced degradation of both proteins, which are primarily located in the nucleus of MCF-7 cells.

Because proteasome-dependent degradation of proteins involves prior conjugation of targeted proteins by ubiquitin or ubiquitin-like molecules, we also investigated formation of ER{alpha}- and cyclin D1-ubiquitin complexes in MCF-7 cells treated with PGJ2 (Fig. 5)Citation . Retinoids also inhibit the growth of breast cancer cells and down-regulate ER{alpha} (46, 47, 48) and cyclin D1 protein (55) in MCF-7 cells. Moreover, retinoic acid induced proteasome-dependent degradation of cyclin D1 in immortalized bronchial epithelial cells (48) , and these responses are similar to those induced by PGJ2 in this study. Results illustrated in Fig. 5Citation identify ubiquitinated cyclin D1 and ER{alpha} bands that are enhanced after treatment of MCF-7 cells with PGJ2, and this was consistent with PPAR{gamma}-mediated proteasome-dependent degradation of cyclin D1 and ER{alpha} in MCF-7 cells. Retinoic acid also induced a pattern of cyclin D1-ubiquitinated bands similar to that observed for PGJ2 (data not shown).

In summary, our studies demonstrate that PPAR{gamma} agonists that inhibit growth and cell cycle progression (G0-G1->S-phase) in MCF-7 cells also induce proteasome-dependent degradation of both cyclin D1 and ER{alpha}. This mechanism complements results of previous studies showing that PPAR{gamma} also inhibits cyclin D1 transcription (39) , and the combination of both transcriptional and posttranscriptional inhibition of cyclin D1 by PPAR{gamma} agonists may contribute to the efficacy of these compounds as inhibitors of breast cancer cell proliferation. Moreover, in ongoing studies, our results indicate that PPAR{gamma} agonists inhibit growth and induce proteasome-dependent degradation of cyclin D1 in multiple cancer cell lines, and this may represent an important PPAR{gamma}-induced tumor growth-inhibitory pathway.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant ES09106 and the Texas Agricultural Experiment Station. Back

2 To whom requests for reprints should be addressed, at Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 Texas A&M University, Veterinary Research Building 409, College Station, Texas 77843-4466. Phone: (979) 845-5988; Fax: (979) 862-4929; E-mail: ssafe{at}cvm.tamu.edu Back

3 The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PGJ2, 15-deoxy-{Delta}12,14-prostaglandin J2; ER, estrogen receptor; cdk, cyclin-dependent kinase; FBS, fetal bovine serum; ß-Gal, ß-galactosidase; TBS, Tris-buffered saline; RIPA, radioimmunoprecipitation assay; WCL, whole cell lysate; PI, propidium iodide; AhR, aryl hydrocarbon receptor; CII, calpain II. Back

Received 5/14/02. Accepted 12/27/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Reddy J. K., Azarnoff D. L., Hignite C. E. Hypolipidaemic hepatic peroxisome proliferators form a novel class of chemical carcinogens. Nature (Lond.), 283: 397-398, 1980.[Medline]
  2. Lock E. A., Mitchell A. M., Elcombe C. R. Biochemical mechanisms of induction of hepatic peroxisome proliferation. Annu. Rev. Pharmacol. Toxicol., 29: 145-163, 1989.[Medline]
  3. Corton J. C., Anderson S. P., Stauber A. Central role of peroxisome proliferator-activated receptors in the actions of peroxisome proliferators. Annu. Rev. Pharmacol. Toxicol., 40: 491-518, 2000.[Medline]
