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CELLULAR AND MOLECULAR

Peroxisome Proliferator-Activated Receptor Ligands Affect Growth-Related Gene Expression in Human Leukemic Cells

Departments of Medicine and Experimental Oncology (S.L., S.P., F.B., A.F., M.M., P.R., M.U.D., G.B.) and Anatomy, Pharmacology, and Forensic Medicine (C.F.), University of Turin, Turin, Italy

Received January 14, 2003; accepted March 7, 2003.

	Abstract

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Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors. Three subtypes of PPARs (, , and ) have been identified in different tissues. PPAR and PPAR ligands inhibit cell proliferation and induce differentiation in several human cell models. We demonstrated that both PPAR (clofibrate and ciprofibrate) and PPAR ligands (troglitazone and 15 deoxy-prostaglandin J2, 15d-PGJ2) inhibited growth, induced the onset of monocytic-like differentiation, and increased the proportion of G₀/G₁ cells in the HL-60 leukemic cell line. Moreover, 3 days after the treatment with 2.5 µM 15d-PGJ2, an increase in sub-G₀/G₁ population occurred, compatible with an induction of programmed cell death. To clarify the mechanisms involved in HL-60 growth inhibition due to the effects of PPAR ligands, we investigated their action on the expression of some genes involved in the control of cell proliferation, differentiation, and cell cycle progression such as c-myc, c-myb, and cyclin D1 and D2. Clofibrate (50 µM), ciprofibrate (50 µM), and 15d-PGJ2 (2.5 µM) inhibited c-myb and cyclin D2 expression, whereas they did not affect c-myc and cyclin D1 expression. Only troglitazone (5 µM) decreased c-myc mRNA and protein levels, besides decreasing c-myb and cyclin D2. The down-regulations of c-myb and cyclin D2 expression represent the first evidence of the inhibitory effect exerted by PPAR ligands on these genes. Moreover, the inhibition of c-myc expression by troglitazone may depend on a PPAR-independent mechanism.

Although the ability of PPAR ligands to inhibit cell growth and to induce cell differentiation has been demonstrated in several cell lines (Demetri et al., 1999; Moore et al., 2001), neither the mechanism by which PPAR ligands inhibit cell growth nor the mechanism involved in differentiation induction has been established conclusively. In particular, the effect displayed by PPAR ligands on c-myc expression was controversial. Troglitazone, a synthetic ligand of PPAR, inhibits c-myc expression in myeloid leukemia cells (Yamakawa-Karakida et al., 2002), and 15-deoxy-prostaglandin J2 inhibits N-myc expression in neuroblastoma cells (Marui et al., 1990), whereas it does not decrease c-myc expression in vascular smooth muscle cells (Okura et al., 2000). No literature data exist regarding the effect of PPAR or PPAR ligands on the expression of c-myb, another important transcription factor involved in the control of proliferation and differentiation (Oh and Reddy, 1999). Moreover, the effect of these substances in inhibiting cell cycle progression has been documented (Scatena et al., 1999; Kawakami et al., 2002). Fibrates, in dose-dependent manner, significantly alter the cell cycle distribution, mainly leading to G₀/G₁ phase increment and G₂/M phase reduction in human leukemic cell lines (Scatena et al., 1999). Troglitazone arrests U937 cells in the G₁ phase of the cell cycle (Asou et al., 1999) and inhibits cyclin D1 expression in MCF7 cells (Yin et al., 2001). However, recent findings demonstrate that some mechanisms in cell growth regulation are affected by PPAR ligands through a PPAR-independent action (Palakurthi et al., 2001; Lennon et al., 2002).

To clarify the mechanisms involved in PPAR-induced HL-60 growth inhibition due to the effects of PPAR ligands, we investigated the action of two PPAR ligands (clofibrate and ciprofibrate) and two PPAR ligands (troglitazone and 15d-PGJ2) on the expression of some genes involved in the control of cell proliferation, differentiation, and cell cycle progression such as c-myc, c-myb, and cyclin D1 and D2. Because PPAR demonstrated opposite action on cell proliferation, it has not been investigated in this study.

	Materials and Methods

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Cells and Culture Conditions. HL-60 cells were cultured at 37°C in a humidified atmosphere of 5% CO₂, air using RPMI 1640 medium supplemented with 2 mM glutamine, antibiotics, and 10% fetal calf serum (Biochrom AG Seromed, Berlin, Germany). Growth rate and cell viability were monitored daily by the trypan blue exclusion test (Sigma-Aldrich, Milano, Italy).

PPAR Ligand Treatments. Clofibrate (Sigma-Aldrich), ciprofibrate (Sigma-Aldrich), troglitazone [generous gift from Dr. Fabio Marra (University of Florence, Florence, Italy)] and 15-deoxy-prostaglandin J2 (Calbiochem, La Jolla, CA) were prepared in stock solutions 100x in ethanol (final concentration of ethanol in flask, 0.8%) and added at different concentrations to cell suspension (200,000 cells/ml). Control cells were treated with the vehicle alone (0.8% ethanol).

