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[Cancer Research 64, 5162-5171, August 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Inhibition of Activator Protein 1 Activation, Vascular Endothelial Growth Factor, and Cyclooxygenase-2 Expression by 15-Deoxy-{Delta}12,14-Prostaglandin J2 in Colon Carcinoma Cells: Evidence for a Redox-Sensitive Peroxisome Proliferator-Activated Receptor-{gamma}-Independent Mechanism

Raquel Grau, Miguel A. Iñiguez and Manuel Fresno

Centro de Biología Molecular "Severo Ochoa," CSIC-UAM, Universidad Autónoma de Madrid, Madrid, Spain


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) are significantly associated with tumor growth and metastasis. Here we show that phorbol ester-mediated induction of VEGF and COX-2 expression in colon carcinoma cells is inhibited by 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2). This cyclopentenone was able to inhibit activator protein1 (AP-1)-dependent transcriptional induction of COX-2 and VEGF promoters induced by phorbol 12-myristate 13-acetate (PMA) or c-Jun overexpression. 15d-PGJ2 interfered with at least two steps within the signaling pathway leading to AP-1 activation. First, 15d-PGJ2 impaired AP-1 binding to a consensus DNA sequence. Second, 15d-PGJ2 selectively inhibited c-Jun NH2 terminal kinase (JNK) but not extracellular signal-regulated kinase or p38 mitogen-activated protein kinase activation induced by PMA. This led to a decreased ability of JNK to phosphorylate c-Jun and to activate its transactivating activity. Inhibition of AP-1 activation and COX-2 or VEGF transcriptional induction by this cyclopentenone was found to be independent of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) because it was not affected by either expression of a dominant negative form of PPAR{gamma} or the use of a PPAR{gamma} antagonist. In contrast, we have found that the effects of 15d-PGJ2 on AP-1 activation may occur through its ability to induce intracellular oxidative stress. The antioxidant N-acetylcysteine significantly reversed the inhibition by 15d-PGJ2 of AP-1 activity and COX-2 or VEGF transcriptional induction. Together, these findings provide new insight into the antitumoral properties of 15d-PGJ2 through the inhibition of the induction of AP-1-dependent genes involved in tumor progression, such as COX-2 and VEGF.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is a growing body of evidence showing a close relationship among expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) and how they contribute to tumor growth and angiogenesis (1 , 2) . Cyclooxygenases catalyze the conversion of arachidonic acid to prostaglandin H2, the first step in the biosynthesis of prostanoids. There are two main COX isoforms: COX-1 and COX-2. Whereas COX-1 is constitutively expressed in most tissues, COX-2 expression is induced by several stimuli, such as mitogens, cytokines, and tumor promoters (3) . Increasing evidence has highlighted the role played by COX-2 in cancer, especially in colon carcinoma, making this enzyme an important therapeutic target for cancer prevention. Many human cancers, most notably of colon origin, display elevated COX-2 expression, and studies in COX-2 null mice have demonstrated the role of this enzyme in tumor progression and metastasis (1 , 4 , 5) . Moreover, epidemiologic studies have revealed a role of selective COX-2 inhibitors in decreasing the risk of developing colon cancer and in suppressing tumor formation and growth in animal models (6, 7, 8) . Accumulating evidence also supports a key role for VEGF expression in tumorigenesis contributing to tumor neovascularization and dissemination. Accordingly, increased VEGF expression has been found in most tumors, and agents neu-tralizing VEGF expression or activity inhibit tumor growth in vivo (9 , 10) .

The prostanoid 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) is a cyclopentenone-type prostaglandin derived by dehydration of prostaglandin D2 produced by many cell types (11) . This cyclopentenone may alter cellular functions by multiple mechanisms. 15d-PGJ2 has been shown to inhibit the expression of a variety of proinflammatory genes, including COX-2 (12, 13, 14, 15) . These effects seem to be mediated by the inhibition of various transcription factors, such as nuclear factor {kappa}B and activator protein 1 (AP-1; Refs. 12 , 14, 15, 16, 17, 18, 19 ). Some of the effects of 15d-PGJ2 are mediated by its ability to bind and activate the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}; Refs. 20 , 21 ). However, 15d-PGJ2 can induce a variety of responses independently of PPAR{gamma} because of the reactive cyclopentenone ring of this cyclopentenone, which may react with proteins containing nucleophilic groups, altering protein function (11 , 22) .

Recent evidence indicates that 15d-PGJ2 and other PPAR{gamma} ligands possess antineoplastic properties because of their inhibitory effects on tumor cell proliferation and angiogenesis (23) . Thus, the ability of 15d-PGJ2 to act as an antitumoral agent has garnered considerable attention, although the precise molecular mechanisms underlying that effect are poorly understood. Here we show that 15d-PGJ2 diminishes transcriptional induction of COX-2 and VEGF in colon carcinoma cells by inhibiting AP-1-mediated transcriptional activation. 15d-PGJ2 interferes with the activation of c-Jun NH2-terminal kinase (JNK) by phorbol 12-myristate 13-acetate (PMA), which results in reduced kinase activity leading to a decrease in c-Jun-transactivating activity. 15d-PGJ2 also inhibits AP-1 binding to DNA. The actions of 15d-PGJ2 on AP-1 activation and COX-2 and VEGF transcriptional induction were found to be independent of PPAR{gamma} and mediated by the induction of intracellular oxidative stress.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
RPMI, Opti-MEM, glutamine, and antibiotics were from Life Technologies (Rockville, MD). Fetal bovine serum was purchased from Sigma (St. Louis, MO). PMA, calcium ionophore A23187 (Io), and N-acetylcysteine (NAC) were from Sigma. The cyclopentenone prostaglandin 15d-PGJ2, the PPAR{gamma} antagonist (GW-9662), the prostaglandin E2 (PGE2) enzyme immunoassay kit, and the monoclonal COX-2 antibody were from Cayman Chemical (Ann Arbor, MI). Antibodies against extracellular signal-regulated kinase (ERK), JNK, p38, and their phosphorylated forms were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-coupled antimouse and antirabbit antibodies and the enhanced chemiluminescence detection system were from Pierce (Rockford, IL). Invitrogen (Carlsbad, CA) synthesized the oligonucleotides. [32P] ATP and [32P] dCTP for radioactive labeling were from Amersham Biosciences (Piscataway, NJ). Reagents for DNA transfection and luciferase assays were from Promega (Madison, WI). The most commonly used chemicals were from Sigma and Merck (Darmstadt, Germany).