  4. Issemann I., Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature (Lond.), 347: 645-650, 1990.[Medline]
  5. Schmidt A., Endo N., Rutledge S. J., Vogel R., Shinar D., Rodan G. A. Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acids. Mol. Endocrinol., 6: 1634-1641, 1992.[Abstract/Free Full Text]
  6. Sher T., Yi H. F., McBride O. W., Gonzalez F. J. cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor. Biochemistry, 32: 5598-5604, 1993.[Medline]
  7. Kliewer S. A., Forman B. M., Blumberg B., Ong E. S., Borgmeyer U., Mangelsdorf D. J., Umesono K., Evans R. M. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc. Natl. Acad. Sci. USA, 91: 7355-7359, 1994.[Abstract/Free Full Text]
  8. Zhu Y., Qi C., Korenberg J. R., Chen X. N., Noya D., Rao M. S., Reddy J. K. Structural organization of mouse peroxisome proliferator-activated receptor {gamma} (mPPAR {gamma}) gene: alternative promoter use and different splicing yield two mPPAR {gamma} isoforms. Proc. Natl. Acad. Sci. USA, 92: 7921-7925, 1995.[Abstract/Free Full Text]
  9. Yoshikawa T., Brkanac Z., Dupont B. R., Xing G. Q., Leach R. J., Detera-Wadleigh S. D. Assignment of the human nuclear hormone receptor, NUC1 (PPAR{Delta}), to chromosome 6p21.1-p21.2. Genomics, 35: 637-638, 1996.[Medline]
  10. Mangelsdorf D. J., Thummel C., Beato M., Herrlich P., Schutz G., Umesono K., Blumberg B., Kastner P., Mark M., Chambon P., Evans R. M. The nuclear receptor superfamily: the second decade. Cell, 83: 835-839, 1995.[Medline]
  11. Perlmann T., Evans R. M. Nuclear receptors in Sicily: all in the famiglia. Cell, 90: 391-397, 1997.[Medline]
  12. Forman B. M., Tontonoz P., Chen J., Brun R. P., Spiegelman B. M., Evans R. M. 15-Deoxy-{Delta}12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR {gamma}. Cell, 83: 803-812, 1995.[Medline]
  13. Kliewer S. A., Lenhard J. M., Willson T. M., Patel I., Morris D. C., Lehmann J. M. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor {gamma} and promotes adipocyte differentiation. Cell, 83: 813-819, 1995.[Medline]
  14. Murphy G. J., Holder J. C. PPAR-{gamma} agonists: therapeutic role in diabetes, inflammation and cancer. Trends Pharmacol. Sci, 21: 469-474, 2000.[Medline]
  15. Fajas L., Debril M. B., Auwerx J. Peroxisome proliferator-activated receptor-{gamma}: from adipogenesis to carcinogenesis. J. Mol. Endocrinol., 27: 1-9, 2001.[Abstract]
  16. Kersten S., Desvergne B., Wahli W. Roles of PPARs in health and disease. Nature (Lond.), 405: 421-424, 2000.[Medline]
  17. Willson R. M., Lambert M. H., Kliewer S. A. Peroxisome proliferator-activated receptor {gamma} and metabolic disease. Annu. Rev. Biochem., 70: 341-367, 2001.[Medline]
  18. Ikezoe T., Miller C. W., Kawano S., Heaney A., Williamson E. A., Hisatake J., Green E., Hofmann W., Taguchi H., Koeffler H. P. Mutational analysis of the peroxisome proliferator-activated receptor {gamma} gene in human malignancies. Cancer Res., 61: 5307-5310, 2001.[Abstract/Free Full Text]
  19. Gupta R. A., Brockman J. A., Sarraf P., Willson T. M., DuBois R. N. Target genes of peroxisome proliferator-activated receptor {gamma} in colorectal cancer cells. J. Biol. Chem., 276: 29681-29687, 2001.[Abstract/Free Full Text]
