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Originally published In Press as doi:10.1074/jbc.M300771200 on April 10, 2003

J. Biol. Chem., Vol. 278, Issue 26, 23360-23368, June 27, 2003
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p21Cip1 Gene Expression Is Modulated by Egr1

A NOVEL REGULATORY MECHANISM INVOLVED IN THE RESVERATROL ANTIPROLIFERATIVE EFFECT*

Fulvio Della Ragione {ddagger}, Valeria Cucciolla, Vittoria Criniti, Stefania Indaco, Adriana Borriello and Vincenzo Zappia

From the Department of Biochemistry and Biophysics "F. Cedrangolo," Second University of Naples, 80138, Naples, Italy

Received for publication, January 23, 2003 , and in revised form, April 9, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidemiological observations indicate that resveratrol, a natural antioxidant stilbene, exerts cardioprotective and chemopreventive effects. Moreover, the molecule induces in vitro cell growth inhibition and differentiation. Using human erythroleukemic K562 cells as model system, we demonstrated that resveratrol induces a remarkable {gamma}-globin synthesis, the erythroid differentiation being linked to impairment of cell proliferation, increased p21Cip1 expression and inhibition of cdk2 activity. The up-regulation of p21Cip1 transcription is prevented by cycloheximide, indicating the requirement of intermediate protein(s), which, in turn, regulate gene expression. The quantitative analysis of some transcription factors involved in the erythroid lineage, namely GATA-1, GATA-2, and Egr1, indicated that resveratrol selectively up-regulates Egr1 by an Erk1/2-dependent mechanism. The presence of an Egr1 consensus sequence in the p21Cip1 promoter suggested the hypothesis that this transcription factor directly regulates the expression of the cdk inhibitor. Transfection studies with deleted gene promoter constructs, as well as EMSA, pull-down, and chromatin immunoprecipitation experiments substantiated this view, demonstrating that Egr1 binds in vitro and in vivo to the identified consensus sequence of the p21Cip1 promoter. Moreover, an Egr1 phosphorothioate antisense hinders p21Cip1 accumulation and the antiproliferative effects of resveratrol. In conclusion, this is the first demonstration that Egr1 controls p21Cip1 expression by directly interacting with a specific sequence on its gene promoter. The identified regulatory mechanism also contributes to the clarification of the complex chemopreventive and antiproliferative properties of resveratrol.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Resveratrol1 (3,5,4'-trihydroxystilbene) is a natural stilbene endowed with antioxidant properties, synthesized in plants to counteract environmental oxidative stress such as UV irradiation or exposure to ozone (1–3). The molecule, distributed at a significant level in the skin of grapes and in a few other spermatophytes, is functionally classified as an antifungine phytoalexin, which confers resistance to pathogen attack (1).

In the last decade, resveratrol has received wide attention since a number of epidemiological observations suggest that it might reduce the risk of cardiovascular diseases and cancer (4, 5). Indeed, the phytoalexin has been reported to prevent atherosclerosis (6) by modulating the synthesis of hepatic apolipoproteins and lipids (7), inhibiting platelet aggregation (8–10) and the synthesis of proatherogenic eicosanoids in human platelets and neutrophils (8, 11).

Jang et al. (12) described the ability of resveratrol to inhibit events associated with the initiation, promotion, and progression of cancer. The molecule is able to: (i) induce phase II drug-metabolizing enzymes, (ii) cause cell growth arrest, and (iii) inhibit the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture and tumori-genesis in a mouse skin cancer model. The antimutagenic activity of resveratrol against the carcinogen 3-amino-1,4-dimethyl-5H-pyrido[4,3-{beta}]indole was also demonstrated by Ames assay using Salmonella typhimurium (13).

Resveratrol exerts in vitro a strong antiproliferative activity on various cell types. When added to cell cultures, the compound reduces the growth rate and DNA synthesis in a dose-dependent fashion (14, 15). It suppresses the proliferation of normal human hepatocytes (16) and keratinocytes (17), as well as the growth of tumor cell lines established from different tissues (breast cancer, cervical tumors, gastric adenocarcinomas, colon cancer, and others) (18–22). In a number of instances, resveratrol induces apoptosis (23, 24), caspase inhibitors being able to block this event (25, 26). Finally, we have recently demonstrated that resveratrol blocks the growth of HL-60 cells at S/G2 phase and induces cell differentiation toward the mielo-monocytic phenotype (27). Such an observation suggests that the molecule might cause, in a specific context, hematological differentiation.

The molecular mechanisms of the in vivo and the in vitro antitumoral properties of resveratrol have been extensively investigated. The molecule is able to inhibit the hydroperoxidase activity of type 1 cyclooxygenase (12) as well as the expression of type 2 of the same enzyme (28). Moreover, recent reports demonstrated that the plant stilbene inhibits ornithine decarboxylase (29), DNA polymerase (30), CYP1A, CYP2E1, and CYP3A (31) and ribonucleotide reductase (32). In this case, resveratrol acts as a radical scavenger by interacting with and destroying the tyrosyl radical of the R2 subunit, which is necessary for the activity of this enzyme (32). Finally, it has been reported that resveratrol, like several other naturally occurring stilbenes, mimics, in some instances, estradiol effects (33). However, despite the large number of reports, the molecular basis of phytoalexin antiproliferative effects has not been definitively clarified.

Several investigations have shown an increased expression of {epsilon}- and {gamma}-globin genes in human erythroleukemic K562 cells treated with a variety of molecules impairing cell proliferation (34). Therefore, this cell line has been widely used as an excellent in vitro model to evaluate the effects of new antiproliferative drugs (35). As resveratrol might cause cell differentiation, we decided to examine its activity on K562 cells. The induced {gamma}-globin expression caused us to investigate the molecular basis of resveratrol effects and to elucidate the possible transduction pathway(s) responsible for erythroid differentiation of K562. This approach provides new insights into the biochemical events leading to cell growth inhibition by resveratrol as well as into novel control mechanisms of the cell division cycle.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Resveratrol, etoposide, and cycloheximide were supplied by Sigma. PD98059 (2'-amino-3'methoxyflavone), Erk1/2 inhibitor; SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), p38 kinase inhibitor; KN62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine), Ca2+/calmodulin kinase inhibitor; H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), cAMP-dependent kinase inhibitor; curcumin (diferuloylmethane), JNK inhibitor were furnished by BioMol Research Laboratories, Inc. StrataScript RT-PCR kit was purchased from Stratagene (La Jolla, CA). FuGENE 6 Transfection Reagent was from Roche Applied Science. LightShift Chemiluminescent EMSA kit was obtained from Pierce.