Detection of Differentiation-Associated Surface Antigens. Expression of the cell surface antigen CD14 was tested by immunofluorescence and detected by fluorescence microscopy. Cells were washed twice with PBS and then incubated with mouse monoclonal fluorescein isothiocyanate-conjugated antibody (Sigma-Aldrich) directed against CD14 (clone UCHM-1). After incubation with the antibodies, 3 x 10⁶ cells/sample were pelleted, resuspended in 1 ml of 0.1% sodium azide in PBS, layered onto a slide, covered with a coverslip, and scored for fluorescence in microscopy (Dialux; Leitz, Wetzlar, Germany). At least 100 cells were counted for each experiment (three separate experiments from three different preparations for each condition).

Flow Cytometric Analysis. HL-60 (10 x 10⁶ cells) were centrifuged at 1000 rpm for 10 min at 4°C, and the cell pellets were fixed in 70% ice-cold ethanol for 1 h at 4°C. The supernatant was centrifuged at 3000 rpm for 10 min at 4°C and fixed in 70% ice-cold ethanol for 1 h at 4°C. After centrifugation, both pellets were washed once with PBS, collected in one tube, and then treated with 0.4 mg/ml RNase (type 1-A; Sigma-Aldrich) for 30 min at 37°C. Propidium iodide (Sigma-Aldrich) was added to a final concentration of 18 µg/ml and incubated for at least 5 min at room temperature before analyzing in a FACScan cytometer (BD Biosciences, San Jose, CA), equipped with an argon ion laser tuned at 488 nm (ModFit LT 3.0 software).

RNA Isolation and Semiquantitative RT-PCR Analysis. RNA analyses were performed by a semiquantitative PCR method as described previously (Pizzimenti et al., 1999). Briefly, the experimental strategy included the following precautions: 1) the number of PCR cycles was kept low to obtain an exponential amplification of PCR products; 2) all results were standardized using the signal obtained with L7 (large ribosomal subunit protein L7); 3) all experiments were performed with at least three independent cDNA preparations; and 4) to control for DNA contamination, primers were designed to span at least one exon-intron boundary. Total RNA was isolated using the TRIzol kit (Invitrogen, Milano, Italy). cDNA synthesis was performed with 4 µg of total RNA in a reaction volume of 40 µl containing 1.25 µg of oligonucleotide (dT) primer; l mM of dATP, dGTP, dCTP, and dTTP (Amersham Biosciences Italia, Cologno Monzese, Italia); 66 units of RNAsin (Promega Italia s.r.l., Milano, Italy); 8 µl of 5x first-strand buffer; 10 mM dithiothreitol; and 300 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen). Samples were incubated for l h at 37°C and the reaction was stopped by heating for l0 min. at 95°C. PCR reactions were performed in a GeneAmp PCR System 9600 (PerkinElmer), with 1 µl of cDNA reaction mixture in a volume of 50 µl containing 200 µM of dATP, dTTP, dGTP, and dCTP; 1 µM of 5' and 3' primer; and 1.25 units of TAQ DNA polymerase (Polymed, Firenze, Italy). Samples were subjected to denaturation at 94°C for 30 s, annealing for 30 s (the annealing temperature was 60°C for L7, D2, and c-myc primers, 63°C for c-myb, and 70°C for D1 primers) and extension at 72°C for 30 s, followed by a final extension at 72°C for 10 min. Negative controls contained water instead of cDNA. The primer pair sequences used for PCR amplification and the numbers of PCR cycles done are indicated as follows: c-myc, 20 cycles: forward primer 5'-GAGACAACGACGGCGGTG-3' and reverse primer 5'-GCTCGTTCCTCCTCTGGC-3', amplifying a 788-bp fragment; c-myb, 18 cycles: forward primer 5'-TGGACAGAAGAGGAAGACAGAA-3' and reverse primer 5'-GCAGAGATGGAGTGGAGTGG-3', amplifying a 633-bp fragment; cyclin D1, 28 cycles: forward primer 5'-GCCAACCTCCTCAACGACCGG-3' and reverse primer 5'-GTCCATGTTCTGCTGGGCCTG-3', amplifying a 743-bp fragment; cyclin D2, 24 cycles: forward primer 5'-CCGCCGGGCTTGGCCAT-3' and reverse primer 5'-CTTTCGGCCCAACTGGCATCC-3', amplifying a 905-bp fragment; and L7, 18 cycles: forward primer 5'-ATGGAGGGTGTAGAAGAGAA-3' and reverse primer 5'-AATCATGGTAGACACCTTAG-3', amplifying a 764-bp fragment.

A l0-µl sample of the PCR reaction mixture was separated on a 1% agarose gel and amplification products were stained with GelStar nucleic acid gel staining (FMC Bioproducts, Rockland, ME). Densitometric analysis was performed by using a software program (Multi-Analyst, version 1.1; Bio-Rad, Segrate, Italy).

Preparation of Total Extracts and Western Blot Analysis. Cells (10 x 10⁶) were washed twice in cold PBS, pH 7.4. Total extracts were prepared by lysis in a buffer containing Tris-HCl buffer, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 0.05% aprotinin. Insoluble proteins were discarded by high-speed centrifugation at 4°C. Protein concentration in the supernatant was measured in triplicate using a commercially available assay (Bio-Rad).