  20. Itami A., Watanabe G., Shimada Y., Hashimoto Y., Kawamura J., Kato M., Hosotani R., Imamura M. Ligands for peroxisome proliferator-activated receptor {gamma} inhibit growth of pancreatic cancers both in vitro and in vivo. Int. J. Cancer, 94: 370-376, 2001.[Medline]
  21. Motomura W., Okumura T., Takahashi N., Obara T., Kohgo Y. 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, 2000.[Abstract/Free Full Text]
  22. Wächtershäuser A., Loitsch S. M., Stein J. PPAR-{gamma} is selectively upregulated in Caco-2 cells by butyrate. Biochem. Biophys. Res. Commun., 272: 380-385, 2000.[Medline]
  23. Sato H., Ishihara S., Kawashima K., Moriyama N., Suetsugu H., Kazumori H., Okuyama T., Rumi M. A., Fukuda R., Nagasue N., Kinoshita Y. Expression of peroxisome proliferator-activated receptor (PPAR){gamma} in gastric cancer and inhibitory effects of PPAR{gamma} agonists. Br. J. Cancer, 83: 1394-1400, 2000.[Medline]
  24. Takahashi N., Okumura T., Motomura W., Fujimoto Y., Kawabata I., Kohgo Y. Activation of PPAR{gamma} inhibits cell growth and induces apoptosis in human gastric cancer cells. FEBS Lett., 455: 135-139, 1999.[Medline]
  25. Inoue K., Kawahito Y., Tsubouchi Y., Kohno M., Yoshimura R., Yoshikawa T., Sano H. Expression of peroxisome proliferator-activated receptor {gamma} in renal cell carcinoma and growth inhibition by its agonists. Biochem. Biophys. Res. Commun., 287: 727-732, 2001.[Medline]
  26. Kitamura S., Miyazaki Y., Hiraoka S., Nagasawa Y., Toyota M., Takakura R., Kiyohara T., Shinomura Y., Matsuzawa Y. 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, 2001.[Medline]
  27. Kubota T., Koshizuka K., Williamson E. A., Asou H., Said J. W., Holden S., Miyoshi I., Koeffler H. P. 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, 1998.[Abstract/Free Full Text]
  28. Elnemr A., Ohta T., Iwata K., Ninomia I., Fushida S., Nishimura G., Kitagawa H., Kayahara M., Yamamoto M., Terada T., Miwa K. PPAR{gamma} ligand (thiazolidinedione) induces growth arrest and differentiation markers of human pancreatic cancer cells. Int. J. Oncol., 17: 1157-1164, 2000.[Medline]
  29. Kitamura S., Miyazaki Y., Shinomura Y., Kondo S., Kanayama S., Matsuzawa Y. Peroxisome proliferator-activated receptor {gamma} induces growth arrest and differentiation markers of human colon cancer cells. Jpn. J. Cancer Res., 90: 75-80, 1999.[Medline]
  30. Elstner E., Muller C., Koshizuka K., Williamson E. A., Park D., Asou H., Shintaku P., Said J. W., Heber D., Koeffler H. P. Ligands for peroxisome proliferator-activated receptor {gamma} and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc. Natl. Acad. Sci. USA, 95: 8806-8811, 1998.[Abstract/Free Full Text]
  31. Suh N., Wang Y., Honda T., Gribble G. W., Dmitrovsky E., Hickey W. F., Maue R. A., Place A. E., Porter D. M., Spinella M. J., Williams C. R., Wu G., Dannenberg A. J., Flanders K. C., Letterio J. J., Mangelsdorf D. J., Nathan C. F., Nguyen L., Porter W. W., Ren R. F., Roberts A. B., Roche N. S., Subbaramaiah K., Sporn M. B. A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative, and anti-inflammatory activity. Cancer Res., 59: 336-341, 1999.[Abstract/Free Full Text]