Antibodies—Monoclonal antibodies to cyclin A, cyclin E, cdc2, Sp1, and phospho-Erk (Tyr-204) were purchased by Santa Cruz Biotechnology (Santa Cruz, CA) and against PARP (poly(ADP-ribose) polymerase) from BioMol. Monoclonal antibodies to p21Cip1 were from Pharmingen (San Diego, CA), while those against p27Kip1 were from Transduction Laboratories (Lexington, UK). Polyclonal antibodies to cdk2, cdk4, cdk6, cyclin D2, cyclin D3, Egr1, Erk1/2, NF-{kappa}B, c-Jun, and phospho-c-Jun (Ser-63) were from Santa Cruz Biotechnology.

K562 Cell Line Treatment and Differentiation and Flow Cytometry Analyses—The K562, HL-60, and LAN-5 cell lines were obtained from the American Type Culture Collection. The cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum and penicillin-streptomycin in a 5% CO2 atmosphere at 37 °C in a humidified incubator. Resveratrol was directly added to cell cultures at the indicated concentrations, while untreated cells contained the solvent alone (dimethyl sulfoxide). In the experiments employing kinase inhibitors, the molecules were added 1 h before the addition of resveratrol. For the analysis of gene expression, the cells were treated, when indicated, with the protein synthesis inhibitor cycloheximide at 36 µM concentration. K562 cell differentiation was evaluated by determining the expression of {gamma}-globin and GpIIb genes (see below) in cells collected 3 days after the addition of 30 µM resveratrol. Flow cytometry analyses were carried out as described in Ref. 36.

RNA Extraction and Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)—Total RNA was extracted and purified as previously described (37). RT-PCR analyses were carried out as described in Ref. 37. The primers and the PCR conditions employed for each gene were reported below. All the reactions had a hot start of 5 min at 95 °C and a final elongation step at 72 °C for 7 min. GAPDH: 5'-GGTATCGTGGAAGGACTCATGAC-3' (sense) and 5'-ATGCCAGTGAGCTTCCCGTCAGC-3' (antisense); 15–20 cycles composed of steps at 95 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min. Egr1: 5'-TGGCTTCCAGGTTCCCATGATCCCC-3' (sense) and 5'-GGCAAGCGTAAGGGCGTTCGTGGG-3' (antisense); 30–35 cycles composed of steps at 95 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min. p21Cip1: 5'-AGGCGCCATGTCAGAACCGGCTGG-3' (sense) and 5'-GGAAGGTAGAGCTTGGGCAGGC-3' (antisense); 23–28 cycles composed of steps at 95 °C for 1 min, 68 °C for 1 min, 72 °C for 1 min. p27Kip1: 5'-ATGTCAAACGTGCGAGTGTCTAAC-3' (sense) and 5'-TTACGTTTGACGTCTTCTGAGGCCA-3' (antisense); 25–30 cycles composed of steps at 95 °C for 1 min, 63 °C for 1 min, 72 °C for 1 min. p57Kip2: 5'-TCCACGATGGAGCGTCTTGT-3' (sense) and 5'-GTCCACTTCGGTCCACTGCA-3' (antisense); 25–30 cycles composed of steps at 95 °C for 1 min, 63 °C for 1 min, 72 °C for 1 min. GATA-1: 5'-GGAGCCCTCTCAGCTCAGC-3' (sense) and 5'-GCCACCAGCTGGTCCTTCAG-3' (antisense); 33–38 cycles composed of steps at 95 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min. GATA-2: 5'-GCGCAGCAAGGCTCGTTCCTGTTCAGAA-3' (sense) and 5'-CGCCATAAGGTGGTGGTTGTCGTCTGACAA-3' (antisense); 32–37 cycles composed of steps at 95 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min. {gamma}-globin: 5'-GTCATTTCACAGAGG-3' (sense) and 5'-TGGATTGCCAAAACG-3' (antisense); 25–30 cycles composed of steps at 95 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min. GpIIb: 5'-GGAAGATGGCCAGAGC-3' (sense) and 5'-GTTCCAGTGCTGCCAGGGGGC-3' (antisense); 33–38 cycles composed of steps at 95 °C for 1 min, 62 °C for 1 min, 72 °C for 1 min.

Before amplification with each specific primer pair, an aliquot of the cDNA preparation was amplified using GAPDH primers to determine the integrity of the generated cDNA. Moreover, we used five different cDNA concentrations to assure that signals (both of GAPDH and of the gene under analysis) were proportional to input mRNA. Finally, each experiment was performed at least in triplicate and, in several cases, in quadruplicate.

Western Blot Analysis and Enzymatic Assays—Cell extracts were prepared as described in Refs. 38 and 39. 10–80 µg of cell extracts were separated by SDS-PAGE employing different percentages of acrylamide resolving gel, transferred to a nitrocellulose membrane, and incubated with different antisera (39). The immunocomplexes were detected by the enhanced chemiluminescent technique (Amersham Biosciences) as described in Refs. 38 and 39. Cdk2 and cdk4 activities were determined as reported in Ref. 40 by using histone H1 (cdk2) or pRb (cdk4) as phosphate acceptors.