Cell Culture.
The human colon carcinoma cell line SW620 was cultured in RPMI medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and antibiotics and maintained at 37°C as monolayer in a humidified atmosphere containing 5% CO2. The Caco-2 cell line was cultured in the same conditions in MEM supplemented with 1 mM sodium pyruvate. Cells were grown to 70% confluence, trypsinized with 0.25% trypsin and 2 mM EDTA, and plated for experimental use. Cells were changed to medium with 0.5% fetal bovine serum before treatment with pharmacologic reagents. There was no evidence of toxicity in any of our experiments as determined by the WST-1 cell viability assay.

Plasmid Constructs.
Human COX-2 promoter constructs in pXP2LUC promoter plasmid have been described previously (24) . The VEGF-LUC plasmid contains the region –1910 to +379 of the human VEGF promoter (25) . The –73col-LUC plasmid includes the 73/+63-bp region of the human collagenase promoter fused to the luciferase gene (26) . The pRSV-c-Jun expression plasmid has been described previously (27) . The dominant negative human PPAR{gamma} (AF2 mut) in pcDNA3 was a gift of Dr. V. K. Chatterjee (28) . The expression plasmid encoding mouse PPAR{gamma} and that containing the transactivation ligand-binding domain of PPAR{gamma} fused to the GAL4-DBD (pCMX-Gal-L-mPPAR{gamma}) were provided by Dr. R. M. Evans (20) . The reporter plasmid P4xACO-LUC containing four copies of the peroxisome-proliferator response element of the acyl-CoA oxidase (ACO) was a gift from Dr. B. Vogelstein (29) . The GAL4-c-Jun plasmid was obtained from Dr. P. Angel (30) . This plasmid expresses the first 166 amino acids of the human c-Jun fused to the DNA binding domain of the yeast GAL4 transcription factor (amino acids 1–147). The GAL4-DBD is the parental vector pABGAL-linker plasmid (31) . The GAL4-LUC reporter plasmid includes five copies of GAL4 DNA binding sites fused to the luciferase gene (32) .

mRNA Analysis.
Total RNA was extracted using the TriZol reagent (Invitrogen) and analyzed by reverse transcription-PCR. One µg of RNA was reverse transcribed into cDNA and used for PCR amplification with human COX-2, VEGF, PPAR{alpha}, PPAR{gamma}, PPAR{delta}, or glyceraldehide 3-phosphate dehydrogenase (GAPDH) specific primers by the RNA PCR Core Kit (Perkin-Elmer, Wellesley, MA) as described previously (33) . Briefly, the PCR reaction was amplified by 20–35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min (55°C for VEGF primers), and extension at 72°C for 1 min. Amplified cDNAs were separated by agarose gel electrophoresis, and bands were visualized by ethidium bromide staining. The sequences of the primers used were as follows: COX-2: sense, 5'-TTCAAAAGAAGTGCTGGAAAAG-GT-3'; antisense, 5'-GATCATCTCTACCTGAGTGTCTTT-3'; VEGF: sense, 5'-GAGTGTGTGCCCACTGAGGAGTCCAAC-3'; antisense, 5'-CTCCT-GCCCGGCTCACCGCCTCGGCTT-3'; PPAR{gamma}: sense, 5'-GAAATGACCA-TGGTTGACACAG-3'; antisense, 5'-CTTGACGTTGGTCTTGTCGG-3'; and GAPDH: sense, 5'-CCACCCATGGCAAATTCCATGGCA-3'; antisense 5'-TCTAGACGGCAGGTCAGGTCCACC-3'.

When indicated, the agarose gels were Southern blotted to a nylon filter and then incubated with a [32P]-labeled human VEGF, COX-2, or GAPDH cDNA probe as described previously (33) . Radioactivity in the radioactive bands was quantified by a phosphorimager and normalized with respect to the GAPDH values from parallel samples.

Immunoblot Analysis.
Cells were disrupted in ice-cold lysis buffer [20 mM Tris-HCl (pH 7.6), 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS, and 0.1% deoxycholate] with protease inhibitors (5 µg/ml leupeptin, 5 µg/ml aprotinin, 5 µg/ml pepstatin, and 0.5 mM phenyl-methylsulphonyl fluoride) and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF). Solubilized extracts (30 µg) were separated by SDS-PAGE on 10% polyacrylamide gel and electrophoretically transferred to nitrocellulose filters. After blocking for 2 h with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated overnight at 4°C with the corresponding antiserum. Monoclonal mouse anti-COX-2 (Cayman Chemical) was used at 1:1,000 dilution. Antibodies against phosphorylated or unphosphorylated mitogen-activated protein kinases (MAPKs; p38, ERK, and JNK) were purchased from Santa Cruz Biotechnology. The filters were washed and incubated for 1 h with secondary antibody linked to horseradish peroxidase (Pierce) at 1:15,000 dilution, and the stained bands were visualized by the SuperSignal Substrate detection system (Pierce).