All proteins were separated by SDS-polyacrylamide gel electrophoresis and electroblotted on nitrocellulose membrane (Bio-Rad Laboratories). Membranes were blocked overnight at 4°C in Trisbuffered saline containing 5% milk plus 0.5% Tween 20 and then incubated at room temperature with primary (anti-c-myc clone 9E10, anti-cyclin D1 clone HD11, anti-cyclin D2 clone C-17; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; anti c-myb clone 1-1; Upstate Biotechnology, Lake Placid, NY; anti -actin clone AC-15; Sigma-Aldrich) and horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Detection was carried out by enhanced chemiluminescence according to the manufacturer's protocol (Amersham Biosciences Inc., Italia, Cologno Monzese, Italy). Densitometric analysis was performed by using a software program (Multi-Analyst, version 1.1, Bio Rad). All results were standardized using the signal obtained with -actin.

	Results

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PPAR and PPAR Ligands Inhibit HL-60 Cell Growth and Induced CD14 Expression. The growth of HL-60 cells treated with clofibrate, ciprofibrate, troglitazone, and 15d-PGJ2 is shown in Fig. 1. The effect on cell growth was dose-dependent for all the substances used, and the effectiveness in inhibiting growth was higher for the PPAR ligands (in particular, for the 15d-PGJ2) than for PPAR ligands. According to previous results (Pizzimenti et al., 2002), the highest doses of PPAR ligands induced the onset of CD14 expression starting from day 4 to day 6 after the treatment. In fact, after clofibrate and ciprofibrate (50 µM), troglitazone (5 µM), and 15d-PGJ2 (2.5 µM) treatments, the values of CD14 positive-cells were between 28 and 42.5% at day 4. These values increased in the following days, except after 50 mM ciprofibrate treatment, where at days 5 and 6 the number of CD14 positive-cells decreased (Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1. A, growth of HL-60 cells treated with clofibrate at the indicated concentrations (10 and 50 µM). B, growth of HL-60 cells treated with ciprofibrate at the indicated concentrations (10 and 50 µM). C, growth of HL-60 cells treated with troglitazone at the indicated concentrations (2.5 and 5 µM). D, growth of HL-60 cells treated with 15d-PGJ2 at the indicated concentrations (1 and 2.5 µM). C, control cultures; C + EtOH: cultures treated with 0.8% ethanol. Data are the mean ± S.D. of five separate experiments. Variance analysis: *, p < 0.05; **, p < 0.01 versus C + EtOH.

The reduction of cell growth by PPAR ligand treatment may depend on growth-related gene modulation or cell death induction. Necrosis has been excluded by the trypan blue exclusion test, which indicated similar number of trypan blue-positive cells in control and treated cell populations. Previous results demonstrated that high doses of clofibrate (100 µM), troglitazone (50 µM), and prostaglandin J2 (10 µM) induced apoptosis in 15 to 20% of the HL-60 cell population at day 1 after the treatment (Pizzimenti et al., 2002). To investigate the possibility that lower PPAR ligand concentrations, although able to inhibit cell growth, may induce programmed cell death in the days after the treatment, we performed a cell cycle analysis with particular regard to the individuation of sub-G₀/G₁ population.

Effect of PPAR and Ligands on Cell Cycle Distribution of HL-60 Cells. Cell cycle analysis demonstrated that both PPAR and PPAR ligands induced an increase of cells in the G₀/G₁ phase of cell cycle (Fig. 2). This phenomenon was more evident at day 3 where the percentage of G₀/G₁ cells was 41% in the control cells, 61% in cells treated with 50 µM clofibrate, 55% in cells treated with 50 µM ciprofibrate, 60% in cells treated with 5 µM troglitazone, and 70% in cells treated with 2.5 µM 15d-PGJ2.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Effect of 50 µM clofibrate (CLOF), 50 µM ciprofibrate (CIPROF), 5 µM troglitazone (TG), and 2.5 µM 15d-PGJ2 on cell cycle distribution at different time points (8, 24, 48, and 72 h), compared with untreated control cells (C). HL-60 cells were stained with propidium iodide, as described under Materials and Methods and analyzed by flow cytometry. Values are the mean ± S.D. of three different cell preparations.

Figure 3 shows that the sub-G₀/G₁ population is 3-fold increased in 15d-PGJ2-treated cells 3 days after the treatment, whereas other PPAR ligands did not increase the sub-G₀/G₁ population. This action of 15d-PGJ2 is already evident at days 1 and 2 (data not shown), where the sub-G₀/G₁ population was 27 and 33%, respectively, whereas the control values were similar to those detected at day 3.

View larger version (19K): [in this window] [in a new window]   Fig. 3. FACScan analysis of cell cycle and apoptosis. A, cytofluorimetric histograms of cells collected at 72 h, representative of different cell preparations, are shown. Cells were treated with 50 µM clofibrate, 50 µM ciprofibrate, 5 µM troglitazone, and 2.5 µM 15d-PGJ2 or untreated (control). B, analysis of hypodiploid cell population. Data represent the sub-G0/G1 population identified on the basis of fluorescence intensity and are the mean ± S.D. from three different cell preparations.

