Wy-14,643–Induced Hypomethylation of the c-myc Gene in Mouse Liver (2024)

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Rongrong Ge

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Wei Wang

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Paula M. Kramer

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Siming Yang

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Lianhui Tao

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Michael A. Pereira

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Toxicological Sciences, Volume 62, Issue 1, July 2001, Pages 28–35, https://doi.org/10.1093/toxsci/62.1.28

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01 July 2001

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18 January 2001

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03 April 2001

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01 July 2001

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    Rongrong Ge, Wei Wang, Paula M. Kramer, Siming Yang, Lianhui Tao, Michael A. Pereira, Wy-14,643–Induced Hypomethylation of the c-myc Gene in Mouse Liver, Toxicological Sciences, Volume 62, Issue 1, July 2001, Pages 28–35, https://doi.org/10.1093/toxsci/62.1.28

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Abstract

The carcinogenic activity of Wy-14,643 in mouse liver appears to be nongenotoxic and could involve a decrease in DNA methylation. The mechanism for Wy-14,643–induced decrease in DNA methylation is proposed to involve increased cell proliferation followed by prevention of the methylation of the newly synthesized DNA. To investigate this mechanism, female B6C3F1 mice were administered daily by oral gavage 50 mg/kg Wy-14,643. Mice were sacrificed at 2, 5, 8, 24, 26, 29, 32, 36, 48, 72, and 96 h after the first dose. Some mice also received 450 mg/kg methionine by ip injection at 30 min after administering Wy-14,643. Hypomethylation of the c-myc gene first occurred at 48 h after the first dose of Wy-14,643. Cell proliferation determined by the Proliferating Cell Nuclear Antigen (PCNA)-Labeling Index started to increase at 36 h and peaked at 72h. Wy14,643 did not affect the liver concentration of either S-adenosyl methionine (SAM) or S-adenosyl hom*ocysteine (SAH). Methionine prevented and reversed the hypomethylation of the c-myc gene induced by Wy-14,643. However, the increased levels of SAM and SAH returned to control levels prior to the prevention by methionine of Wy-14,643–induced hypomethylation. Furthermore, methionine did not prevent Wy-14,643–induced increase in the PCNA-Labeling Index. The activity of nuclear DNA methyltransferase (DNA MTase) was increased at 72 and 96 h after administering Wy14,643. Wy14,643 also increased the activity of DNA MTase when added in vitro to nuclear extracts. The results are consistent with Wy-14,643 decreasing the methylation of the c-myc gene by a mechanism that includes enhancement of cell proliferation followed by prevention of the methylation of the newly synthesized DNA. However, the results indicate that Wy-14,643 does not prevent methylation by decreasing either the availability of SAM or the activity of DNA MTase.

S-adenosyl methionine, cell proliferation, DNA methylation, DNA methyltransferase, liver, c-myc, Wy-14,643.

Wy-14,643 (4-chloro-6-[2,3-xylidino]-2-pyrimidinylthioacetic acid) is a hypolipidemic drug and potent peroxisome proliferator. Many peroxisome proliferators including Wy-14,643 have been reported to induce hepatocellular tumors in mice and rats (Cattley et al., 1995; Gonzalez et al., 1998; Holden and Tugwood, 1999; Marsman and Popp, 1994; Roberts-Thomson, 2000). However, Wy-14,643 and many of the other peroxisome proliferators lack significant genotoxic activity (Glauert et al., 1984; Goodman et al., 1991; Moody et al., 1991; Roberts et al., 1997; Roberts, 1999). Thus, a nongenotoxic mechanism involving increased cell proliferation in the liver has been proposed for their carcinogenic activity (Cattley et al., 1991; Marsman et al., 1988; Peters et al., 1997).