Transient Transfection and Luciferase Activity Assay—The firefly luciferase reporter gene plasmids employed were kindly given by Dr. T. Sakai, Kyoto Prefectural University of Medicine, Japan, and their preparation and features described in Ref. 41. Particularly, one plasmid contains the full-length (2320 bases, pWWP) p21Cip1 promoter, while the other two plasmids contain either –124 bases (pWP124) or –60 bases (pWP60) of the promoter relative to the transcription start site. Mutagenesis of the Egr1 site in the p21Cip1 promoter region of the pWP124 plasmid (mpWP124) was performed with the QuickChange TM polymerase chain reaction-based method (Stratagene), using the following two primers 5'-AGGCGGGCCCGGGCTTTTCGGTTGTATATCAGG-3' and 5'-CCTGATATACAACCGAAAAGCCCGGGCCCCGGT -3' (from –70 to –38 base position of the promoter), which replaced four guanosine with four thymines (underlined). The plasmids were prepared and purified as reported in Ref. 42. The reported gene constructs were transiently transfected in K562 cells (60 ng/1.0 x 106 cells) by using the FuGENE 6 reagent. Five hours following transfection, cells were pelleted by centrifugation, washed, and resuspended in fresh medium. Each preparation was separated into 12 aliquots. 6 aliquots were treated with 30 µM resveratrol for 24 h, and 6 aliquots represent the relative controls. Luciferase activity was then determined on the cell lysate by using a luciferase kit from Promega. The activity was expressed as luciferase relative units per milligram of protein. The vacant vector pGL2-Basic was purchased from Promega and used for control reporter plasmid.

Biotin-Streptavidin Pull-down Assay—Four oligonucleotides, containing biotin on the nucleotide at 5'-position, were used in the pull-down assays. The sequences of these oligonucleotides were as follows: wild-type sense oligo, biotin-5'-AGGCGGGCCCGGGCGGGGCGGTTG-3' and wild-type antisense oligo, biotin-5'-CAACCGCCCCGCCCGGGCCCGCCT-3'. These sequences correspond to positions –70 to –53 of the human p21Cip1 promoter. Two scramble oligos were synthesized, which contain the same base composition of those reported above, but different sequences. The sequences were: biotin-5'-GGAGCGGTGGCGGTGGCGCGGCCG-3' (sense) and biotin-5'-CGGCCGCGCCACCGCCACCGCTCC–3' (antisense). These oligonucleotides were annealed, and 24-bp double-stranded oligonucleotides were gel purified and used. Nuclear proteins were extracted as described in the next paragraph. One microgram of each double-stranded oligonucleotide was incubated with 300 µg of nuclear proteins for 20 min at room temperature in a binding buffer containing 12% glycerol, 12 mM HEPES (pH 7.9), 4 mM Tris (pH 7.9), 150 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 10 µg of poly(dI-dC) competitor. Following the incubation, 30 µl of streptavidin-agarose beads (Pierce) were added to the reaction and incubated at 4 °C for 4 h. Prior to this step, 300 µl of the original streptavidinagarose bead preparation were preadsorbed with 500 µl of bovine serum albumin (1 mg/ml), 50 µg of poly(dI-dC), and 50 µg of sheared salmon sperm DNA for 30 min at 25 °C. The beads were washed three times and resuspended in 300 µl of the binding buffer. The protein-DNA-streptavidin-agarose complex was washed three times with binding buffer and loaded onto a SDS gel. Detection of Sp1 and Egr1 proteins was performed as described under immunoblotting.

Preparation of Nuclear Extracts and EMSAs—Nuclear extracts were prepared as follows. Briefly, the cells were suspended in three packed cell volumes of hypotonic buffer, 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol with protease inhibitors (0.05 mM phenylmethylsulfonyl fluoride, 1 mM each of pepstatin, leupeptin, and aprotinin) and allowed to swell for 10 min on ice. The cells were homogenized, transferred to new tubes, and centrifuged for 30 min at 10,000 x g. The released nuclei were suspended in half the packed cell volume of low salt buffer (20 mM HEPES, pH 7.9, 20 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 25% glycerol, 0.2 mM dithiothreitol, and the mixture of protease inhibitors), followed by the dropwise addition of high salt buffer (20 mM HEPES, pH 7.9, 0.6 M KCl, 1.5 mM MgCl2, 25% glycerol, 0.2 mM dithiothreitol, and the mixture of protease inhibitors). The nuclear suspensions were extracted for 30 min at 4 °C with gentle agitation, and centrifuged for 30 min at 14,000 x g. The supernatants (nuclear extracts) were stored at –80 °C in aliquots. EMSA was performed as described below. Prior to the addition of biotin-labeled double-strand DNA probe (the same employed in the pull-down experiments), 4 µg of K562 nuclear extracts were incubated for 20 min on ice in 20 µl of reaction buffer containing 10 mM HEPES, pH 7.5, 2.5 mM MgCl2, 50 mM NaCl, 0.5 mM dithiothreitol, 4% glycerol, 1 µg of double-stranded poly(dI-dC), and 1 µg of bovine serum albumin. Biotin-labeled probe was added (0.5 ng), and the incubation was continued for 20 min at room temperature. In competition experiments, the nuclear extracts were preincubated with the indicated molar excess of unlabeled, double-stranded oligonucleotides for 20 min on ice. In super-shift experiments, the extracts were preincubated with antibodies (anti-Egr1 or anti-Sp1) for 60 min on ice. Protein-DNA complexes were separated on nondenaturing polyacrylamide gels and observed by the LightShift Chemiluminescent EMSA kit following the manufacturer's procedure.