  32. Brockman J. A., Gupta R. A., DuBois R. N. Activation of PPAR{gamma} leads to inhibition of anchorage independent growth of human colorectal cancer cells. Gastroenterology, 115: 1049-1055, 1998.[Medline]
  33. Chang T. H., Szabo E. Induction of differentiation and apoptosis by ligands of peroxisome proliferator-activated receptor {gamma} in non-small cell lung cancer. Cancer Res., 60: 1129-1138, 2000.[Abstract/Free Full Text]
  34. Demetri G. D., Fletcher C. D., Mueller E., Sarraf P., Naujoks R., Campbell N., Spiegelman B. M., Singer S. Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-{gamma} ligand troglitazone in patients with liposarcoma. Proc. Natl. Acad. Sci. USA, 96: 3951-3956, 1999.[Abstract/Free Full Text]
  35. Suh N., Wang Y., Williams C. R., Risingsong R., Gilmer T., Willson T. M., Sport M. B. A new ligand for the peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}), GW7845, inhibits rat mammary carcinogenesis. Cancer Res., 59: 5671-5673, 1999.[Abstract/Free Full Text]
  36. Lefebvre A., Chen I., Desreumaux P., Najib J., Fruchart J., Geboes K., Briggs M., Heyman R., Auwerx J. Activation of the peroxisome proliferator-activated receptor {gamma} promotes the development of colon tumors in C57BL/6J-APCMin/+ mice. Nat. Med., 4: 1053-1057, 1998.[Medline]
  37. Sarraf P., Mueller E., Jones D., King F. J., DeAngelo D. J., Partridge J. B., Holden S. A., Chen L. B., Singer S., Fletcher C., Spiegelman B. M. Differentiation and reversal of malignant changes in colon cancer through PPAR{gamma}. Nat. Med., 4: 1046-1052, 1998.[Medline]
  38. Clay C. E., Namen A. M., Atsumi G., Willingham M. C., High K. P., Kute T. E., Trimboli A. J., Fonteh A. N., Dawson P. A., Chilton F. H. Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells. Carcinogenesis (Lond.), 20: 1905-1911, 1999.[Abstract/Free Full Text]
  39. Wang C. G., Fu M. F., D’Amico M., Albanese C., Zhou J. N., Brownlee M., Lisanti M. P., Chatterjee V. K. K., Lazar M. A., Pestell R. G. Inhibition of cellular proliferation through I{kappa}B kinase-independent and peroxisome proliferator-activated receptor {gamma}-dependent repression of cyclin D1. Mol. Cell. Biol., 21: 3057-3070, 2001.[Abstract/Free Full Text]
  40. Ito Y., Pandey P., Sporn M. B., Datta R., Kharbanda S., Kufe D. The novel triterpenoid CDDO induces apoptosis and differentiation of human osteosarcoma cells by a caspase-8 dependent mechanism. Mol. Pharmacol., 59: 1094-1099, 2001.[Abstract/Free Full Text]
  41. Patel L., Pass I., Coxon P., Downes C. P., Smith S. A., Macphee C. H. Tumor suppressor and anti-inflammatory actions of PPAR{gamma} agonists are mediated via upregulation of PTEN. Curr. Biol., 11: 764-768, 2001.[Medline]
  42. Pignatelli M., Cortes-Canteli M., Lai C., Santos A., Perez-Castillo A. The peroxisome proliferator-activated receptor {gamma} is an inhibitor of ErbBs activity in human breast cancer cells. J. Cell Sci., 114: 4117-4126, 2001.
  43. Wormke M., Stoner M., Saville B., Safe S. Crosstalk between estrogen receptor {alpha} and the aryl hydrocarbon receptor in breast cancer cells involves unidirectional activation of proteosomes. FEBS Lett., 478: 109-112, 2000.[Medline]
  44. McDougal A., Wormke M., Calvin J., Safe S. Tamoxifen-induced antitumorigenic/antiestrogenic action synergized by a selective Ah receptor modulator. Cancer Res., 61: 3901-3907, 2001.