Decrease in the 5-methylcytosine (5-MeC) content of DNA and the hypomethylation of genes are common early events found in many human and animal tumors (Baylin et al., 1998; Bird, 1996; Counts and Goodman, 1995c; Gama-Sosa et al., 1983; Jones and Buckley, 1990; Lapeyre et al., 1981; Vogelstein et al., 1988; Wainfan and Poirier, 1992). 5-MeC is rapidly formed after DNA replication by the addition of the methyl group to cytosine at hemimethylated sites of the newly synthesized strands of DNA. S-Adenosyl methionine (SAM) is the methyl donor and DNA methyltransferase (DNA MTase) catalyzes the reaction resulting in 5-MeC in DNA and S-adenosyl hom*ocysteine (SAH) (Hergersberg, 1991). Methylation of genes, especially in their promoter region, can control their transcription (Bird, 1996; Garcea et al., 1989; Herman et al., 1994; Jones, 1996; Wainfan and Poirier, 1992). Thus, hypomethylation of protooncogenes including the c-myc gene could increase their expression. Increased expression of many protooncogenes has been associated with increased cell proliferation. This includes the immediate to early protooncogenes, c-fos, c-jun, and c-myc whose expressions are increased soon after a stimulus to increase cell proliferation in the liver (Cherkaoui Malki et al., 1990; Goldsworthy et al., 1994). Therefore, hypomethylation of DNA and more specifically of protooncogenes has been hypothesized to be part of the nongenotoxic mechanism for carcinogens (Counts and Goodman, 1995b,c; Gonzalgo and Jones, 1997).

Peroxisome proliferators including Wy-14,643, dibutyl phthalate, dichloroacetic acid (DCA), 2,4-dichlorophenoxyacetic acid (2,4-D), gemfibrozil, trichloroacetic acid (TCA), and trichloroethylene have been shown to decrease the methylation of the c-myc protooncogene and to increase its expression (Tao et al., 1999, 2000). Since Wy-14,643 is a very potent peroxisome proliferator, it was chosen for investigation of the mechanism by which this class of carcinogens caused hypomethylation of the gene. The mechanism evaluated was that peroxisome proliferators induced cell proliferation and then prevented the methylation of newly synthesized DNA. Since, the liver has a very low level of DNA replication, we propose that Wy-14,643 would induce cell proliferation prior to preventing the methylation of the gene. Thus, we report here that Wy-14,643 enhanced cell proliferation prior to inducing hypomethylation of the c-myc gene. Furthermore, we report that the hypomethylation was not the result of decreased availability of SAM or decreased activity of DNA MTase.

MATERIALS AND METHODS

Animals and treatment.

VAF (viral antibody-free) 6-week-old female B6C3F1 mice were obtained from Charles River Laboratories (Frederick, MD). The mice were housed and maintained in our AAALAC accredited laboratory animal facility and in accordance to the U.S. Public Health Service “Guide for the Care and Use of Laboratory Animals.” The mice were administered daily by oral gavage 50 mg/kg Wy-14,643 (Chemsyn Laboratories, Lenexa, KS) in corn oil (4 ml/kg). Mice were sacrificed at 2, 5, 8, 24, 26, 29, 32, 36, 48, 72, and 96 h after the first dose of Wy-14,643. The effect of methionine on Wy-14,643–induced hypomethylation was determined by administering 450 mg/kg methionine or its saline vehicle by ip injection at 30 min after each dose of Wy-14,643. Some mice received only the saline vehicle after the first 2 doses (0 and 24 h) of Wy-14,643 and then received 450 mg/kg methionine after the remaining doses. At sacrifice, each mice was weighed and its liver excised and weighed. A portion of the left lobe of the liver was fixed overnight in 10% formalin, transferred to 70% ethanol, and embedded in paraffin. The remainder of the liver was rapidly frozen in liquid nitrogen and stored at –70°C.

Southern blot analysis of c-myc methylation.

The methylation of the c-myc gene was evaluated by digestion with the methyl-sensitive restriction enzyme, Hpa II followed by Southern blot analysis as previously described (Tao et al., 1999, 2000). Briefly, 50 mg of liver were hom*ogenized in 1.5 ml extraction buffer (10 mM Tris–HCl pH 8.0, 0.1 M EDTA, SDS 0.5%) and digested with RNAase and proteinase K. The DNA was extracted 3 times with phenol, once with phenol:chloroform (1:1), and once with chloroform. It was then precipitated with ethanol, washed twice with 70% ethanol, and dissolved in TE buffer (10 mM Tris–HCl, pH 8.0 and 1mM EDTA).