Chromatin Immunoprecipitation Assays—K562 cells (1.2 x 107 cells) were treated with 30 µM resveratrol for 6 h, collected by low speed centrifugation, and resuspended in 1% formaldehyde solution in 5 ml of RPMI 1640 fresh medium. After 15 min of incubation (with occasional inversion), the cross-linking was stopped by the addition of glycine to a final concentration of 0.125 M. Then, cells were washed with ice-cold TBS (150 mM NaCl, 20 mM Tris-HCl, pH 7.6), placed on ice and lysed with 1 ml radioimmune precipitation assay buffer (RIPA; 10 mM Tris-HCl, pH 8, 140 mM NaCl, 0.025% NaN3, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid), containing the protease inhibitors leupeptin (10 µg/ml), trypsin inhibitor (10 µg/ml), chymostatin (10 µg/ml), phenylmethylsulfonyl fluoride (1 mM), and phosphatase inhibitors Na3VO4 (0.2 mM), NaF (50 mM), NaPyr (50 mM), and incubated on ice for 1 h. Further disruption of the cells was obtained by passing them through a 21-gauge syringe needle. Cell lysates were then sonicated to yield chromatin fragments of ~500 bp, as assessed by agarose gel electrophoresis. Insoluble materials were removed by two centrifugations, the first for 5 min and the second for 15 min at 4 °C at 13,000 x g and the supernatants transferred to new tubes. 2-mg extracts were precleared by incubating them with 30 µl of packed protein A-agarose beads (pretreated with sheared DNA salmon sperm). After centrifugation for 2 min at 7500 x g, supernatants were transferred to fresh tubes. Immunoprecipitation was performed by rocking overnight at 4 °C the precleared extracts with the chosen antiserum, and then, by precipitating the immunocomplexes with protein A-agarose also pretreated with sheared DNA salmon sperm. The immunoprecipitates were washed once with 1 ml of RIPA buffer, once with 1 ml lysis buffer and once with 1 ml LiCl/detergent solution (0.5% deoxycholic acid, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 10 mM Tris-HCl, pH 8). Then, the beads were washed twice with 1 ml TBS and centrifuged at 7500g for 2 min. Elution of immunoprecipitates was performed in two steps: the beads were mixed with 100 µl of 1% SDS/TE and incubated at 65 °C for 10 min, centrifuged briefly and the eluates transferred to fresh tubes. The beads were then washed with 150 µl of 0.67% SDS/TE, briefly centrifuged and the washes were added to eluates. To reverse the formaldehyde cross-links, the samples were then incubated overnight at 65 °C in the presence of NaCl to a final concentration of 0.05 M. After this step, the samples were treated with 50 µg of proteinase K and incubated for 2 h at 37 °C. DNA was phenol-chloroform-extracted, ethanol-precipitated, allowed to air dry, and dissolved in 20 µl of sterile H2O. 5-µl DNA samples were then subjected to amplification by employing primers (5'-GCTGGCCTGCTGGAACTC-3', sense; 5'-GGCTCCACAAGGAACTGACT-3', antisense), which amplified the promoter region of p21Cip1 (from –190 to +25 respective to the transcription start).

Antisense Experiments—Two phosphorothioate antisense oligonucleotides were synthesized. One (5'-TCGGCCTTGGCCGCGGCCAT-3') was directed against the region (from 0 to 19) of Egr1 mRNA, whereas the other (5'-GCTCGTCCGAGTCGTCCTGC-3') was the control scramble oligonucleotide. 20 µl of FuGENE 6 were mixed with 0.5 ml of RPMI 1640 and then added dropwise to 20 µg of the antisense oligos. The sample was then added to 10 ml of K562 cells (400,000 cells/ml). A control experiment contains only FuGENE 6. After 12 h, 30 µM resveratrol was added, and cell growth continued: (i) for another 8 h after which cells were pelleted and analyzed for Egr1 and p21Cip1 expression or (ii) for another 24 h to evaluate cell growth.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Resveratrol Induces Growth Arrest and Differentiation of K562 Cells—When K562 cells were cultured in the presence of different amounts of resveratrol, a remarkable inhibition of the proliferation rate was observed (Fig. 1A). As low as a 30 µM concentration resulted in complete growth impairment, while at higher concentrations (100 µM) the molecule appeared slightly toxic, causing a small decrease in cell number. From these experiments, the estimated resveratrol I50 value was about 10 µM. Incubation of cells with stilbene did not cause a definite commitment toward a non-proliferative state. Indeed, when the cells were incubated for 24 or 48 h with 30 µM resveratrol and, thereafter, the drug was removed, a normal rate of proliferation was restored (data not shown). These results indicated that, under the conditions employed, the growth inhibition was powerful but rapidly reversible, and that the molecule interferes, although not irreversibly, with molecular process(es) required for cell proliferation.



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  		FIG. 1.
Effect of resveratrol on K562 cell proliferation, PARP proteolysis, and expression of {gamma}-globin and GpIIb genes. Panel A, K562 cells were plated at 300,000 cells/ml and incubated with or without different amounts of resveratrol. Cells were then counted daily. Panel B, K562 cells were incubated without (Con) and with different amounts of resveratrol for 24 h. A control experiment was performed by incubating the cells with etoposide (20 µM) for the reported time intervals. Cell extracts from 3 x 105 cells were then prepared as described under "Experimental Procedures" and separated by denaturing polyacrylamide gel electrophoresis. The proteins were then transferred to a nitrocellulose membrane and incubated with the specific antibody to PARP. The cleavage of PARP was demonstrated by the accumulation of the 85-kDa fragment with respect to the intact protein (signal at 115 kDa). Panel C, the expression of {gamma}-globin and GpIIb genes was performed by RT-PCR as reported under "Experimental Procedures." In this experiment, K562 cells were treated with 30 µM resveratrol. Con, control; Res, resveratrol.

 

In order to rule out the possibility that the cytostatic activity of resveratrol was caused by apoptosis, the resveratrol-treated K562 cells were analyzed by flow cytometry. The results indicated that only at 100 µM concentration a very low percentage of apoptotic cells was detectable (less than 10%) (data not shown). The flow cytometry analysis also indicated a remarkable decrease of G2/M peak and the accumulation of cells in G1/S phases (data not reported). The absence of the apoptotic process was also demonstrated by estimating the cleavage rate of PARP. The lack of PARP cleavage (namely the absence of the 85-kDa fragment in immunoblotting analysis) represents a further proof of the absence of (or almost undetectable) apoptosis (Fig. 1B). A control experiment was performed employing etoposide, a well known inducer of apoptosis (Fig. 1B).

Subsequently, we evaluated the differentiation activity of resveratrol. Since K562 cells might differentiate toward two different phenotypes, we analyzed the expression of genes specific for erythroid and megakaryocytic phenotypes, namely {gamma}-globin and GpIIb, respectively (43). As shown in Fig. 1C, only the expression of the {gamma}-globin gene was up-regulated by 30 µM reveratrol, while GpIIb gene transcription was completely unmodified or slightly down-regulated.

In summary, resveratrol, at concentrations comparable to those occurring in wine and grapes, causes a complete arrest of K562 cell proliferation. This effect is fully reversible, not due to the induction of apoptosis, and associated with the differentiation toward the erythroid phenotype.

Biochemical Analysis of Cell Division Cycle Engine in Resveratrol-treated K562 Cells—The results obtained by flow cytometry prompted us to clarify the precise transition of cell division cycle hindered by resveratrol addition. As is well known, the progression through the cell cycle is due to the timely regulated activation of cdks, a class of enzymes whose active form requires the binding between a catalytic subunit and an activating subunit, i.e. cyclin. Besides cyclin interaction, other regulatory events, including positive and negative phosphorylations and binding to cdk inhibitors (cki), control cdk enzymatic activity.