  45. Wijayaratne A. L., McDonnell D. P. The human estrogen receptor-{alpha} is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators. J. Biol. Chem., 276: 35684-35692, 2001.[Abstract/Free Full Text]
  46. Rubin M., Fenig E., Rosenauer A., Menendez-Botet C., Achkar C., Bentel J. M., Yahalom J., Mendelsohn J., Miller W. H., Jr. 9-cis Retinoic acid inhibits growth of breast cancer cells and down-regulates estrogen receptor RNA and protein. Cancer Res., 54: 6549-6556, 1994.[Abstract/Free Full Text]
  47. Fitzgerald P., Teng M., Chandraratna R. A., Heyman R. A., Allegretto E. A. Retinoic acid receptor {alpha} expression correlates with retinoid-induced growth inhibition of human breast cancer cells regardless of estrogen receptor status. Cancer Res., 57: 2642-2650, 1997.[Abstract/Free Full Text]
  48. Langenfeld J., Kiyokawa H., Sekula D., Boyle J., Dmitrovsky E. Posttranslational regulation of cyclin D1 by retinoic acid: a chemoprevention mechanism. Proc. Natl. Acad. Sci. USA, 94: 12070-12074, 1997.[Abstract/Free Full Text]
  49. Pickart C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem., 70: 503-533, 2001.[Medline]
  50. Baumeister W., Walz J., Zuhl F., Seemuller E. The proteasome: paradigm of a self-compartmentalizing protease. Cell, 92: 367-380, 1998.[Medline]
  51. DeMartino G. N., Slaughter C. A. The proteasome, a novel protease regulated by multiple mechanisms. J. Biol. Chem., 274: 22123-22126, 1999.[Free Full Text]
  52. Kornitzer D., Ciechanover A. Modes of regulation of ubiquitin-mediated protein degradation. J. Cell. Physiol., 182: 1-11, 2000.[Medline]
  53. Carlson B., Lahusen T., Singh S., Loaiza-Perez A., Worland P. J., Pestell R., Albanese C., Sausville E. A., Senderowicz A. M. Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol. Cancer Res., 59: 4634-4641, 1999.[Abstract/Free Full Text]
  54. Choi Y. H., Lee S. J., Nguyen P., Jang J. S., Lee J., Wu M. L., Takano E., Maki M., Henkart P. A., Trepel J. B. Regulation of cyclin D1 by calpain protease. J. Biol. Chem., 272: 28479-28484, 1997.[Abstract/Free Full Text]
  55. Teixeira C., Pratt M. A. CDK2 is a target for retinoic acid-mediated growth inhibition in MCF-7 human breast cancer cells. Mol. Endocrinol., 11: 1191-1202, 1997.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
Y. Yin, H. Yuan, X. Zeng, L. Kopelovich, and R. I. Glazer
Inhibition of Peroxisome Proliferator-Activated Receptor {gamma} Increases Estrogen Receptor-Dependent Tumor Specification
Cancer Res., January 15, 2009; 69(2): 687 - 694.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Wei, H.-C. Yang, H.-C. Chuang, J. Yang, S. K. Kulp, P.-J. Lu, M.-D. Lai, and C.-S. Chen
A Novel Mechanism by Which Thiazolidinediones Facilitate the Proteasomal Degradation of Cyclin D1 in Cancer Cells
J. Biol. Chem., September 26, 2008; 283(39): 26759 - 26770.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. U. Mertens-Talcott, S. Chintharlapalli, X. Li, and S. Safe
The Oncogenic microRNA-27a Targets Genes That Regulate Specificity Protein Transcription Factors and the G2-M Checkpoint in MDA-MB-231 Breast Cancer Cells
Cancer Res., November 15, 2007; 67(22): 11001 - 11011.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. York, M. Abdelrahim, S. Chintharlapalli, S. D. Lucero, and S. Safe
1,1-Bis(3'-Indolyl)-1-(p-Substitutedphenyl)methanes Induce Apoptosis and Inhibit Renal Cell Carcinoma Growth
Clin. Cancer Res., November 15, 2007; 13(22): 6743 - 6752.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. Wei, L.-F. Lin, C.-C. Yang, Y.-C. Wang, G.-D. Chang, H. Chen, and C.-S. Chen