The isolated DNA (30 μg) was digested overnight with Hpa II (10 U/μg DNA) at 37°C and electrophoresed on 1% agarose gel. Equal loading of the gels was indicated by equal ethidium bromide fluorescence. The gels were washed with 2X SSC and transferred to Hybond™-+ nylon membranes. The membranes were prehybridized at 42°C for 1 h in 20 ml prehybridization solution (50% formamide, 5X Dehardt's Reagent, 6X SSPE, 10% dextran sulfate, 1% SDS, and 100 μg/ml denatured nonhom*ologous DNA). Random 32P-labeled c-myc probe (65 ηg) was added to the prehybridization solution and hybridization continued for 12 h at 42°C. The c-myc probe was produced by PCR amplification of mouse liver DNA using sense 5′-TCTAGAACCAATGCACAGAGCAAAAG-3′ and antisense 5′-GCCTCAGCCCGCAGTC CAGTACTCC-3′ primers (Tao et al., 1999). After hybridization, the membranes were stringently washed 5 times with 4X SSC containing 0.5% SDS at 65°C, 3 times with 2X SSC containing 0.5% SDS at 37°C, and finally once with 2X SSC at 37°C. After drying, autoradiography was processed using Kodak Biomax MR X-ray film with a Kodak intensifying screen at –70°C.

PCNA-labeling index.

Liver sections were deparaffinized and placed in 2N HCl at 50°C for 20 min. Endogenous peroxidase was quenched with 3% hydrogen peroxide, blocked with diluted horse serum (Vector Laboratories, Burlingame, CA), and incubated with monoclonal mouse anti-PCNA (dilution of 1:300, Sigma Chemical Co., St. Louis, MO) for 1 h. After washing, the sections were incubated with dilute biotinylated anti-mouse IgG (Vector Laboratories) and then with Vectastain ABC reagent (Vector Laboratories). Stain was developed with 3,3′-diaminobenzidine tetrahydrochloride for 45 s and the slides counter stained with hematoxylin. The nuclei of PCNA-positive cells stained brown while unlabeled nuclei were blue. Approximately 4,000 hepatocytes/section were evaluated and the PCNA-Labeling Index determined as the number of PCNA-positive cells divided by the total number of hepatocytes evaluated times 100.

Quantitation of SAM and SAH.

Liver tissue (100 mg) was hom*ogenized in 400 μl of 400 mM perchloric acid. After centrifugation, the supernatant was filtered through a 0.2 μm pore size polypropylene syringe filter. A Waters HPLC system (Milford, MA) was used that consisted of a Model 510 pump, a Model U6K universal injector, a Model 481 Lambda Max UV/ visible LC spectrophotometer, and a Model 730 Data Module. Separation was carried out with a Whatman PartiSphere C18 reverse-phase analytical column (250 mm × 4.6 mm I.D., 5 μm particle) (Clifton, NJ). The mobile phase consisted of Solvent A (8 mM octanesulfonic acid sodium salt and 50 mM NaH2PO4 adjusted to pH 3.0 with H3PO4) and Solvent B (100% methanol). The HPLC column was equilibrated with 80% Solvent A and 20 % Solvent B. The sample was then injected. This was followed by a step gradient consisting of 8 min at the equilibration condition, 30 s to increase Solvent B to 40%, 12.5 min at the new condition, 30 s to return to the equilibration condition, and 10 min before a subsequent injection. The flow rate was 1ml/min and detection was monitored at 254 nm. The HPLC was performed at room temperature. SAM and SAH were identified according to their retention times and co-chromatography with SAM and SAH standards. Quantification was based on integration of peak areas. The results are expressed in nmol/gm liver.

DNA MTase activity assay.