Effect of PPAR Ligands on Oncogene Expression. The effect of 50 µM clofibrate on c-myc, c-myb, and cyclin D1 and D2 mRNA levels is shown in Fig. 4. Clofibrate inhibited c-myb and cyclin D2 expression starting from 8 h after its addition, whereas it did not affect c-myc and cyclin D1 expression. A similar effect was displayed by 50 µM ciprofibrate (Fig. 5).

View larger version (37K): [in this window] [in a new window]   Fig. 4. A, c-myc, c-myb, cyclin D1 (D1), and cyclin D2 (D2) mRNA levels were determined by RT-PCR in HL-60 cells treated with 50 µM clofibrate and collected at the indicated times after the beginning of treatment. B, quantification of RT-PCR products was performed by densitometric scanning. Data are normalized using the L7 (large ribosomal subunit protein 7) signal and represent the mean ± S.D. from three independent experiments. Values are expressed as a percentage of control value.

View larger version (41K): [in this window] [in a new window]   Fig. 5. A, c-myc, c-myb, cyclin D1 (D1), and cyclin D2 (D2) mRNA levels were determined by RT-PCR in HL-60 cells treated with 50 µM ciprofibrate and collected at the indicated times after the beginning of treatment. B, quantification of RT-PCR products was performed by densitometric scanning. Data are normalized using the L7 (large ribosomal subunit protein 7) signal and represent the mean ± S.D. from three independent experiments. Values are expressed as a percentage of control value.

PPAR ligands (troglitazone and 15d-PGJ2) displayed different patterns in the modulation of mRNA levels. Troglitazone (5 µM) transiently inhibited both c-myc and c-myb oncogene expression, mainly at 8 to 24 h after the treatments, and cyclin D2 not until 48 h after the treatment (Fig. 6). On the contrary, 2.5 µM 15d-PGJ2 did not inhibit c-myc expression. This substance, similarly to PPAR ligands, affected c-myb and cyclin D2 expression (Fig. 7). The inhibition of c-myb expression was transient (the nadir was observed after 8 h from the treatment), such as observed after troglitazone treatment. In all cases cyclin D1 expression was not affected.

View larger version (37K): [in this window] [in a new window]   Fig. 6. A, c-myc, c-myb, cyclin D1 (D1), and cyclin D2 (D2) mRNA levels were determined by RT-PCR in HL-60 cells treated with 5 µM troglitazone and collected at the indicated times after the beginning of treatment. B, quantification of RT-PCR products was performed by densitometric scanning. Data are normalized using the L7 (large ribosomal subunit protein 7) signal and represent the mean ± S.D. from three independent experiments. Values are expressed as a percentage of control value.

View larger version (38K): [in this window] [in a new window]   Fig. 7. A, c-myc, c-myb, cyclin D1 (D1), and cyclin D2 (D2) mRNA levels were determined by RT-PCR in HL-60 cells treated with 2.5 µM 15d-PGJ2 and collected at the indicated times after the beginning of treatment. B, quantification of RT-PCR products was performed by densitometric scanning. Data are normalized using the L7 (large ribosomal subunit protein 7) signal and represent the mean ± S.D. from three independent experiments. Values are expressed as a percentage of control value.

The analysis of the protein content after PPAR ligands treatment of HL-60 cells was performed by Western blot, at the same times of mRNA content analysis. Clofibrate (50 µM) induced a complete disappearance of the c-myb protein 8 to 24 h after addition, as well as strong inhibition of cyclin D2 expression starting from 8 to 48 h (Fig. 8). Similar effects were displayed by 50 µM ciprofibrate, except that cyclin D2 inhibition was transient (Fig. 9). Troglitazone (5 µM) transiently decreased the protein concentration of c-myc and c-myb (from 8 to 24 h for c-myc and only at 8 h for c-myb), and progressively decreased, starting from 24 h, the level of cyclin D2 protein (Fig. 10). Results obtained in protein extracts derived from 15d-PGJ2-treated cells, confirmed the patterns obtained by PCR. 15d-PGJ2 (2.5 µM) transiently reduced the c-myb protein level (from 8 to 24 h) and induced a progressive reduction of cyclin D2 protein (Fig. 11).

View larger version (35K): [in this window] [in a new window]   Fig. 8. A, Western blot analysis of c-MYC, c-MYB, cyclin D1 (D1), and cyclin D2 (D2) protein levels in HL-60 cells treated with 50 µM clofibrate and collected at the indicated times after the beginning of treatment. Equal protein loading was confirmed by exposure of the membranes to the anti--actin antibody. B, relative densitometric values of c-MYC, c-MYB, cyclin D1, and cyclin D2 protein levels. Quantification of protein products was performed by densitometric scanning. Data are normalized using the -actin signal and are indicated as mean ± S.D. from three independent experiments and are expressed as a percentage of control value.

View larger version (36K): [in this window] [in a new window]   Fig. 9. A, Western blot analysis of c-MYC, c-MYB, cyclin D1 (D1), and cyclin D2 (D2) protein levels in HL-60 cells treated with 50 µM ciprofibrate and collected at the indicated times after the beginning of treatment. Equal protein loading was confirmed by exposure of the membranes to the anti--actin antibody. B, relative densitometric values of c-MYC, c-MYB, cyclin D1, and cyclin D2 protein levels. Quantification of protein products was performed by densitometric scanning. Data are normalized using the -actin signal and are indicated as mean ± S.D. from three independent experiments and are expressed as a percentage of control value.