Thiazolidinediones Modulate the Expression of beta-Catenin and Other Cell-Cycle Regulatory Proteins by Targeting the F-Box Proteins of Skp1-Cul1-F-box Protein E3 Ubiquitin Ligase Independently of Peroxisome Proliferator-Activated Receptor {gamma}
Mol. Pharmacol., September 1, 2007; 72(3): 725 - 733.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
F.-S. Chou, P.-S. Wang, S. Kulp, and J. J. Pinzone
Effects of Thiazolidinediones on Differentiation, Proliferation, and Apoptosis
Mol. Cancer Res., June 1, 2007; 5(6): 523 - 530.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H.-J. Kim, J.-Y. Kim, Z. Meng, L. H. Wang, F. Liu, T. P. Conrads, T. R. Burke, T. D. Veenstra, and W. L. Farrar
15-Deoxy-{Delta}12,14-Prostaglandin J2 Inhibits Transcriptional Activity of Estrogen Receptor-{alpha} via Covalent Modification of DNA-Binding Domain
Cancer Res., March 15, 2007; 67(6): 2595 - 2602.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. Chintharlapalli, S. Papineni, and S. Safe
1,1-Bis(3'-Indolyl)-1-(p-substitutedphenyl)methanes Inhibit Growth, Induce Apoptosis, and Decrease the Androgen Receptor in LNCaP Prostate Cancer Cells through Peroxisome Proliferator-Activated Receptor {gamma}-Independent Pathways
Mol. Pharmacol., February 1, 2007; 71(2): 558 - 569.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
A. Ciucci, P. Gianferretti, R. Piva, T. Guyot, T. J. Snape, S. M. Roberts, and M. G. Santoro
Induction of Apoptosis in Estrogen Receptor-Negative Breast Cancer Cells by Natural and Synthetic Cyclopentenones: Role of the I{kappa}B Kinase/Nuclear Factor-{kappa}B Pathway
Mol. Pharmacol., November 1, 2006; 70(5): 1812 - 1821.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. Aiello, G. Pandini, F. Frasca, E. Conte, A. Murabito, A. Sacco, M. Genua, R. Vigneri, and A. Belfiore
Peroxisomal Proliferator-Activated Receptor-{gamma} Agonists Induce Partial Reversion of Epithelial-Mesenchymal Transition in Anaplastic Thyroid Cancer Cells
Endocrinology, September 1, 2006; 147(9): 4463 - 4475.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
P. Lei, M. Abdelrahim, and S. Safe
1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit ovarian cancer cell growth through peroxisome proliferator-activated receptor-dependent and independent pathways.
Mol. Cancer Ther., September 1, 2006; 5(9): 2324 - 2336.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
L. Tosca, P. Solnais, P. Ferre, F. Foufelle, and J. Dupont
Metformin-Induced Stimulation of Adenosine 5' Monophosphate-Activated Protein Kinase (PRKA) Impairs Progesterone Secretion in Rat Granulosa Cells
Biol Reprod, September 1, 2006; 75(3): 342 - 351.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
J-R Weng, C-Y Chen, J J Pinzone, M D Ringel, and C-S Chen
Beyond peroxisome proliferator-activated receptor {gamma} signaling: the multi-facets of the antitumor effect of thiazolidinediones.
Endocr. Relat. Cancer, June 1, 2006; 13(2): 401 - 413.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
S. Chintharlapalli, S. Papineni, and S. Safe
1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell and tumor growth through PPAR{gamma}-dependent and PPAR{gamma}-independent pathways
Mol. Cancer Ther., May 1, 2006; 5(5): 1362 - 1370.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Katzenellenbogen, O. Pappo, H. Barash, N. Klopstock, L. Mizrahi, D. Olam, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, L. A. Mitchell, et al.
Multiple adaptive mechanisms to chronic liver disease revealed at early stages of liver carcinogenesis in the mdr2-knockout mice.
Cancer Res., April 15, 2006; 66(8): 4001 - 4010.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
C. M. Banwell, D. P. MacCartney, M. Guy, A. E. Miles, M. R. Uskokovic, J. Mansi, P. M. Stewart, L. P. O'Neill, B. M. Turner, K. W. Colston, et al.