The activity of DNA MTase was determined as described by Adams et al. (1991). Nuclei were isolated and incubated on ice with 0.8 M KCl, 50 mM Tris–HCl (pH 7.8) for 10 min. After dilution to 0.3 M KCl with 10 mM Tris–HCl (pH 7.8), the nuclei extract was incubated for 30 min and centrifuged. The supernatant was brought to 10% glycerol using 50% glycerol in the original lysis buffer. The protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). The nuclei extract (12.5 μg protein) was incubated with 2 μg of polydeoxy inosine-polydeoxycytidine (Pharmacia Biotechnology, Piscataway, NJ) and 4.5 μCi of [methyl-3H] S-adenosyl-L-methionine (75 Ci/mmol; Amersham, Arlington Heights, IL) in a total volume of 100 μl, at 37°C and for 2 h. The reaction was stopped by the addition of 300 μl of a solution containing 1% SDS, 2 mM EDTA, 5% isopropanol, 125 mM NaCl, 0.25 mg/ml SS-DNA (Salmon Testis DNA), and 1 mg/ml Proteinase K. After an additional 1h incubation at 37°C, the mixture was then extracted sequentially with phenol, phenol/chloroform, and chloroform. The DNA was precipitated with ethanol and RNA was removed by incubating in 0.3 M sodium hydroxide at 37°C for 10 min. The DNA was then spotted on a Whatman GF/C filter, dried, and washed 3 times with ice-cold 5% trichloroacetic acid. The filter was placed in a vial and 10 ml of Cytoscint scintillation fluid (ICN Radiochemicals, Irvine, CA) was added. Radioactivity was counted in a Beckman scintillation counter. Results are expressed as cpm/μg protein/2 h incubation.

The ability of Wy-14,643 to inhibit DNA MTase activity in nuclei extracts was determined. The nuclei extracts were obtained from control female mice. Wy-14,643 (0, 1, 3, or 10 mM) was dissolved in 0.01% DMSO, neutralized with sodium hydroxide and added to the incubation. The incubation, extraction, and determination of incorporated radioactivity were performed as described above. As a control for the salt formed during neutralization of the Wy-14,643, 10 mM sodium chloride was demonstrated not to alter the DNA MTase activity in the nuclei extracts.

Statistical evaluation.

SigmaStat software, version 2.0 (Jandel Corp., San Rafael, CA), was used to perform the statistical analysis. The results were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by the Tukey test using a p-value < 0.05.

RESULTS

Effect of Wy-14,643 on the Methylation of the c-myc Gene*

After Hpa II digestion of DNA from mice administered Wy-14,643, 4 bands of 0.5, 1.0, 1.4, and 2.2 kb were presented when the Southern gels were probed for the promoter region of the c-myc gene (Fig. 1). The ethidium bromide fluorescence depicted in Figure 1A demonstrates that the lanes of the gel were loaded equally. All the gels of this study were demonstrated by ethidium bromide staining to have equal loading of their lanes. Furthermore, we have previously demonstrated that these bands were present after Hpa II digestion of DNA from mice administered other peroxisome proliferators (i.e., DCA, TCA, or trichloroethylene) and that they were the result of hypomethylation of CCGG sites (Tao et al., 2000). The 4 bands after Hpa II digestion of DNA from mice administered Wy-14,643 first appeared at 48 h after the first dose of the peroxisome proliferator (Fig. 1B). Wy-14,643 treatment of longer duration (i.e., 72 and 96 h) resulted in increased intensity of the bands indicating that the extent of hypomethylation continued to increase with treatment. The 4 bands were not present in Hpa II-digested DNA from mice sacrificed at 36 h or earlier from the first dose of Wy-14,643 and from mice administered the corn oil vehicle. The bands were also absent in DNA that was not digested with Hpa II. Thus, 48 h was required for Wy-14,643 to start to hypomethylate the c-myc gene.