Analysis of cell extracts by immunoblotting, carried out at various time intervals (from 4 h to 2 days), demonstrated that the levels of several components of the G1 phase cell cycle machinery (i.e. p27Kip1, cyclin D2, cyclin D3, cdk4, cdk6, and cdk2) were totally unmodified by resveratrol addition (Fig. 2A and data not shown). Conversely, a remarkable increase of p21Cip1 was observed (Fig. 2A). The levels of p16INK4A and p15INK4B were not evaluated because our previous investigations demonstrated that the genes encoding these proteins are homozygously deleted in K562 cells (38).



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  		FIG. 2.
Effect of resveratrol on cki and cdc2 content of K562 cells. Panel A, K562 cells were incubated with 30 µM resveratrol for different time intervals. Cell extracts from 3 x 105 cells were then prepared as described under "Experimental Procedures" and p27Kip1 (p27) and p21Cip1 (p21) content determined by immunoblotting. Panel B, K562 cells were incubated for 5 h with different amounts of resveratrol (Res). p27Kip1 (p27), p57Kip2 (p57), p21Cip1 (p21) gene expression were determined by RT-PCR as reported under "Experimental Procedures." Panel C, an experiment similar to that reported in panel B was performed except that p21Cip1 (p21) expression was determined in cells incubated with resveratrol in the presence or absence of 36 µM cycloheximide (cyclohex). Panel D, K562 cells were incubated for 24 h without (Con) and with the reported amounts of resveratrol. The content of cdc2 was determined by immunoblotting as described under "Experimental Procedures."

 

Furthermore, we analyzed the mRNA level of the cdk inhibitors at 5 h after the addition of different amounts of resveratrol. As shown in Fig. 2B, a clear up-regulation of p21Cip1 expression was demonstrable, while the expression of p27Kip1 and p57Kip2 was totally unmodified. Surprisingly, the presence of cycloheximide completely abolished the induction of p21Cip1 gene transcription (Fig. 2C). The finding indicates that the resveratrol-dependent accumulation of p21Cip1 requires one (or more) newly synthesized protein(s), which, in turn, activate(s) cki gene expression.

Subsequently, we analyzed proteins controlling the successive phases of cell division cycle, namely cyclin E, cyclin A, cyclin B, and cdc2. No variation of cyclins was observed except a decrease of cyclin B (data not shown). Contemporaneously, a remarkable down-regulation of cdc2 level was observed in a time- (not shown) and dose-dependent fashion (Fig 2D). Finally, we analyzed cdk4 and cdk2 activities in the specific immunoprecipitates. Both the enzymatic activities decreased remarkably following resveratrol addition (data not reported).

In summary, the biochemical analysis of cell cycle engine demonstrates that: (i) resveratrol-treated K562 cells are unable to entry into S phase and (ii) the inhibition of growth and the accumulation of cells in G1 phase are associated with an early, but indirect, increase of p21Cip1 gene transcription.

Resveratrol Induces the Expression of the Egr1 Gene—Although the early p21Cip1 accumulation might be the cause of the resveratrol-dependent G1 arrest, the expression of the gene does not show a direct (i.e. in cis) regulation by pathways activated by the stilbene (Fig. 2C). Thus, we studied the effect of resveratrol on the expression of three genes encoding transcription factors, namely Egr1, GATA-1, and GATA-2. These genes were chosen because data demonstrate that they play pivotal roles in inducing cell growth arrest and differentiation during hematopoietic cell lineage (44–46). Moreover, data from our laboratory suggested that Egr1 is up-regulated by radical scavenger molecules (77).

We also evaluated the nuclear level of NF-{kappa}B protein in resveratrol-treated K562 cells. Indeed, the modulation of this transcription factor has been proposed as an early and important cellular response to the phytoalexin treatment (47).

As shown in Fig. 3A, a strong increase of Egr1 expression was observed after the addition of resveratrol, while the transcription of GATA-1 and GATA-2 was not influenced by the grape alexin (Fig. 3B). Moreover, the K562 nuclear content of NF-{kappa}B, i.e. the active form of this transcription factor, was unmodified by the stilbene treatment, thus suggesting that this protein is not involved in the resveratrol-induced growth arrest of erythroleukemic cells (Fig. 3C).



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  		FIG. 3.
Expression of GATA-1 and -2, and Egr1 in resveratroltreated K562 cells. K562 cells were incubated for 5 h with different amounts of resveratrol (Res). Egr1 (panel A), GATA-1, and GATA-2 (panel B) expression were determined by RT-PCR as reported under "Experimental Procedures." Panel C, K562 cells were incubated for 8 h without (Con) and with 30 µM resveratrol (Res). Then, nuclear extracts from 3 x 105 cells were prepared (see "Experimental Procedures") and NF-{kappa}B content determined by immunoblotting.

 

Time course experiments, performed in the presence or absence of cycloheximide, demonstrated that resveratrol induces an early and direct transcription of the Egr1 gene (after 1 h), which is followed by a second round of expression, observable only in the absence of cycloheximide (Fig. 4A). Therefore, this second peak of expression is due to the synthesis of a protein that further stimulates Egr1 gene transcription. A Western blot analysis also showed an identical pattern with two phases of protein accumulation (Fig. 4B). Finally, the resveratrol-induced Egr1 protein is completely localized in the nucleus, suggesting that the transcription factor is fully active (Fig. 4C).



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  		FIG. 4.
Egr1 content and localization in K562 cells grown in the presence of resveratrol. Panel A, K562 cells were incubated with 30 µM resveratrol (Res) for different time intervals in the presence or absence of 36 µM cycloheximide (Cyclohex). Then, Egr1 gene expression was determined by RT-PCR as reported under "Experimental Procedures." Panel B, K562 cells were incubated with 30 µM resveratrol for different time intervals. The content of Egr1 was determined by immunoblotting as described under "Experimental Procedures." Panel C, K562 cells were incubated with or without 30 µM resveratrol for 8 h. Then, nuclear and cytosolic extracts were prepared as described under "Experimental Procedures." The contents of Egr1 and histone deacetylase 1 (HDAC1) were determined by immunoblotting. HDAC1, a nuclear protein, was employed to confirm the purity of the cellular fractions.