Altered nuclear receptor corepressor expression attenuates vitamin d receptor signaling in breast cancer cells.
Clin. Cancer Res., April 1, 2006; 12(7): 2004 - 2013.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
T Suzuki, S Hayashi, Y Miki, Y Nakamura, T Moriya, A Sugawara, T Ishida, N Ohuchi, and H Sasano
Peroxisome proliferator-activated receptor {gamma} in human breast carcinoma: a modulator of estrogenic actions.
Endocr. Relat. Cancer, March 1, 2006; 13(1): 233 - 250.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
M. Konopleva, W. Zhang, Y.-X. Shi, T. McQueen, T. Tsao, M. Abdelrahim, M. F. Munsell, M. Johansen, D. Yu, T. Madden, et al.
Synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest in HER2-overexpressing breast cancer cells.
Mol. Cancer Ther., February 1, 2006; 5(2): 317 - 328.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
W. Kassouf, S. Chintharlapalli, M. Abdelrahim, G. Nelkin, S. Safe, and A. M. Kamat
Inhibition of Bladder Tumor Growth by 1,1-Bis(3'-Indolyl)-1-(p-Substitutedphenyl)Methanes: A New Class of Peroxisome Proliferator-Activated Receptor {gamma} Agonists
Cancer Res., January 1, 2006; 66(1): 412 - 418.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. Chintharlapalli, S. Papineni, S. J. Baek, S. Liu, and S. Safe
1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes Are Peroxisome Proliferator-Activated Receptor {gamma} Agonists but Decrease HCT-116 Colon Cancer Cell Survival through Receptor-Independent Activation of Early Growth Response-1 and Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1
Mol. Pharmacol., December 1, 2005; 68(6): 1782 - 1792.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
C.J. Loy, S. Evelyn, F.K. Lim, M.H. Liu, and E.L. Yong
Growth dynamics of human leiomyoma cells and inhibitory effects of the peroxisome proliferator-activated receptor-{gamma} ligand, pioglitazone
Mol. Hum. Reprod., August 1, 2005; 11(8): 561 - 566.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. Chintharlapalli, S. Papineni, M. Konopleva, M. Andreef, I. Samudio, and S. Safe
2-Cyano-3,12-dioxoolean-1,9-dien-28-oic Acid and Related Compounds Inhibit Growth of Colon Cancer Cells through Peroxisome Proliferator-Activated Receptor {gamma}-Dependent and -Independent Pathways
Mol. Pharmacol., July 1, 2005; 68(1): 119 - 128.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
J.-W. Huang, C.-W. Shiau, Y.-T. Yang, S. K. Kulp, K.-F. Chen, R. W. Brueggemeier, C. L. Shapiro, and C.-S. Chen
Peroxisome Proliferator-Activated Receptor {gamma}-Independent Ablation of Cyclin D1 by Thiazolidinediones and Their Derivatives in Breast Cancer Cells
Mol. Pharmacol., April 1, 2005; 67(4): 1342 - 1348.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Fu, C. Wang, Z. Li, T. Sakamaki, and R. G. Pestell
Minireview: Cyclin D1: Normal and Abnormal Functions
Endocrinology, December 1, 2004; 145(12): 5439 - 5447.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. Hong, I. Samudio, S. Liu, M. Abdelrahim, and S. Safe
Peroxisome Proliferator-Activated Receptor {gamma}-Dependent Activation of p21 in Panc-28 Pancreatic Cancer Cells Involves Sp1 and Sp4 Proteins
Endocrinology, December 1, 2004; 145(12): 5774 - 5785.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
X. Zhang, M. C. Rodriguez-Galan, J. J. Subleski, J. R. Ortaldo, D. L. Hodge, J.-M. Wang, O. Shimozato, D. A. Reynolds, and H. A. Young
Peroxisome proliferator-activated receptor-{gamma} and its ligands attenuate biologic functions of human natural killer cells
Blood, November 15, 2004; 104(10): 3276 - 3284.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Chintharlapalli, R. Smith III, I. Samudio, W. Zhang, and S. Safe