Effect of Wy-14,643 on Cell Proliferation

The effect of Wy-14, 643 on the PCNA-Labeling Index in the liver is presented in Figure 2. The PCNA-Labeling Index started to increase at 32 h after the first dose of Wy-14,643, continued to increase until 72 h, and remained high at 96 h. Thus, cell proliferation started to increase prior to the occurrence at 48 h of hypomethylation of the c-myc gene. Figure 2 also presents the effect of methionine on cell proliferation induced by Wy-14,643. Administering methionine 30 min after each dose of Wy-14,643 did not prevent the ability of Wy-14,643 to increase the PCNA-Labeling Index. In fact methionine increased the PCNA-Labeling Index in both control mice and mice administered Wy-14,643 (p-value < 0.05).

Effect of Methionine on Wy-14,643–Induced Hypomethylation of the c-myc Gene

Methionine prevented the hypomethylation of the c-myc gene induced by Wy-14,643 (Fig. 3). Similar to Figure 1, the 4 bands present after Hpa II digestion of the DNA from Wy-14,643 treated mice did not occur until 48 h after the first dose and increased in intensity with longer duration of treatment. Also, similar to Figure 1 these bands were absent in Hpa II-digested DNA from corn oil-treated mice. When methionine (450 mg/kg) was administered 30 min after each dose of Wy-14,643, it appeared to completely prevent the occurrence of the 4 bands demonstrating prevention of Wy-14,643–induced hypomethylation of the c-myc gene.

To determine whether methionine had to be administered prior to the enhancement of cell proliferation, mice were administered the water vehicle instead of methionine at 30 min after the first 2 doses of Wy-14,643 (0 and 24 h). The mice then received methionine 30 min after each subsequent dose of Wy-14,643 (48 and 72 h). Starting methionine treatment at 48 h resulted at 72 and 96 h in the absence of the 4 bands that were previously present at 48 h (labeled SM in Fig. 3). Thus, methionine reversed the hypomethylation induced by Wy-14,643 at 48 h back to the more methylated state found in the liver of control mice.

Effect of Wy-14,643 and Methionine on the Liver Concentration of SAM and SAH

Wy-14,643 did not effect the liver concentrations of SAM and SAH (Figs. 4A and 4B). For all the time points evaluated, including 72 and 96 h after the first dose of Wy-14,643 (not presented in Figs. 4A and 4B), the liver concentrations of SAM and SAH were not significantly altered by Wy-14,643 when compared to the concentrations in the livers of mice administered the corn oil vehicle. Methionine administered after each dose of Wy-14,643 or the corn oil vehicle increased the liver concentrations of both SAM and SAH approximately 10-fold by 2 h. The concentration of both SAM and SAH then returned to control levels by 8 h. Therefore, there was a rapid increase followed by a similarly rapid decrease in SAM and SAH levels after each dose of methionine whether or not Wy-14,643 preceded it. Thus, by 8 h after the second dose of Wy-14,643 (32 h after the first dose), the concentrations of SAM and SAH were no longer increased by methionine administered at 24 h. This would indicate that the methionine-induced increase in the concentrations of SAM and SAH had returned to control levels prior to its prevention of Wy-14,643–induced hypomethylation of the c-myc gene at 48 h.

Effect of Wy-14,643 on DNA MTase Activity

DNA MTase activity in nuclei extracts from mice administered Wy-14,643 was significantly increased at 72 and 96 h after the first dose (Fig. 5). Although, DNA MTase activity was also increased at 48 h, it was not statistically significant. Wy-14,643 also increased DNA MTase activity when added in vitro to incubations containing nuclei extract from control mice(Fig. 6). The increase in DNA MTase activity was dose dependent between 1–10 mM Wy-14,643. Thus, 10 mM Wy-14,643 resulted in a 2.4-fold increase in enzyme activity (i.e., 71.0 ± 3.5 and 241 ± 1.5 cpm/μg protein for 0 and 10 mM Wy-14,643, respectively).