Immunoprecipitation and in Vitro Kinase Assay.
Whole cell extracts from SW620 cells were obtained using ice-cold lysis buffer [20 mM Tris-HCl (pH 7.6), 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS, and 0.1% deoxycholate] with protease inhibitors (5 µg/ml leupeptin, 5 µg/ml aprotinin, 5 µg/ml pepstatine, and 0.5 mM phenyl-methylsulphonyl fluoride) and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF). In immunoprecipitation assays, whole cell extracts were incubated with 1 µl of the anti-JNK antibody (Santa Cruz Biotechnology) and 30 µl of protein A-Sepharose (Amersham Biosciences). Immunocomplexes were washed seven times with lysis buffer and three times with kinase reaction buffer [20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM ß-glycerophosphate, 0.5 mM DTT, 5 mM NaF, and 100 µM Na3VO4]. Immunoprecipitates and crude cell extracts were resolved by SDS-PAGE and transferred to nitrocellulose filters (Bio-Rad, Hercules, CA) to detect the presence of JNK by immunoblot analysis. For in vitro kinase assays, immunoprecipitated JNK was incubated with 0.7 µg of recombinant glutathione S-transferase c-Jun protein (GST-c-Jun; amino acids 1–79) fusion protein in kinase reaction buffer with 20 µM ATP and 1 µCi [32P]-ATP (specific activity, 3000 Ci/mol). Reactions were stopped by addition of 25 µl of Laemmli buffer and resolved in 10% SDS-PAGE. Gels were fixed with 10% acetic acid and 50% methanol and exposed to autoradiographic films to detect protein phosphorylation. The density of the bands was quantified with a computer densitometer (IS-1000 Digital Imaging System; Alpha Innotech, San Leandro, CA).

Transfection and Luciferase Assays.
Transcriptional activity was measured using luciferase reporter gene assays in transiently transfected carcinoma cells by LipofecAMINE Plus reagent as recommended by the manufacturer (Invitrogen). Briefly, exponential growing cells (0.5 x 106 cells/ml) were incubated for 4 h at 37°C with a mixture of 0.5–1 µg of the correspondent reporter plasmid, 2 µl of LipofectAMINE, and 3 µl of Plus agent in OptiMEM. In cotransfection experiments, 0.15–1.5 µg/ml of the correspondent expression plasmid were included. The total amount of DNA in each transfection was kept constant by using the corresponding empty expression vectors. Cells then were resuspended in complete medium with 0.5% fetal bovine serum and incubated at 37°C for an additional 16 h. Transfected cells were exposed to different stimuli as indicated. Cells then were harvested and lysed. Luciferase activity was determined by using the luciferase assay system (Promega) with a luminometer Monolight 2010 (Analytical Luminescence Laboratory, San Diego, CA). The data presented are expressed as the mean of the determinations in relative luciferase units (RLUs) ± SD or as fold induction (observed experimental RLU/basal RLU in absence of any stimulus).

Determination of COX Activity.
To evaluate COX activity in intact cells, the media were aspirated after treatments, and cells were rinsed with HBSS (pH 7.4) supplemented with 0.1% BSA. Cells then were incubated for 30 min at 37°C in the same buffer with an excess of arachidonic acid (10 µM). Prostanoids in the supernatant were purified by solid phase extraction, and levels of PGE2 were determined using a PGE2 enzyme immunoassay kit (Cayman Chemical). Cells were disrupted in lysis buffer, and protein concentration was determined. Results are expressed as pg of PGE2/µg of protein assayed. All of the samples were tested in triplicate.

Electrophoretic Mobility Shift Assays.
Nuclear extracts were prepared from SW620 cells as described previously (24) . Briefly, cells were collected by centrifugation and resuspended in 400 µl of ice-cold buffer A [10 mM HEPES (pH 7.6), 10 mM KCl, 0.1 mM EDTA, 0.1 EGTA, 0.75 mM spermidine, 0.15 mM spermine, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM Na2MoO4, 1 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml aprotinin]. After 15 min on ice, NP40 was added to a final concentration of 0.6% v/v, and cells were vortexed and centrifuged for 20 min at 650 x g. The nuclear pellet was extracted with 50 µl of buffer C [20 mM HEPES (pH 7.6), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM Na2MoO4, 1 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml aprotinin] for 30 min on a rocking platform and further centrifuged at 15,000 x g for 10 min. Protein concentration was determined by the Bradford assay (Bio-Rad). For the gel retardation assay, 5 µg of nuclear protein were incubated with 1 µg of poly(dI-dC) DNA carrier in DNA-binding buffer [2% (w/v) polyvinyl ethanol, 2.5% (v/v) glycerol, 10 mM Tris (pH 8), 0.5 mM EDTA, and 0.5 mM DTT] with 6 mM MgCl2 in a final volume of 10.5 µl for 10 min. Fifty thousand cpm of 32P-labeled double-stranded AP-1 consensus oligonucleotide (Promega) then were added, and the reaction was incubated at room temperature for 15 min. For competition experiments, a 30-fold molar excess of unlabeled oligonucleotide was added before the addition of the probe. When indicated, nuclear extracts were incubated with 10 µM 15d-PGJ2 before the addition of the probe. DNA-protein complexes were resolved by electrophoresis on 4% nondenaturing polyacrylamide gels. The sequence of the AP-1 consensus oligonucleotide was (5' to 3') CGCTTGATGAGTCAGCCGGAA.

Spectrofluorometric Measurement of Intracellular ROS.
Intracellular reactive oxygen species (ROS) were measured by the CM-H2DCFDA assay as described previously (34) . Cells were cultured at 37°C in the presence or absence of reagents as indicated in the corresponding figures, washed with PBS, and incubated with the peroxide sensitive fluorescent probe 5-(and-6)-carboxy-2,7,dichlorodihydro fluorescein diacetate (carboxy-H2DCFDA; Molecular Probes, Eugene, OR; 20 µM) for an additional 30 min at 37°C. After two washes with PBS, cells were solubilized with 1% SDS and 5 mM Tris HCl (pH 7.4). The fluorescent intensity of the lysate was determined using a spectrofluorometer (Aminco-Bowman Series 2; Thermo Electron, Waltham, MA) with excitation and emission wavelengths of 502 nm and 530 nm, respectively. Samples were assayed in triplicate. Data are shown as arbitrary units of fluorescence ± SD.