 

Since the expression of Egr1 was observed as early as 15 min after the addition of resveratrol (data not shown), we were interested in identifying the phytoalexin-dependent transduction pathway(s) leading to the expression of the transcription factor. Thus, K562 cells were incubated with resveratrol plus a panel of kinase inhibitors, and then the Egr1 mRNA content was evaluated. As shown in Fig. 5A, the addition of PD98059 prevented the resveratrol-dependent expression of the Egr1 gene, thus suggesting that the phytoalexin activates the Erk1/2 pathway. This observation was then directly demonstrated evaluating Erk1/2 activation by immunoblot detection of the kinase phosphorylated forms (Fig. 5B). The other kinase inhibitors were inactive (i.e. SB203580, KN62, H89, and curcumin), allowing the exclusion of p38 kinase, Ca2+/calmodulin kinase, cAMP-dependent kinase, and JNK in the resveratrol-dependent Egr1 up-regulation. Importantly, all the employed kinase inhibitors are believed to be highly specific except curcumin, which in addition to interfering with the JNK pathway, has been reported to exert additional other metabolic effects. Thus, we decided to evaluate directly the activity of the stilbene on JNK activity by analyzing the level of phosphorylation of c-Jun, the major substrate of this kinase, after incubation of K562 cells with resveratrol at different time intervals. As shown in Fig. 5C, the natural antioxidant does not modify JNK activity, thus allowing the exclusion of this pathway in the up-regulation of Egr1 gene transcription.



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  		FIG. 5.
Effect of resveratrol on transduction pathways. Panel A, K562 cells were incubated for 1 h with (or without) the reported kinase inhibitors. Then, 30 µM resveratrol (Res) was added and the incubation continued for 5 h. Finally, Egr1 gene expression was determined by RT-PCR. Panel B, K562 cells were incubated with 30 µM resveratrol for the reported times. Then, the content of phospho-Erk1 and phospho-Erk2 (pErk1 and pErk2) was determined by immunoblotting. Subsequently, the same blot was stripped and analyzed for Erk1 and Erk2 level. Panel C, K562 cells were incubated for different time intervals with 30 µM resveratrol (Res). Then, the content of phosphoc-Jun and c-Jun was determined by immunoblotting. Panel D, effect of resveratrol on Egr1 content in HL-60 and LAN-5 cells determined by immunoblotting.

 

We also evaluated the effect of resveratrol on the expression of Egr1 in two other cellular model systems, namely HL-60 (a promyelocitic cell line) and LAN-5 (a neuroblastoma cell line) cells. As reported in Fig. 5D, the transcription factor level increased in both cell lines, indicating that resveratrol-dependent Egr1 accumulation is not restricted to K562 cells.

Egr1 Regulates the Expression of p21Cip1 Gene and K562 Proliferation—The finding that the transcription of p21Cip1 occurs in trans moved us to investigate further the direct involvement of Egr1 in the regulation of the cdk inhibitor gene expression. First, we wondered whether the phytoalexin-related expression of p21Cip1 was hindered by preincubation of cells with the kinase inhibitors described above. We observed that PD98059 prevented the up-regulation of p21Cip1 (Fig. 6A), while all the other kinase inhibitors did not (data not reported).



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  		FIG. 6.
Effect of resveratrol on p21Cip1 expression. Panel A, K562 cells were preincubated for 1 h with (or without) PD98059 (PD). Then, 30 µM resveratrol (Res) was added and the incubation continued for 5 h. Finally, p21Cip1 (p21) expression was determined by RT-PCR. Panel B, K562 cells were incubated for 5 h with (or without) 30 µM resveratrol (Res). Then, the content of Sp1 was determined by immunoblotting. Panel C, promoter sequence of the p21Cip1 gene. The putative Egr1 consensus sequence is shown in a box. The TATA sequence is also shown.

 

Subsequently, we analyzed the effect of resveratrol on the content of Sp1, a transcription factor thought to be a major regulator of p21Cip1 gene expression (41, 49–50). Immunoblotting analysis of Sp1 nuclear level in control and treated cells showed that the amount of the protein is not modified by the phytoalexin (Fig. 6B). This finding allowed us to rule out, at least in part, the hypothesis that resveratrol acts by stimulating the known Sp1-responsive elements localized on the p21Cip1 promoter region (41, 49–50).

A computer-aided analysis of the p21Cip1 promoter sequence (41, 49–50) resulted in the identification of a putative Egr1 consensus sequence (Fig. 6C). This finding, along with the above reported data, suggested to us the possibility of a direct linkage between Egr1 up-regulation and enhanced p21Cip1 gene transcription. In order to evaluate this possibility, we carried out a series of experiments that were aimed to confirm the interaction between Egr1 and the sequence identified on the cki gene promoter.

Initially, we evaluated in K562 cells the effect of resveratrol on the luciferase expression driven by constructs containing the wild-type or two deleted constructs of the p21Cip1 gene promoter. As shown in Fig. 7A, the induction-fold were identical in constructs containing the wild-type (2320 bases), 124, and 60 bases of promoter. Importantly, the –60-base plasmid (pWP60) includes the putative Egr1 binding sequence. In addition, the mutation of the Egr1 consensus sequence in the pWP124 plasmid (mpWP124) causes the loss of resveratrol activity.