DISCUSSION

Many peroxisome proliferators are mouse liver carcinogens for which a nongenotoxic mechanism involving increased cell proliferation has been proposed (Goodman and Counts, 1993; Moody et al., 1991; Roberts et al., 1997; Roberts, 1999). DNA hypomethylation has also been hypothesized as being involved in the nongenotoxic mechanism of mouse liver carcinogens (Counts and Goodman, 1995a,b; Gonzalgo and Jones, 1997; Jones and Gonzalgo, 1997). DNA methylation, especially in the promoter region of a gene, can control its expression (Bird, 1996; Garcea et al., 1989; Herman et al., 1994; Jones, 1996; Wainfan and Poirier, 1992). Hypomethylation of DNA, including protooncogenes such as c-myc, is an early event in most cancers, including liver (Baylin et al., 1998; Bird, 1996; Counts and Goodman, 1995b; Gama-Sosa et al., 1983; Jones and Buckley, 1990; Lapeyre et al., 1981; Vogelstein et al., 1988; Wainfan and Poirier, 1992). Thus, numerous chemicals including chloroform and other trihalomethanes, phenobarbital and the peroxisome proliferators, Wy-14,643, DCA, TCA, and trichloroethylene that are carcinogenic in mouse liver have been reported to decrease the methylation of DNA and genes (Coffin et al., 2000; Counts et al., 1996; Ray et al., 1994; Tao et al., 1999, 2000; Vorce and Goodman, 1990, 1991).

Peroxisome proliferators could decrease the methylation of DNA and the c-myc gene by preventing the methylation of hemimethylated sites formed during DNA replication. DNA methylation occurs shortly after the start of DNA replication and continues for a short time afterwards. For example, when DNA replication is induced in rats by partial hepatectomy, the greatest rate of DNA methylation occurred during DNA synthesis (i.e., 20–24 h) after the operation (Lutz et al., 1973). Thus, the most sensitive period for prevention of DNA methylation would be during DNA replication. However, the liver has a very low rate of DNA replication. Therefore, the proposed mechanism for DNA hypomethylation of preventing the methylation of newly synthesized DNA requires enhancement of cell proliferation to increase the rate of DNA replication. The chemicals that have been reported to decrease the methylation of DNA and genes have also been shown to enhance the level of cell proliferation in mouse liver (Cattley et al., 1995; Marsman et al., 1988; Pereira and Phelps, 1996; Wada et al., 1992).

The ability of Wy-14, 643 to increase cell proliferation was determined by the PCNA-Labeling Index. Increase in the PCNA-Labeling Index did not start until 32 h after the first dose of Wy-14,643. The PCNA-Labeling Index continued to increase until 72 h and remained elevated at 92 h. Decreased methylation of the c-myc gene started to occur between 36 and 48 h after the start of treatment with Wy-14,643 (i.e., it was not found at 36 h but was at 48 h). The extent of hypomethylation increased further between 48 and 96 h. Thus, the temporal relationship between increased cell proliferation and hypomethylation of the c-myc gene was consistent with the hypothesis that Wy-14,643 first increases cell proliferation (DNA replication) and then prevents the methylation of the newly synthesized strands of DNA. Furthermore, although the increase in cell proliferation was somewhat low at the times sampled (PCNA-Labeling Indices of 3–4.5%), the increase was maintained from 36 h until the end of the study at 96 h. During this time cells continued to enter the cell cycle and to proliferate resulting in the synthesis of more unmethylated DNA requiring methylation. This was consistent with the further decrease in the methylation of the c-myc gene from 48 to 96 h. However, this correlation of increased cell proliferation with hypomethylation does not preclude the possibility that there was demethylation of the gene independent of cell proliferation.

Wy-14,643 could prevent the methylation of hemimethylated sites in newly replicated DNA by (1) decreasing the activity of DNA MTase, (2) decreasing the availability of SAM for methylation of DNA, (3) increasing the concentration in the liver of SAH, an inhibitor of DNA MTase, or (4) altering the structure of chromatin at CCGG sites resulting in blockage of the access of DNA MTase (Wolffe et al., 1999). Nuclei DNA MTase activity was not decreased in the liver of mice administered Wy-14,643 but was rather increased. DNA MTase activity could be increased in response to the DNA hypomethylation induced by Wy-14,643. Thus, in tumors increased DNA MTase activity has been proposed to result from feedback stimulation by the hypomethylation of their DNA (Baylin et al., 1998; Lapeyre et al., 1981). However, Wy-14,643 also increased DNA MTase activity when added in vitro to incubations containing nuclei extracts. Therefore, the increased DNA MTase activity could have resulted from a direct effect of Wy-14,643.