Cell Viability Assay.
The viability of the cells was measured using tetrazolium salt WST-1 (Roche, Basel, Switzerland). SW620 cells were incubated for 3, 6, and 24 h in the presence of increasing doses of 15d-PGJ2 (2, 5, and 10 µM) and incubated with 10% WST-1 reagent. Quantification of the formazan dye produced by metabolically active cells was spectrophotometrically measured at 450 nm and a reference wavelength of 630 nm.

All of the experiments shown are either representative or the mean of at least three performed to guarantee the reproducibility of the results.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of 15d-PGJ2 on Phorbol Ester-Induced COX-2 and VEGF Expression.
To explore the influence of 15d-PGJ2 on gene expression in colon carcinoma cells, we studied the effect of the treatment with this cyclopentenone on the expression of COX-2 and VEGF in the SW620 cell line. SW620 cells express little basal COX-2 mRNA or protein as detected by reverse transcription-PCR and Western blot analysis (Fig. 1, A and B)Citation . However, on induction with PMA + Io, COX-2 mRNA and protein levels were strongly induced. Although 15d-PGJ2 (10 µM) treatment significantly increased COX-2 mRNA and protein levels, addition of this cyclopentenone resulted in a strong inhibition of the induction of COX-2 expression by PMA (Fig. 1, A and B)Citation . The inhibitory effects of this cyclopentenone on COX-2 expression correlated with those observed with COX activity. Thus, 15d-PGJ2 abrogated PMA/Io induction of PGE2 synthesis in SW620 cells (Fig. 1C)Citation . Expression of the noninducible COX-1 isoform was almost negligible, being not affected by any of these treatments (data not shown).



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  		Fig. 1. 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) regulates cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) expression. SW620 cells were incubated for 1 h with 15d-PGJ2 (10 µM) before addition of phorbol 12-myristate 13-acetate (PMA; 15 ng/ml) plus ionophore (Io; 1 µM) as indicated. A, COX-2 and glyceraldehide 3-phosphate dehydrogenase (GAPDH) mRNA levels were analyzed by reverse transcription-PCR in cells treated with PMA + Io for 6 h in the presence or absence of 15d-PGJ2. Results of the scanning of the upper panels are shown in the graphic below as the ratio of arbitrary densitometric units of COX-2 mRNA to GAPDH mRNA. B, analysis of COX-2 protein levels. Protein extracts from cells treated with PMA + Io for 16 h in the presence or absence of 15d-PGJ2 were separated by SDS-PAGE and analyzed by Western blot analysis. C, COX activity in SW620 cells. Prostaglandin E2 (PGE2) production in cell supernatants after 30 min incubation with arachidonic acid (10 µM) was measured by a standard enzyme immunoassay as described in "Materials and Methods." Results are shown as the mean ± SD of the ratio of pg of PGE2 to µg of total protein of two determinations conducted in duplicate. D, mRNA levels of VEGF and GADPH detected by reverse transcription-PCR as described in A. Results of the scanning are shown in the lower panel as the ratio of VEGF mRNA to GAPDH mRNA levels in arbitrary densitometric units.

 
We also analyzed the effects of 15d-PGJ2 on VEGF expression in these cells. The predominant VEGF-A splice variants in SW620 cells are VEGF165 and VEGF121. As shown in Fig. 1DCitation , PMA + Io-mediated induction of VEGF mRNA was inhibited by 15d-PGJ2 treatment. Similar results were found with other colon carcinoma cell lines as Caco-2 (data not shown).

15d-PGJ2 Inhibits Transcriptional Activity of VEGF and COX-2 Promoters.
In SW620 cells, PMA or PMA/Io strongly induced the transcriptional activities of human COX-2 (pCOX-2-LUC) or VEGF (pVEGF-LUC) promoters to almost comparable levels (Fig. 2, A and B)Citation . Pretreatment with 15d-PGJ2 blunted PMA or PMA/Io induction of COX-2-LUC or VEGF-LUC activity. Because induction driven by PMA was enough to promote a strong transcriptional activation of both promoters in a 15d-PGJ2-sensitive manner, we next analyzed the mechanism by which this cyclopentenone interferes with PMA-mediated events in the transcriptional induction of COX-2 and VEGF.



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  		Fig. 2. Effects of 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) on cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) promoter activity. SW620 cells transfected with pCOX-2-LUC or pVEGF-LUC plasmids were preincubated for 1 h with 15d-PGJ2 (5 µM) and then stimulated with phorbol 12-myristate 13-acetate (PMA; 15 ng/ml) or PMA plus ionophore (Io; 1 µM) for 16 h. Results are the mean ± SD of two determinations expressed as relative luciferase units (RLUs)/µg of total protein in the cell extract. Results are representative of at least two independent experiments.

 
15d-PGJ2 Inhibits AP-1-Dependent Transcriptional Induction of VEGF or COX-2.
Involvement of AP-1 in the transcriptional induction of COX-2 and VEGF was analyzed by cotransfection of promoter luciferase constructs along with an expression vector for c-Jun (AP-1 member). As shown in Fig. 3Citation , c-Jun strongly transactivated COX-2 and VEGF promoters in a similar extent that it activates the AP-1-driven p-73Col-LUC reporter gene used as a control. Furthermore, PMA stimulation produced a cooperative effect with c-Jun overexpression on pCOX-2-LUC and pVEGF-LUC transcription and on the AP-1-dependent luciferase reporter.



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  		Fig. 3. Involvement of activator protein (c-Jun) in the transcriptional induction of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2). SW620 cells were cotransfected with reporter plasmids p-73-Col-LUC, pVEGF-LUC, or pCOX-2-LUC along with 10–50 ng of RSV-c-Jun expression plasmid. Cells then were incubated in the absence (white bars) or presence of phorbol 12-myristate 13-acetate (15 ng/ml; black bars) for 16 h, and luciferase activity then was determined. Results are the mean ± SD of two determinations shown as relative luciferase units (RLUs)/µg of total protein. In all of the cases, results are representative of at least two independent experiments.