1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes Induce Peroxisome Proliferator-Activated Receptor {gamma}-Mediated Growth Inhibition, Transactivation, and Differentiation Markers in Colon Cancer Cells
Cancer Res., September 1, 2004; 64(17): 5994 - 6001.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. J. Schlezinger, G. J. Howard, C. H. Hurst, J. K. Emberley, D. J. Waxman, T. Webster, and D. H. Sherr
Environmental and Endogenous Peroxisome Proliferator-Activated Receptor {gamma} Agonists Induce Bone Marrow B Cell Growth Arrest and Apoptosis: Interactions between Mono(2-ethylhexyl)phthalate, 9-cis-Retinoic Acid, and 15-Deoxy-{Delta}12,14-prostaglandin J2
J. Immunol., September 1, 2004; 173(5): 3165 - 3177.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
C. J. Nicol, M. Yoon, J. M. Ward, M. Yamashita, K. Fukamachi, J. M. Peters, and F. J. Gonzalez
PPAR{gamma} influences susceptibility to DMBA-induced mammary, ovarian and skin carcinogenesis
Carcinogenesis, September 1, 2004; 25(9): 1747 - 1755.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Sharma, A. Pradeep, L. Wong, A. Rana, and B. Rana
Peroxisome Proliferator-activated Receptor {gamma} Activation Can Regulate {beta}-Catenin Levels via a Proteasome-mediated and Adenomatous Polyposis Coli-independent Pathway
J. Biol. Chem., August 20, 2004; 279(34): 35583 - 35594.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
Z. Yang, C. J. Barnes, and R. Kumar
Human Epidermal Growth Factor Receptor 2 Status Modulates Subcellular Localization of and Interaction with Estrogen Receptor {alpha} in Breast Cancer Cells
Clin. Cancer Res., June 1, 2004; 10(11): 3621 - 3628.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. J. Kim, T. E. Akiyama, F. S. Harman, A. M. Burns, W. Shan, J. M. Ward, M. J. Kennett, F. J. Gonzalez, and J. M. Peters
Peroxisome Proliferator-activated Receptor {beta} ({delta})-dependent Regulation of Ubiquitin C Expression Contributes to Attenuation of Skin Carcinogenesis
J. Biol. Chem., May 28, 2004; 279(22): 23719 - 23727.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Sharma, A. Pradeep, R. G. Pestell, and B. Rana
Peroxisome Proliferator-activated Receptor {gamma} Activation Modulates Cyclin D1 Transcription via {beta}-Catenin-independent and cAMP-response Element-binding Protein-dependent Pathways in Mouse Hepatocytes
J. Biol. Chem., April 23, 2004; 279(17): 16927 - 16938.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
C. Qin, D. Morrow, J. Stewart, K. Spencer, W. Porter, R. Smith III, T. Phillips, M. Abdelrahim, I. Samudio, and S. Safe
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-substituted phenyl)methanes
Mol. Cancer Ther., March 1, 2004; 3(3): 247 - 260.
[Abstract] [Full Text]

Home page
 J. Nutr.Home page
P. Tanmahasamut, J. Liu, L. B. Hendry, and N. Sidell
Conjugated Linoleic Acid Blocks Estrogen Signaling in Human Breast Cancer Cells
J. Nutr., March 1, 2004; 134(3): 674 - 680.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Lapillonne, M. Konopleva, T. Tsao, D. Gold, T. McQueen, R. L. Sutherland, T. Madden, and M. Andreeff
Activation of Peroxisome Proliferator-activated Receptor {gamma} by a Novel Synthetic Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic Acid Induces Growth Arrest and Apoptosis in Breast Cancer Cells
Cancer Res., September 15, 2003; 63(18): 5926 - 5939.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qin, C.
Right arrow Articles by Safe, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qin, C.
Right arrow Articles by Safe, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online