View larger version (35K): [in this window] [in a new window]   Fig. 10. A, Western blot analysis of c-MYC, c-MYB, cyclin D1 (D1), and cyclin D2 (D2) protein levels in HL-60 cells treated with 5 µM troglitazone and collected at the indicated times after the beginning of treatment. Equal protein loading was confirmed by exposure of the membranes to the anti--actin antibody. B, relative densitometric values of c-MYC, c-MYB, cyclin D1, and cyclin D2 protein levels. Quantification of protein products was performed by densitometric scanning. Data are normalized using the -actin signal and are indicated as mean ± S.D. from three independent experiments and are expressed as a percentage of control value.

View larger version (37K): [in this window] [in a new window]   Fig. 11. A, Western blot analysis of c-MYC, c-MYB, cyclin D1 (D1), and cyclin D2 (D2) protein levels in HL-60 cells treated with 2.5 µM 15d-prostaglandin J2 and collected at the indicated times after the beginning of treatment. Equal protein loading was confirmed by exposure of the membranes to the anti--actin antibody. B, relative densitometric values of c-MYC, c-MYB, cyclin D1, and cyclin D2 protein levels. Quantification of protein products was performed by densitometric scanning. Data are normalized using the -actin signal and are indicated as mean ± S.D. from three independent experiments and are expressed as a percentage of control value.

	Discussion

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Among the gene expressions tested in this study, only c-myb and cyclin D2 gene expression were inhibited by both PPAR and PPAR ligands. However, the effective concentrations are higher for PPAR ligands with respect to PPAR ligands, according to that observed on cell growth inhibition and cell differentiation induction.

Cell cycle analysis indicates that an increase of G₀/G₁ cells occurs in the culture treated with PPAR ligands, and in particular with 15d-PGJ2, according to data reported by others (Scatena et al., 1999; Kawakami et al., 2002). 15d-PGJ2 also increases the sub-G₀/G₁ population 2 and 3 days after the treatment.

The down-regulations of c-myb and cyclin D2 expression represent the first evidence of the inhibitory effect exerted by PPAR ligands on these genes. The myb gene family (whose members are A-myb, B-myb, and c-myb) encodes nuclear protein that functions as a transcriptional transactivator (Oh and Reddy, 1999). Expression of these genes is cell cycle-regulated, and inhibition of their expression with antisense oligonucleotides has been found to affect cell cycle progression, cell division, and/or differentiation (Raschella et al., 1992). Inhibition of c-myb expression by compounds inducing differentiation has been widely studied in leukemic cells (Kuehl et al., 1988) and c-myb down-regulation accompanied the cessation of growth and the onset of differentiation markers (Yen et al., 1992). Our results also indicate that PPAR ligands induce the monocytic differentiation of HL-60 cells, as measured by CD14 expression, at the same dose effective in decreasing c-myb mRNA and protein, suggesting that these two phenomena may be linked.

Cyclin D2 expression is also inhibited by the PPAR ligands. According to previous observation in the HL-60 cell model, cyclin D1 expression was not affected by PPAR ligand treatment, in contrast to that observed in pancreatic (Toyota et al., 2002) and in ras-transformed rat intestinal epithelial cells (Kitamura et al., 2001). In cell culture D-type cyclins, which show tissue specific expression, do not seem to functionally overlap (Sherr, 1995). In HL-60 cells, cyclin D1 and D2 are down-regulated during differentiation, whereas cyclin D3 is up-regulated (Bartkova et al., 1998). We restricted our observation to the D1 and D2 cyclins, because their role in differentiation is better defined. Despite their importance in the control of growth, cell cycle progression, and development, the exact role played by each cyclin D-type is not yet understood. Individual knockout of D1 or D2 genes in mice does not affect the overall development of the animal but rather affects the development of specialized tissues and cell lineages (Fantl et al., 1995; Sicinski et al., 1995).

According to previous observations in the HL-60 cell model (Pizzimenti et al., 1999), our results suggest that the inhibition of cyclin D2 expression induced by PPAR ligands contributes to the cessation of proliferation and to the onset of differentiation.

The major part of PPAR actions in stimulating gene expression depends on the binding between PPAR (after dimerization with retinoic X receptor ) and the PPRE sequences located on the promoter of target genes. Agonists stimulate binding of PPAR to PPRE (Schlezinger et al., 2002). Some PPRE sequences are identical for PPAR and PPAR (i.e., the UDP-glucuronosyltransferase 1A9 enzyme) (Barbier et al., 2003); others are differentially regulated by PPAR and PPAR ligands (i.e., the expression of uncoupling proteins, UCP 1) (Teruel et al., 2000). A PPAR indirectly dependent mechanism has been postulated for the FAT/CD36, which is activated by PPAR and PPAR ligands in absence of PPRE in the responding upstream promoter region (Sato et al., 2002).