 
Interestingly, 15d-PGJ2 inhibited the PMA and the c-Jun-mediated transactivation of COX-2 promoter, further supporting that some of the effects of this cyclopentenone occurred through the inhibition of AP-1-dependent transcription (Fig. 4A)Citation . Moreover, pretreatment of SW620 cells with 15d-PGJ2 before PMA stimulation inhibited the induction of the AP-1-dependent –73Col-LUC reporter (Fig. 4B)Citation . Once established that 15d-PGJ2 was inhibiting AP-1-dependent transcriptional activation, we next addressed the mechanism by which 15d-PGJ2 was inhibiting AP-1-mediated responses. As shown in Fig. 4CCitation , in an electrophoretic mobility shift analysis assay, PMA induced the appearance of a retarded protein complex that binds specifically to the AP-1 consensus sequence because it was completely inhibited by an excess of cold DNA probe (comp). The formation of this complex was severely impaired in cells pretreated with 15d-PGJ2 before PMA stimulation. Interestingly, the inhibitory effect of 15d-PGJ2 on AP-1 binding was even more pronounced when nuclear extracts of cells stimulated with PMA were incubated in vitro with 15d-PGJ2 for 30 min than when added to intact cells (Fig. 4D)Citation . These results suggest that inhibition of AP-1 binding to its cognate response elements in the DNA can be attributed to a direct interaction of this cyclopentenone with the components of the AP-1 factor.



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  		Fig. 4. 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) inhibits activator protein 1 (AP-1)-mediated signaling. A, SW620 cells were cotransfected with pCOX-2-LUC along with increasing quantities of pRSV-c-Jun expression vector. Cells were grown in the presence or absence of 15d-PGJ2 (10 µM) and stimulated with phorbol 12-myristate 13-acetate (PMA; 15 ng/ml) for 16 h. Results are the mean ± SD shown as relative luciferase units (RLUs)/µg of total protein. B, cells were transfected with the –73col-LUC reporter and then preincubated with 15d-PGJ2 (5 µM) before stimulation with PMA for additional 16 h. C, nuclear extracts were obtained from cells incubated with 15d-PGJ2 (10 µM) for 1 h and then stimulated with PMA (15 ng/ml) for 90 min as indicated. AP-1 binding to a consensus AP-1 probe then was evaluated by electrophoretic mobility shift analysis (EMSA). A 30-fold molar excess of unlabeled AP-1 consensus oligonucleotides was added to determine the specific binding (comp). The graphics below show the densitometry of the radioactive bands in arbitrary units. D, nuclear extracts were isolated from SW620 cells grown in the presence or absence of PMA (15 ng/ml) for 90 min. 15d-PGJ2 (10 µM) was added to the extracts 30 min before incubation with the consensus AP-1-labeled probe, and DNA binding was evaluated by EMSA. The bottom panel shows the densitometry of the radioactive bands in arbitrary units.

 
15d-PGJ2 Inhibits JNK Activation.
Activation of the AP-1 component c-Jun is strongly dependent on phosphorylation at specific residues by JNK (35) . To determine whether 15d-PGJ2 also affected JNK activation, we examined changes in JNK phosphorylation by immunoblot analysis with specific antiphospho-JNK antibodies. The activation of SW620 cells with PMA induced a time-dependent phosphorylation of both JNK isoforms (JNK-1 and JNK-2) without changes in their total protein levels (Fig. 5A)Citation . 15d-PGJ2, although somewhat increased JNK (mostly JNK-1) activation, was able to inhibit the induction on JNK phosphorylation mediated by PMA. PMA treatment also induced ERK and p38 activation in these cells. Whereas 15d-PGJ2 did not affect PMA increase in ERK1/2 phosphorylation, it increased phosphorylation of p38 MAPK (Fig. 5B)Citation .



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  		Fig. 5. Effects of 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) on the activation of mitogen-activated protein kinases (MAPKs). A, SW620 cells were preincubated or not with 15d-PGJ2 (10 µM) for 1 h and then stimulated with phorbol 12-myristate 13-acetate (PMA) for the period indicated (min). Levels of phosphorylated (P) forms of c-Jun NH2 terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) were detected by immunoblot analysis with specific antibodies. The same blot was stripped and reprobed with anti-JNK antibody. B, cells preincubated or not with 15d-PGJ2 (10 µM) for 1 h were stimulated for 30 min with PMA. Phosphorylated (P) p38, MAPK, ERK, and JNK were detected by immunoblot analysis with specific antiphospho-specific antibodies. The results are representative of three independent experiments.

 
Accordingly, inhibition of JNK phosphorylation by 15d-PGJ2 resulted in a decrease in its enzymatic activity. As shown in Fig. 6ACitation , untreated cells have no basal JNK activity detectable by an in vitro phosphorylation assay using immunoprecipitated JNK and GST-c-Jun (amino acids 1–79) as substrate. After PMA treatment, a strong increase in the phosphorylation of GST-c-Jun was observed. 15d-PGJ2 reduced in a dose-response manner the induction of GST-c-Jun phosphorylation by PMA. The results of the in vitro phosphorylation assay paralleled the amount of phosphorylated JNK in the cells (Fig. 6A, a)Citation , suggesting that the decrease in c-Jun phosphorylation was caused by decreased JNK activation. Moreover, this decrease cannot be ascribed to a different amount of JNK in the immunoprecipitates (Fig. 6A, b)Citation . To address the possibility of a direct effect of 15d-PGJ2 on JNK enzymatic activity, this kinase was immunoprecipitated from cells treated with PMA, and immunoprecipitates then were incubated for 30 min with 15d-PGJ2 before the kinase assay. In this case, we did not observe any significant effect of 15d-PGJ2 on JNK activity (Fig. 6C)Citation , indicating that this cyclopentenone did not directly affect the kinase activity of activated JNK.