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  		FIG. 7.
Interaction beween Egr1 and p21Cip1 promoter. Panel A, pWWP, pWP124, pWP60, and mpWP124 plasmids were transiently transfected into K562 cells, and luciferase activities were analyzed after 24 h of treatment with 30 µM resveratrol. Relative luciferase activity is shown as luciferase units in cell lysate/mg of protein. On the left, the schemes of each construct are reported, highlighting the Egr1 site and the TATA box. Data are shown as means (bars, S.D.) of at least three different experiments. Panel B, EMSAs were carried out as reported under "Experimental Procedures." Nuclear extracts were prepared from control cells (Con) and cells treated with 30 µM resveratrol (Res). A control experiment was performed with the addition of unlabeled oligos to the EMSA reaction mix. Supershift experiments were carried out employing anti-Sp1 ({alpha}Sp1) and anti-Egr1 ({alpha}Egr1) antibodies. Panel C, biotin-labeled oligonucleotide was incubated with nuclear extracts from control (Con) and resveratrol-treated (Res) K562 cells. The protein-DNA complexes were recovered as under "Experimental Procedures" and analyzed by immunoblotting using antibodies directed against Egr1. Panel D, K562 cells were treated for 24 h with or without 30 µM resveratrol. Then, chromatin was cross-linked with nuclear proteins and broken by ultrasound into small fragments. After immunoprecipitation with Egr1 antibodies, DNA was released from proteins and the occurrence of Egr1-bound p21Cip1 promoter was evaluated by PCR employing primers that amplified the Egr1 consensus sequence (see also "Experimental Procedures"). p21Cip1 prom, p21Cip1 promoter; {alpha}Egr1, samples immunoprecipitated by anti-Egr1 antibodies: IgG, control antiserum; Res, resveratrol; input, PCR of DNA occurring in control samples (diluted 100-fold); MW, molecular weight.

 

Then, we performed an EMSA analysis by incubating nuclear extracts from control and resveratrol-treated K562 cells with a biotinylated double-stranded oligonucleotide containing the sequence observed in Fig. 6C. As shown in Fig. 7B, a clear electrophoretic shift was observable only in the sample containing extracts from cells grown in the presence of the phytoalexin. A subsequent supershift analysis demonstrated that the resveratrol-induced transcription factor, interacting with the oligonucleotide, is Egr1 and not Sp1.

A further proof that Egr1 binds the promoter region of the p21Cip1 gene was obtained by means of the pull-down approach. In this experiment, we used the biotinylated double-stranded oligonucleotide as a bait. As reported in Fig. 7C, the DNA sequence precipitated specifically a remarkable amount of Egr1 protein, particularly in extracts from resveratrol-treated cells. Conversely, the binding of Sp1 factor to the sequence was neither significantly observed nor increased by resveratrol (data not shown).

Finally, chromatin immunoprecipitation experiments demonstrated that Egr1 protein binds in vivo to the p21Cip1 promoter. Indeed, the DNA immunoprecipitated by anti-Egr1 antibodies contains the –70 to –53 sequence of the p21Cip1 promoter (Fig. 7D). Importantly, the amount of promoter sequence precipitated by the antibodies increased remarkably after treatment of cells with resveratrol. A control rabbit antiserum did not precipitate this sequence (Fig. 7D).

Because the above reported experiments demonstrated clearly that Egr1 recognizes in vivo and in vitro a sequence occurring in the promoter region of the p21Cip1 gene, we wondered whether Egr1 is the major transcription factor responsible for the cdk inhibitor accumulation driven by resveratrol. Thus, we evaluated the effect of an Egr1 antisense phosphorothioate oligonucleotide on the expression of Egr1 and p21Cip1 after the addition of resveratrol. We observed that the antisense clearly hindered stilbene-dependent Egr1 mRNA up-regulation, thus demonstrating the efficacy of the molecule (Fig. 8A). A scramble phosphorothioate with the same base composition but different sequence was completely ineffective (Fig. 8A). Importantly, the antisense-dependent decrease of Egr1 mRNA also caused a down-regulation of p21Cip1 mRNA (Fig. 8A). This observation demonstrated a functional interplay between the two genes. Finally, the addition of Egr1 antisense also caused a significant reduction of the antiproliferative effect of resveratrol, while the control antisense was inactive (Fig. 8B). This result demonstrates a direct mechanistic connection not only between Egr1 and p21Cip1 gene expression, but also between Egr1 and resveratrol-dependent growth restraining activity.



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  		FIG. 8.
Effect of Egr1 antisense oligonucleotide on Egr1 and p21Cip1 expression level and resveratrol antiproliferative activity. Panel A, K562 cells were treated for 12 h with an Egr1 antisense phosphorothioate oligonucleotide (Egr1 AS) or a scramble antisense oligonucleotide (Scramble AS) and then, for 8 h, with or without 30 µM resveratrol. Subsequently, Egr1 and p21Cip1 (p21) expression was determined by RT-PCR. Panel B, K562 cells were plated at 3 x 105 cells/ml and treated for 12 h with Egr1 phosphorothioate antisense oligonucleotide or a scramble antisense oligonucleotide and then, for 24 h, with or without 30 µM resveratrol. The cell number was then counted and expressed as percentage of the initial value. Four independent experiments were performed, and the reported value is the mean. Bars represent S.D. of at least three independent experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study, devoted to investigate the molecular mechanism(s) of growth arrest and differentiation induced by resveratrol, yielded two main conclusions: (i) up-regulation of Egr1 transcription is a major effect of resveratrol activity and (ii) Egr1 is able to control p21Cip1 expression. This novel regulatory pattern is of particular interest since both Egr1 and p21Cip1 modulate a variety of cellular events endowed with relevant biological significance.

As reported in the Introduction, resveratrol is a natural stilbene able to induce growth impairment, differentiation, or apoptosis. Thus, the molecule has been considered a promising drug in cancer chemoprevention (51). Following oral administration in red wine, resveratrol pharmacokinetics has been described by an open one- or two-compartment model (52). Additional studies revealed that the stilbene is rapidly adsorbed at the intestinal level and that, during long term treatment, its potential concentration in plasma reaches values comparable to those efficacious in vitro (53). However, extensive investigations appear necessary to evaluate the plasma content of resveratrol, and of additional structurally similar antioxidant stilbenes, with different types of diets and pharmacological treatments. It is important to extrapolate in vivo the results obtained from in vitro molecular studies.

Initially, we observed that resveratrol inhibits the growth of K562 cells and induces their erythroid differentiation, in agreement with similar findings recently reported by others (54). A detailed analysis of the major components of cell cycle engine demonstrated that its antiproliferative activity is associated with an increased expression of the p21Cip1 gene, detectable both at the mRNA and protein level. The build-up of p21Cip1 results in a decrease of cdk2 and cdk4 activities, followed by accumulation of cells in G1 phase. Surprisingly, cycloheximide prevented the induction of p21Cip1 gene expression, suggesting the requirement of new protein(s) for the cki accumulation. This finding was totally unexpected, since the effect of a large number of molecules on the cki gene expression is associated with a direct control of gene transcription (i.e. in cis mechanism) (49, 50).