PPAR indirectly dependent mechanisms have also been demonstrated for the inhibitory action displayed by PPAR on some growth regulatory genes, i.e., cyclin D1 repression by PPAR involved competition for limiting the abundance of p300 through a c-Fos binding site of the cyclin D1 promoter; 15d-PGJ2 enhanced recruitment of p300 to PPAR but reduced the binding to c-Fos (Wang et al., 2001). Other authors reported that PPAR ligands attenuated the mitogen-induced degradation of p21 and p27, two important cyclin/cyclin-dependent kinase inhibitory proteins (Wakino et al., 2000). In spite of the amount of evidences accumulated in these past years about the PPAR antiproliferative action, the mechanism whereby PPAR mediates growth inhibition and, in particular, growth-related gene expression inhibition, has yet to be elucidated. Certainly, it seems to be different in relation to cell type (Berger and Moller, 2002). Our results demonstrated that both PPAR and ligands inhibited c-myb and cyclin D2 expression in human leukemic cells. Because no PPRE sequences have been found on the promoter of c-myb and cyclin D2 gene, we can hypothesize a PPAR indirectly dependent mechanism that involved the modulation of transcription factor activity. Recently, it has been demonstrated that signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of the cyclin D2 gene (Friedrichsen et al., 2003), and PPAR ligands suppress Janus tyrosine kinase-signal transducer and activator of transcription signaling (Park et al., 2003). Likewise, PPAR (Pahan et al., 2002) and PPAR (Strauss et al., 2000) ligands inhibited activation of nuclear factor-B and AP-1, two transcription factor involved in the regulation of c-myb expression (Suhasini et al., 1997).

Among the PPAR ligands tested, only troglitazone affects c-myc mRNA and protein levels. The inhibition of c-myc expression, observed after troglitazone treatment, has been also confirmed by other works (Shimada et al., 2002; Yamakawa-Karakida et al., 2002). Some authors (Yamakawa-Karakida et al., 2002) suggest that the down-regulation of c-myc expression by this ligand can be linked to apoptosis induction. However, our data suggest that the inhibition of c-myc expression by this ligand can contribute to growth inhibition and differentiation induction rather than apoptosis induction, because troglitazone was used at nonapoptotic doses. Interestingly, from our results, it arises that neither the PPAR ligands (clofibrate and ciprofibrate) nor the natural PPAR ligand, 15d-PGJ2, inhibited c-myc expression. Thus, it is possible that the inhibition of c-myc mRNA and protein expression, in troglitazone-treated cells, may depend on a PPAR-independent mechanism, through the recruitment of free Tcf-4 and thus the inhibition of Tcf-4 binding to c-myc promoter, as suggested by Yamakawa-Karakida et al. (2002). On the other hand a PPAR-independent mechanism has been demonstrated for other important cell functions modulated by troglitazone, such as the activation of mitogen-activated protein kinase cascade (Lennon et al., 2002) and the inhibition of translation initiation (Palakurthi et al., 2001).

In conclusion, our results demonstrate that PPAR ligands inhibit HL-60 cell proliferation and induce differentiation through the down-modulation of nuclear transcription factors (c-myc and c-myb) and cyclin D2 expression. The greater effect on cell growth inhibition, displayed by 15d-PGJ2, can be also ascribed to the induction of programmed cell death, as indicated by the increase in sub-G₀/G₁ cell population. Moreover, we cannot exclude that PPAR ligands, which affect cell growth and gene expression at lower doses, may also affect other growth-regulatory gene expressions and thus inhibit the cell growth with higher effectiveness.

	Acknowledgements

 
We thank Ronald Medina for the careful revision of the article.

	Footnotes

 
This work was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica (Cofin. 1999) and Turin University (EX-60% 2002).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.049098.

ABBREVIATIONS: PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferation responsive element; 15d-PGJ2, 15 deoxy-prostaglandin J2; RT-PCR, reverse transcription-polymerase chain reaction; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; bp, base pair(s).

Address correspondence to: Prof. Giuseppina Barrera, Dipartimento di Medicina e Oncologia Sperimentale, Sezione di Patologia Generale, Corso Raffaello, 30; 10125 Torino, Italy. E-mail: giuseppina.barrera{at}unito.it

	References

Top Abstract Materials and Methods Results Discussion References

 

Asou H, Verbeek W, Williamson E, Elstner E, Kubota T, Kamada N, and Koeffler HP (1999) Growth inhibition of myeloid leukaemia cells by troglitazone, a ligand for peroxisome proliferator activated receptor and retinoids. Int J Oncol 15: 1027–1031.[Medline]

Barbier O, Villeneuve L, Bocher V, Fontaine C, Pineda Torra I, Duhem C, Kosykh V, Fruchart JC, Guillemette C, and Stael B (2003) The UDP-glucoronosyltransferase 1A9 enzyme is a peroxisome proliferator-activated receptor and target gene. J Biol Chem, in press.