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  		Fig. 6. Effects of 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) on c-Jun NH2 terminal kinase (JNK) activity. A, SW620 cells were preincubated with 15d-PGJ2 at the concentrations indicated (µM) for 1 h and then stimulated or not with phorbol 12-myristate 13-acetate (PMA) for 30 min. JNK was immunoprecipitated with an anti-JNK-specific antibody. In vitro kinase reactions were performed on immunoprecipitates using glutathione S-transferase c-Jun protein (GST-c-Jun; amino acids 1–79) as the substrate. The proteins were resolved by SDS-PAGE, and phosphorylation was detected by autoradiography. Top, phosphorylation of GST-c-Jun by immunoprecipitated JNK. The presence of phosphorylated and total JNK in the extracts was determined by immunoblot analysis (a and b). B, cells were stimulated with PMA for 30 min, and JNK then was immunoprecipitated. The immunoprecipitates were incubated with 15d-PGJ2 at the concentrations indicated (µM) for 30 min. In vitro radioactive kinase reactions then were performed using GST-c-Jun (amino acids 1–79) as the substrate. GST-c-Jun phosphorylation was detected by autoradiography. C, cells were transiently cotransfected with the reporter plasmid GAL4-LUC along with a GAL4-c-Jun expression vector. Cells then were treated with PMA for 16 h in the absence or presence of 15d-PGJ2 (5 µM). Luciferase activity then was determined. Results are the mean ± SD shown as relative luciferase units (RLUs)/µg of total protein. In all of the cases, the results are representative of at least two independent experiments.

 
The reduction in JNK activity may lead to a diminished c-Jun transactivation because it is strongly dependent on the phosphorylation by this kinase of Ser 63 and Ser 73, which are located in the transactivation domain (35) . Thus, we next tested whether 15d-PGJ2 affected c-Jun transactivating activity. As shown in Fig. 6BCitation , 15d-PGJ2 significantly reduced (an average of 50% in the several experiments performed) the basal transactivating activity of the chimeric protein GAL4-c-Jun, which contains the c-Jun transactivation domain (amino acids 1–166) fused to the DNA binding domain of the GAL4 yeast transcription factor. Moreover, 15d-PGJ2 also reduces in a similar extent the increase in transactivation induced by PMA stimulation (Fig. 6B)Citation .

Involvement of PPAR{gamma} on the Effects of 15d-PGJ2.
Although 15d-PGJ2 has been described as a ligand of PPAR{gamma}, mechanisms independent of PPAR{gamma} binding also have been described for this cyclopentenone (12 , 18 , 19 , 36 , 37) . Thus, we tested whether the effects of 15d-PGJ2 observed previously were dependent or independent of PPAR{gamma} activation. SW620 cells expressed basal levels of PPAR{gamma} mRNA that were not altered in response to PMA treatment (Fig. 7A)Citation . Moreover, 15d-PGJ2 was able to transactivate a peroxisome-proliferator response element-dependent luciferase reporter (ACO-LUC). This induction was increased by simultaneous cotransfection of a wild-type PPAR{gamma} expression vector and blocked by coexpression of a PPAR-{gamma} truncated form that acts as a dominant negative (Ref. 28 ; Fig. 7BCitation ). Furthermore, GW9662, a specific PPAR{gamma} antagonist, inhibited luciferase activity of the ACO-LUC reporter gene and the GAL4-PPAR{gamma} construct, in which luciferase expression is mediated by the ligand-dependent transactivation domain of this receptor (Refs. 20 , 29 ; Fig. 7CCitation ). These results demonstrate that 15d-PGJ2 was able to regulate transcription through PPAR{gamma} in these cells. However, the transfection of wild-type PPAR{gamma} did not potentiate 15d-PGJ2 inhibition of the activity of COX-2 or VEGF promoters (Fig. 8A)Citation . Even more, cotransfection with dominant negative PPAR{gamma} or addition of the PPAR{gamma} antagonist GW-9662 did not revert 15d-PGJ2 inhibition of pCOX-2-LUC or pVEGF-LUC (Fig. 8B)Citation . These results indicate that 15d-PGJ2 regulates COX-2 and VEGF transcription via PPAR{gamma}-independent mechanisms.



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  		Fig. 7. 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) mediates peroxisome proliferator-activated receptor (PPAR)-dependent transcriptional activation in SW620 cells. A, reverse transcription-PCR analysis of PPAR{gamma} mRNA expression in SW620 cells. An aliquot of the amplified DNA was separated on an agarose gel and stained with ethidium bromide for qualitative comparison. Cells were cultured in the absence or presence of phorbol 12-myristate 13-acetate (PMA; 15 ng/ml) for 16 h. B, SW620 cells were transfected with the PPAR responsive reporter plasmid acyl-CoA oxidase (ACO)-LUC along with wild-type or dominant negative PPAR{gamma} expression plasmids. After transfection, cells were treated for 16 h with 15d-PGJ2 (5 µM), and luciferase activity was determined. C, cells were transiently transfected with the ACO-LUC reporter or with a GAL4-LUC reporter plus an expression vector for GAL4-PPAR{gamma} chimera. Cells were incubated with the PPAR{gamma} antagonist GW-9662 (1 µM) for 1 h before treatment with 15d-PGJ2 for an additional 16 h and assayed for luciferase activity.

 


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  		Fig. 8. Analysis of the role of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) on the inhibition of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) promoter activity by 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2). A, SW620 cells were transiently transfected with the COX-2-LUC or VEGF-LUC reporters along with empty vector or wild-type or dominant negative PPAR{gamma} expression plasmids. After transfection, cells were pretreated for 1 h with 15d-PGJ2 (5 µM) before phorbol 12-myristate 13-acetate (PMA) treatment. Cells were lysed, and luciferase activity was determined. Results are shown as percentage of activation by PMA considering 100% the induction of promoter activity in the absence of 15d-PGJ2. B, cells transfected with the COX-2-LUC or VEGF-LUC constructs were preincubated with the PPAR{gamma} antagonist GW-9662 (1 µM) and/or with 15d-PGJ2 (5 µM) before incubation with PMA for 16 h. Results are the mean ± SD of two determinations shown as relative luciferase units (RLUs)/µg of total protein. In all of the cases, results are representative of at least two independent experiments with similar results.