Although the increase of the p21Cip1 level might account for G1 phase arrest, the molecular mechanism(s) regulating its expression remained to be explained. Therefore, we evaluated the effects of the molecule on three transcription factors that play selective roles in growth arrest and differentiation of hematopoietic cells, namely Egr1, GATA-1, and GATA-2 (44, 46). Moreover, the nuclear levels of NF-{kappa}B were analyzed, since this transcription factor has been reported to be modulated by resveratrol in U937 cells (47). However, our results indicate that the expression of GATA-1 and -2, as well as the nuclear NF-{kappa}B content, are not influenced by resveratrol, while the phytoalexin induces uniquely a remarkable nuclear accumulation of Egr1. The Egr1 up-regulation is detectable as early as 15 min after resveratrol addition and follows a complex biphasic kinetics (Fig. 4B).

Egr1 (also known as NGFI-A, TIS8, Krox-24, and Zif268) is a member of the immediate early gene family and encodes a nuclear phosphoprotein involved in the regulation of cell growth and differentiation in response to a variety of growth factors and stress stimuli (55–59). Moreover, Egr1 is down-regulated in several types of neoplasias, suggesting that it can act as a tumor suppressor gene in analogy with WT-1, another immediate early gene (60, 61). Indeed, Egr1 protein is decreased or undetectable in human breast and small cell lung tumors (62, 63) as well as in an array of tumor cell lines (60, 64, 65). Gene deletions or mutations have also been reported in sporadic cancer cases (66). It is worth mentioning that a recent study demonstrated that Egr1 expression is strongly reduced in brain tumors compared with normal brain tissue, where the basal expression is high (67). Particularly, in brain cancer, Egr1 expression is suppressed in 87% of cases, independently of other alterations of cell cycle genes. These results indicate that the loss of Egr1 transcription might represent an important event of glial cell malignancy development and/or progression (67). In addition, forced re-expression of Egr1 suppresses the growth of transformed cells, both in soft agar and in athymic mice (63). Accordingly, studies with antisense vectors indicated that the transformed phenotype is enhanced by the inhibition of Egr1 expression (68). Altogether these findings indicate a consistent growth suppression role for Egr1, consistent with the described function of resveratrol chemopreventive activity.

Regarding the mechanism of resveratrol activity, we reached the conclusion that phytoalexin-dependent Egr1 buildup is exclusively attributed to the activation of the Erk1/Erk2 pathway. This finding: (i) confirms previous studies that demonstrate that Egr1 gene transcription is predominantly controlled by Erk1/2 kinases (69–71) and (ii) suggests that resveratrol might modulate the Ras -> Raf -> Erk1/2 pathway.

Overall, these data supported the hypothesis that up-regulation of Egr1 was functionally linked to the enhancement of p21Cip1 transcription. Two observations indirectly confirmed this view: namely, a computer-aided analysis of the p21Cip1 gene promoter region shows the presence of one Egr1 consensus sequence, and the inhibition of Erk1/2 activity prevents p21Cip1 gene transcription. The putative consensus sequence should be gcggggcg (from –58 to –51 relative to the starting transcription site), which lies in a region previously suggested to contain two Sp1 consensus sequences (41). It must be emphasized that, however, very frequently Egr1 consensus sequences overlap Sp1 consensus sequences (72, 73).

Several experimental approaches allowed us to convincingly demonstrate that our hypothesis is true. Indeed (i) transfection studies with the deleted gene promoter indicate that the resveratrol responding region of the p21Cip1 gene promoter is localized from the –60 to –1 position, (ii) pull-down experiments show a remarkable binding of Egr1 to its putative consensus sequences on the p21Cip1 promoter, (iii) EMSA experiments confirm that nuclear Egr1 interacts with the p21Cip1 promoter region, and (iv) chromatin immunoprecipitation analyses demonstrate that Egr1 binds in vivo to the cki promoter and that the interaction of the transcription factor increases after resveratrol treatment.

Finally, the Egr1 phosphorothioate antisense oligonucleotide used in our experiments prevents resveratrol-dependent Egr1 and p21Cip1 cellular accumulation and, at the same time, hinders the antiproliferative effect of the natural phytoalexin. This allows us to infer a mechanistic link not only between resveratrol-dependent K562 growth inhibition and Egr1 increase, but also between Egr1 activation and p21Cip1 up-regulation.

The control of p21Cip1 expression represents a central and critical mechanism for driving cells toward different paths, such as proliferation, growth arrest, differentiation, or apoptosis. Thus, Egr1 can be added to the few transcription factors regulating p21Cip1 expression, including p53, Sp1, AP2, Smad, and the Myc-Miz complex (48, 74–76).

In conclusion, the present study contributes to insights into the molecular mechanisms of resveratrol-dependent growth impairment and, in general, of its chemopreventive activity. Indeed, a number of intervention trials are investigating its potential usefulness in the treatment of cardiovascular diseases, as well as cancer and AIDS. In addition, our data indicate that Egr1 might represent an interesting molecular target for therapy and chemoprevention and that the level of the transcription factor might be manipulated by resveratrol, a molecule devoid of significant toxic side effects.


   FOOTNOTES
 
* This work was supported in part by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC) and from MURST (Progetti di Rilevante Interesse Nazionale). 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. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics F. Cedrangolo, Second University of Naples, Via Costantinopoli, 16, 80138, Naples, Italy. Tel.: 39-081-5665812; Fax: 39-081-441688; E-mail: fulvio.dellaragione{at}unina2.it.

1 The abbreviations used are: resveratrol, 3,5,4'-trihydroxystilbene; EMSA, electrophoretic mobility shift assay; PARP, poly(ADP-ribose) polymerase; JNK, c-Jun N-terminal kinase; Erk, extracellular signal-regulated kinase; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; oligo, oligonucleotide; cdk, cyclin-dependent kinase; cki, cdk inhibitor. Back


   ACKNOWLEDGMENTS
 
We thank Dr. T. Sakai, Kyoto Prefectural University of Medicine, Japan, for the firefly luciferase reporter gene plasmids.



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