Bartkova J, Lukas J, Strauss M, and Bartek J (1998) Cyclin D3: requirement for G₁/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation. Oncogene 17: 1027–1037.[CrossRef][Medline]

Berger J and Moller DE (2002) The mechanisms of action of PPARs. Annu Rev Med 53: 409–435.[CrossRef][Medline]

Demetri GD, Fletcher CD, Mueller E, Sarraf P, Naujoks R, Campbell N, Spiegelman BM, and Singer S (1999) Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor ligand troglitazone in patients with liposarcoma. Proc Natl Acad Sci USA 96: 3951–3956.[Abstract/Free Full Text]

Fantl V, Stamp G, Andrews A, Rosewell I, and Dickson C (1995) Mice lacking cyclin D1 are small and show defects in eye and mammary gland development. Genes Dev 9: 2364–2372.[Abstract/Free Full Text]

Friedrichsen BN, Richter HE, Hansen JA, Rhodes CJ, Nielsen JH, Billestrup N, and Moldrup A (2003) STAT5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic -cells. Mol Endocrinol, in press.

Hansen JB, Zhang H, Rasmussen TH, Petersen RK, Flindt EN, and Kristiansen K (2001) Peroxisome proliferator-activated receptor (PPAR)-mediated regulation of preadipocyte proliferation and gene expression is dependent on cAMP signaling. J Biol Chem 276: 3175–3182.[Abstract/Free Full Text]

Hellemans K, Michalik L, Dittie A, Knorr A, Rombouts K, De Jong J, Heirman C, Quartier E, Schuit F, Wahli W, et al. (2003) Peroxisome proliferator-activated receptor- signaling contributes to enhanced proliferation of hepatic stellate cells. Gastroenterology 124: 184–201.[CrossRef][Medline]

Kawakami S, Arai G, Hayashi T, Fujii Y, Xia G, Kageyama Y, and Kihara K (2002) PPAR ligands suppress proliferation of human urothelial basal cells in vitro. J Cell Physiol 191: 310–319.[CrossRef][Medline]

Kitamura S, Miyazaki Y, Hiraoka S, Nagasawa Y, Toyota M, Takakura R, Kiyohara T, Shinomura Y, and Matsuzawa Y (2001) PPAR agonists inhibit cell growth and suppress the expression of cyclin D1 and EGF-like growth factors in ras-transformed rat intestinal epithelial cells. Int J Cancer 94: 335–342.[CrossRef][Medline]

Kuehl WM, Bender TP, Stafford J, McClinton D, Segal S, and Dmitrovsky E (1988) Expression and function of the c-myb oncogene during hematopoietic differentiation. Curr Top Microbiol Immunol 141: 318–323.[Medline]

Lennon AM, Ramauge M, Dessouroux A, and Pierre M (2002) MAP kinase cascade are activated in astrocytes and preadipocytes by 15-deoxy-prostaglandin J2 and the thiazolidinedione ciglitazone through peroxisome proliferator activated receptor -independent mechanism involving reactive oxygenated species. J Biol Chem 277: 29681–29685.[Abstract/Free Full Text]

Marui N, Sakai T, Hosokawa N, Yoshida M, Aoike A, Kawai K, Nishino H, and Fukushima M (1990) N-myc suppression and cell cycle arrest at G₁ phase by prostaglandins. FEBS Lett 270: 15–18.[CrossRef][Medline]

Moore KJ, Rosen ED, Fitzgerald ML, Rondow F, Andersson LP, Altshuler D, Milstone DS, Mortensen RM, Spiegelman BM, and Freeman MW (2001) The role of PPAR in macrophage differentiation and cholesterol uptake. Nat Med 7: 41–47.[CrossRef][Medline]

Oh IH and Reddy EP (1999) The myb gene family in cell growth, differentiation and apoptosis. Oncogene 18: 3017–3033.[CrossRef][Medline]

Okura T, Nakamura M, Takata Y, Watanabe S, Kitami Y, and Hiwada K (2000) Troglitazone induces apoptosis via the p53 and Gadd45 pathway in vascular smooth muscle cells. Eur J Pharmacol 407: 227–235.[CrossRef][Medline]

Pahan K Jana M, Liu X, Taylor BS, Wood C, and Fischer SM (2002) Gemfibrozil, a lipid-lowering drug, inhibits the induction of nitric-oxide synthase in human astrocytes. J Biol Chem 277: 45984–45991.[Abstract/Free Full Text]

Palakurthi SS, Aktas H, Grubissich LM, Mortensen RM, and Halperin JA (2001) Anticancer effects of thiazolidinediones are independent of peroxisome proliferator-activated receptor and mediated by inhibition of translation initiation. Cancer Res 61: 6213–6218.[Abstract/Free Full Text]

Park BH, Volgestein B, and Kinzler KW (2001) Genetic disruption of PPAR decreases the tumorigenicity of human colon cancer. Proc Natl Acad Sci USA 98: 2598–2603.[Abstract/Free Full Text]

Park EJ, Park SY, Joe EH, and Jou I (2003) 15d-PGJ2 and rosiglitazone suppress JAK-STAT inflammatory signaling through induction of SOCS1 and SOCS3 in glia. J Biol Chem, in press.

Pizzimenti S, Barrera G, Dianzani MU, and Brusselbach S (1999) Inhibition of D1, D2 and A-cyclin expression in HL-60 cells by the lipid peroxidation product 4-hydroxynonenal. Free Radic Biol Med 26: 1578–1586.[CrossRef][Medline]

Pizzimenti S, Laurora S, Briatore F, Ferretti C, Dianzani MU, and Barrera G (2002) Synergistic effect of 4-hydroxynonenal and PPAR ligands in controlling human leukemic cell growth and differentiation. Free Radic Biol Med 3: 233–245.