 
Involvement of the Redox State on 15d-PGJ2 Inhibition of AP-1 Activation.
It has been shown that 15d-PGJ2 may act by a mechanism independent of PPAR{gamma} by modulating the redox status of the cells (38) . To determine ROS accumulation, SW620 cells were loaded with the peroxide-sensitive fluorescent probe carboxy-H2DCFDA in the absence or presence of 15d-PGJ2 or the antioxidant NAC, and then 2',7'- dichlorofluorescin fluorescence was quantified by spectrofluorometry. As shown in Fig. 9ACitation , treatment of SW620 cells with 15d-PGJ2 increased intracellular ROS, which was reversed by the antioxidant NAC. Interestingly, treatment with increasing doses of this antioxidant also prevented 15d-PGJ2-mediated inhibition of COX-2 transcriptional activation by PMA (Fig. 9B)Citation . Similar results were obtained when analyzing the activity of the VEGF promoter or the –73Col-LUC construct (Fig. 9, C and D)Citation . The inhibitory effect of 15d-PGJ2 on AP-1 binding to DNA also was partially reverted by NAC (Fig. 10)Citation . Furthermore, the inhibitory effect of 15d-PGJ2 on c-Jun-mediated transcriptional induction of COX-2 also was reversed by NAC (Fig. 11)Citation . These results suggested that inhibition of AP-1 activation by 15d-PGJ2 in colon carcinoma cells occurred by a redox-dependent mechanism.



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  		Fig. 9. Involvement of the redox state on the effects of 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2). A, 15d-PGJ2 induces reactive oxygen species production in SW620 cells that is ameliorated by N-acetylcysteine (NAC) pretreatment. Cells were incubated in the presence of vehicle (DMSO) or increasing concentrations of 15d-PGJ2 (µM) for 15 min. Cells were lysed after 30 min incubation with the peroxide sensitive compound carboxy-H2DCFDA (20 µM), and 2',7'-dichlorofluorescin fluorescence was determined. When indicated, NAC (5 mM) was added to the cells 2 h before 15d-PGJ2. B, effect of NAC on 15d-PGJ2 inhibition of cyclooxygenase-2 (COX-2) promoter activity. SW620 cells were transfected with pCOX-2-LUC and then preincubated with NAC at the indicated concentration (mM) for 2 h before 15d-PGJ2 treatment. Cells then were stimulated or not with phorbol 12-myristate 13-acetate (PMA) for 16 h. C and D, cells were transfected with pVEGF-LUC or p-73col-LUC, preincubated with NAC (5 mM) for 2 h, treated with 15d-PGJ2 for an additional 1 h, and finally stimulated with PMA for 16 h. Results are the mean ± SD of two determinations shown as relative luciferase units (RLUs)/µg of total protein. Results are representative of the several performed.

 


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  		Fig. 10. N-acetylcysteine (NAC) reverses 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) inhibition of activator protein (AP-1) binding to DNA. SW620 cells were pretreated with NAC (5 mM) for 2 h before 15d-PGJ2 (10 µM) for 1 h and before being stimulated with phorbol 12-myristate 13-acetate (PMA) for 90 min as indicated. Nuclear extract were obtained, and binding to an AP-1 consensus probe was evaluated by an electrophoretic mobility shift analysis assay. The graphics below show the densitometry of the radioactive bands in arbitrary units. These results are representative of three independent experiments.

 


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  		Fig. 11. N-acetylcysteine (NAC) prevents inhibition by 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) of c-Jun-mediated activation of the cyclooxygenase-2 (COX-2) promoter. SW620 cells were transiently transfected with the pCOX-2-LUC reporter together with 50 ng of c-Jun expression plasmid. Cells then were incubated with NAC (5 mM) for 2 h, then with 15d-PGJ2 (5 µM) for 1 h when indicated, and subsequently activated with phorbol 12-myristate 13-acetate for another 16 h. Cells were lysed, and luciferase activity was determined. Results are shown as the mean ± SD of observed relative luciferase units (RLUs)/µg of total protein.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Accumulating evidence shows that COX-2 and VEGF are crucial agents in cell proliferation and tumor growth, promoting tumor-associated angiogenesis (1 , 2) . Conversely, recent studies have suggested an antitumoral role of cyclopentenones such as 15d-PGJ2 in a variety of cancers by its ability to inhibit cell proliferation and angiogenesis (23 , 39 , 40) .

In the present study, we have shown that 15d-PGJ2 severely diminished phorbol ester and c-Jun-mediated induction of VEGF and COX-2 expression in colon carcinoma cells. Contradictory effects of 15d-PGJ2 and other PPAR{gamma} agonists on COX-2 and VEGF expression have been described in the literature. Whereas some authors report that these agents suppress COX-2 or VEGF expression in a variety of cell lines after cell stimulation (16 , 17 , 41 , 42) , other results indicate that 15d-PGJ2 and other PPAR{gamma} agonists are inductors of COX-2 or VEGF expression (41 , 43, 44, 45) . Although these apparent discrepancies may have multiple causes, it is likely that 15d-PGJ2 displays a dual effect; whereas this cyclopentenone induces basal COX-2 or VEGF expression, in most cases, presumably by PPAR{gamma} activation, it also is able to reduce the up-regulation of these genes by PPAR{gamma}-dependent or -independent mechanisms. It is important to notice that increases in basal COX-2 expression by 15d-PGJ2 did not correlate with a significant increase in prostaglandin synthesis (Fig. 1C)Citation . Thus, the PPAR{gamma}-independent inhibitory actions on the activation of the AP-1 pathway prevail over the stimulatory, PPAR{gamma}-dependent effects of 15d-PGJ2 on COX-2 or VEGF gene expression.

Our results demonstrate that 15d-PGJ2-mediated inhibition of COX-2 and VEGF transcriptional activation occurs by interfering with AP-1-mediated activation events. Most of the inhibitory effects of 15d-PGJ2 on gene transcription have been ascribed to inhibition of nuclear transcription factor activation (12 , 14, 15, 16, 17, 18, 19) . Here we show that 15d-PGJ2 inhibition of AP-1 activation in colon cancer SW620 cells occurs at several steps as summarized in Fig. 12Citation . 15d-PGJ2 inhibits JNK activation, resulting in a reduced kinase activity, therefore diminishing c-Jun phosphorylation and transactivating activity. This cyclopentenone also diminishes DNA binding of AP-1 components. AP-1 activity depends on the transcriptional and post-transcriptional activation of its components, members of Fos and Jun families (35) . c-Jun is considered the main component of AP-1, and its activity is regulated post-transcriptionally by JNK phosphorylation at Ser 63 and Ser 73 on its transactivating domain (35) . JNK activity also is regulated by phosphorylation by various upstream MAPK kinases (46) . Positive and negative effects of 15d-PGJ2 on ERK, JNK, and p38 MAPK activities have been reported (37 , 47, 48, 49, 50) . These effects result in the regulation of the level of phosphorylation and activation of AP-1 components. The fact that 15d-PGJ2 inhibited JNK activation induced by PMA but it did not have a direct effect on kinase activity of activated JNK in a cell-free system in vitro (Fig. 6)Citation point to an impairment of the activation of MAPK kinases leading to JNK activation (46) . We also have found a decrease in AP-1 binding to DNA after 15d-PGJ2 treatment in stimulated cells, which is consistent with previous observations (Fig. 4CCitation ; Refs. 15 , 17 , 42 , 51 ). Interestingly, this inhibitory effect also could be observed after in vitro incubation with 15d-PGJ2 of cell extracts isolated from stimulated cells (Fig. 4D)Citation , which suggests that 15d-PGJ2 also may act directly on AP-1 components. As recently shown by Perez-Sala et al. (51) , 15d-PGJ2 can form a covalent adduct with c-Jun, thus inhibiting the DNA binding activity of AP-1.



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  		Fig. 12. Model for the mechanisms by which 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) inhibits cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) transcriptional activation. 15d-PGJ2 interferes with activator protein (AP-1)-dependent induction of COX-2 and VEGF transcription at several steps. 15d-PGJ2 reduces the activation/phosphorylation of c-Jun NH2 terminal kinase (JNK) induced on cell stimulation (1), which results in diminished activation and phosphorylation of c-Jun (2), reducing its transactivation activity. This cyclopentenone also inhibits the binding of AP-1 proteins to DNA (3). These actions result in the inhibition of the transcriptional induction of AP-1-dependent genes such as COX-2 and VEGF (4). Some of these effects occur through alteration of the cellular redox state and can be reverted by precursors of reduced glutathione (GSH) such as N-acetylcysteine (NAC).

 
Despite being a ligand of PPAR{gamma} (20 , 21) , 15d-PGJ2 also can act through mechanisms independent of the binding to this receptor (11 , 22) . Our data show that transcriptional inhibition of PMA or c-Jun-mediated induction of COX-2 and VEGF genes and AP-1 activation are not mediated by PPAR{gamma}. Instead, our results indicate that the effects of 15d-PGJ2 on the inhibition of AP-1-dependent transcriptional regulation of genes are mediated by a redox-sensitive mechanism. 15d-PGJ2 is a potential inducer of intracellular oxidative stress, depleting reduced glutathione and inducing pro-oxidant enzymes, that can mediate cell degeneration, which explains its antiproliferative and antitumor effects (38 , 52) . Antioxidants such as NAC increase levels of reduced glutathione in many cell types, including cancer cells, thus preventing the effects of 15d-PGJ2 caused by induction of oxidative stress (38 , 53 , 54) . Furthermore, levels of intracellular reduced glutathione are key to regulate AP-1 transcription factor activation (55) . Thus, the pro-oxidant actions of this cyclopentenone are responsible for the induction of cellular oxidative stress, which may explain some of its antitumoral properties. These actions will finally depend on the status of activation of the cells and how efficiently they respond to oxidative stress induced by 15d-PGJ2.

Together, these findings provide new insights into the antitumoral and antiangiogenic properties of the cyclopentenone 15d-PGJ2 through the inhibition of the induction of AP-1 activation and transcriptional up-regulation of genes involved in tumor progression, such as COX-2 and VEGF in colon carcinoma cells. However, one important issue regarding many of the reported effects of 15d-PGJ2 is whether they can be considered of physiologic or pharmacologic significance. Whereas the levels of 15d-PGJ2 measured in several experimental models of inflammation are in the nanomolar range, most of the biological effects of 15d-PGJ2, including those we describe in this manuscript, have been observed to occur at micromolar concentrations (56) . Thus, in addition to defining the physiologic role of 15d-PGJ2, additional work is needed to establish the in vivo relevance of the antitumoral and anti-inflammatory effects elicited in vitro by micromolar doses of this cyclopentenone.


   ACKNOWLEDGMENTS
 
We thank those who have helped us with different reagents as mentioned in "Materials and Methods," and Lucia Horrillo, Gloria Escribano, María Chorro, María Cazorla, and Carmen Punzón for their excellent technical assistance. We also thank Elena Bogonez for her helpful assistance with the fluorometric determinations.


   FOOTNOTES
 
Grant support: Ministerio de Ciencia y Tecnología (BMC2001–0177), Fondo de Investigaciones Sanitarias (G0/173), EICOSANOX integrated project (6th EU Framework Programme), and Laboratorios del Dr. ESTEVE to M. Fresno and Comunidad de Madrid (08.3/0007/1) to M. A. Iñiguez. M. A. Iñiguez is a recipient of the Ramon y Cajal Program of the Ministerio de Ciencia y Tecnología of Spain.

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.

Requests for reprints: Manuel Fresno, Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-497-8413; Fax: 34-91-497-4799; E-mail: mfresno{at}cbm.uam.es

Received 3/ 9/04. Revised 6/ 2/04. Accepted 6/ 2/04.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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