Raschella G, Negroni A, Skorski T, Pucci S, Nieborowska-Skorska M, Romeo A, and Calabretta B (1992) Inhibition of proliferation by c-myb antisense RNA and oligodeoxynucleotides in transformed neuroectodermal cell lines. Cancer Res 52: 4221–4226.[Abstract/Free Full Text]

Sato O, Kuriki C, Fukui Y, and Motojima K (2002) Dual promoter structure of mouse and human fatty acid translocase/CD36 genes and unique transcriptional activation by peroxisome proliferator-activated receptor and ligands. J Biol Chem 277: 15703–15711.[Abstract/Free Full Text]

Scatena R, Nocca G, Sole PD, Rumi C, Puggioni P, Remiddi F, Bottoni P, Ficarra S, and Giardina B (1999) Benzafibrate as differentiating factor of human myeloid leukemia cells. Cell Death Diff 6: 781–787.[CrossRef][Medline]

Schlezinger JJ, Jensen BA, Mann KK, Ryu HY, and Scherr DH (2002) Peroxisome proliferator-activated receptor -mediated NF-B activation and apoptosis in pre-B cells. J Immunol 169: 6831–6841.[Abstract/Free Full Text]

Sherr CJ (1995) D-Type cyclins. Trends Biochem Sci 20: 187–190.[CrossRef][Medline]

Shimada T, Kojima K, Yoshiura K, Hiraishi H, and Terano A (2002) Characteristics of the peroxisome proliferator activated receptor (PPAR) ligand induced apoptosis in colon cancer cells. Gut 50: 658–664.[Abstract/Free Full Text]

Sicinski P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner H, Haslam SZ, Bronson RT, Elledge SJ, and Weinberg RA (1995) Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82: 621–630.[CrossRef][Medline]

Strauss DS, Pascual G, Li M, Welch JS, Ricote M, Hsiang CH, Sengchanthalangsy LL, Ghosh G, and Glass CK (2000) 15-Deoxy- 1,14-prostaglandin J2 inhibits multiple steps in the NF-B signaling pathway. Proc Natl Acad Sci USA 97: 4844–4849.[Abstract/Free Full Text]

Suhasini M, Reddy CD, Reddy EP, DiDonato JA, and Pilz RB (1997) cAMP-induced NF-B (p50/relB) binding to a c-myb intronic enhancer correlates with c-myb up-regulation and inhibition of erythroleukemia cell differentiation. Oncogene 15: 1859–1870.[CrossRef][Medline]

Teruel T, Smith SA, Peterson J, and Clapham JC (2000) Synergistic activation of UCP-3 expression in cultured fetal rat brown adipocytes by PPAR and PPAR ligands. Biochem Biophys Res Commun 273: 560–564.[CrossRef][Medline]

Toyota M, Miyazaki Y, Kitamura S, Nagasawa Y, Kiyohara T, Shinomura Y, and Matsuzawa Y (2002) Peroxisome proliferator-activated receptor reduces the growth rate of pancreatic cancer cells through the reduction of cyclin D1. Life Sci 70: 1565–1575.[CrossRef][Medline]

Wang C, Fu M, D'Amico M, Albanese C, Zhou JN, Brownlee M, Lisanti MP, Chatterjee VK, Lazar MA, and Pestell RG (2001) Inhibition of cellular proliferation through IB kinase-independent and peroxisome proliferator-activated receptor -dependent repression of cyclin D1. Mol Cell Biol 21: 3057–3070.[Abstract/Free Full Text]

Wakino S, Kintscher U, Kim S, Yin F, Hsueh WA, and Law RE (2000) Peroxisome proliferator-activated receptor ligands inhibit retinoblastoma phosphorylation and G₁ S transition in vascular smooth muscle cells. J Biol Chem 275: 22435–22441.[Abstract/Free Full Text]

Willson TM and Wahli W (1997) Peroxisome proliferators-activated receptor agonists. Curr Opin Chem Biol 1: 235–241.[CrossRef][Medline]

Yamakawa-Karakida N, Sugita K, Inukai T, Goi K, Nakamura M, Uno K, Sato H, Kagami K, Barker N, and Nakazawa S (2002) Ligand activation of peroxisome proliferator-activated receptor induces apoptosis of leukemia cells by down-regulating the c-myc gene expression via blockade of the Tcf-4 activity. Cell Death Diff 9: 513–526.[CrossRef][Medline]

Yen A, Samuel V, and Forbes M (1992) Regulation of cell proliferation: late down-regulation of c-myb preceding myelo-monocytic cell differentiation. J Cell Physiol 153: 147–156.[Medline]

Yin F, Wakino S, Liu Z, Kim S, Hsueh WA, Collins AR, Van Herle AJ, and Law RE (2001) Troglitazone inhibits growth of MCF-7 breast carcinoma cells by targeting G₁ cell cycle regulators. Biochem Biophys Res Commun 286: 916–922.[CrossRef][Medline]

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