The possibility was investigated that Wy-14,643 caused DNA hypomethylation by decreasing the availability of SAM or by increasing the concentration of SAH. Wy-14,643 did not alter the liver concentration of either SAM or SAH. Methionine, a precursor for SAM did prevent the hypomethylation of the c-myc gene induced by Wy-14,643. We have also found that methionine prevented DCA, TCA, and trichloroethylene-induced hypomethylation of the c-myc gene (Tao et al., 2000). However, the liver concentrations of both SAM and SAH peaked by 2 h after administering methionine and by 8 h had returned to control levels. This rapid increase followed by a rapid decrease in the concentrations of SAM and SAH occurred each time methionine was administered irrespective of whether the mice also received Wy-14,643. Thus, the increase in the concentrations of SAM and SAH that resulted from administering methionine at 24 h (relative to the first dose of Wy-14,643) had returned by 32 h to the levels present prior to administering the methionine. However, hypomethylation of the c-myc gene did not occur until 48 h, being undetectable at 36 h. Thus, by the time hypomethylation of the c-myc gene occurred, the liver concentrations of SAM and SAH were no longer elevated by methionine, so that methionine does not appear to prevent Wy-14,643–induced hypomethylation by increasing the availability of SAM for methylation.

Methionine could prevent the hypomethylation of the c-myc gene by preventing the increased cell proliferation induced by Wy-14,643. However, methionine did not prevent Wy-14,643 induced increase in the PCNA-Labeling Index. In fact, methionine increased the PCNA-Labeling Index in both Wy-14,643–treated and control mice. Thus, methionine appeared not to prevent the formation of hemimethylated DNA that occurs during cell proliferation.

It is possible that Wy-14,643 decreased the methylation of the c-myc gene by altering the chromatin structure around CCGG sites so as to block the access of DNA MTase. Wy-14,643 and other peroxisome proliferators (2,4-D, dibutyl phthalate, gemfibrozil, DCA, TCA, and trichloroethylene) caused hypomethylation at only a very few of the 12 CCGG sites in the probed region of the c-myc gene (Tao et al., 2000). Digestion of DNA from mice administered Wy-14,643 or the other peroxisome proliferators with Hpa II that cuts only unmethylated CCGG sites resulted in 4 distinct bands of 500–2,200 bp. In contrast, digestion with the methyl-insensitive Msp I resulted in a smear of activity in the 100–600 bp range. This differential sensitivity to Hpa II and Msp I indicates that most of the CCGG sites in DNA from mice administered Wy-14,643 or one of the other peroxisome proliferators still had their internal cytosine methylated. Hence, peroxisome proliferators prevented the methylation at only a limited number of the 12 CCGG sites in the region probed.

The specificity of the CCGG sites that are hypomethylated in response to Wy-14,643 could involve the chromatin structure around the sites including polyamines, histones, transcription factors, receptors, and other proteins. Thus, Wy-14,643 could either directly or through a receptor interact with the chromatin around CCGG sites to block methylation by DNA MTase. Methionine could prevent and reverse Wy-14,643–induced hypomethylation by increasing the concentration of SAM resulting in increased polyamine synthesis and methylation of histones. SAM is decarboxylated by S-adenosylmethionine decarboxylase to supply the aminopropyl group for the synthesis of the polyamines, spermidine and spermine (Chiang et al., 1996; Duranton et al., 1998; Heby, 1995). Increased synthesis of polyamines and/or methylation of histones could alter the chromatin structure around CCGG sites so that their methylation is no longer blocked by Wy-14,643.

In summary, Wy-14,643 was shown to decrease the methylation in the promoter region of the c-myc gene. The results were consistent with a mechanism that includes Wy-14,643 enhancement of cell proliferation followed by prevention of the methylation of the newly synthesized DNA. Thus, the enhancement of cell proliferation by Wy-14,643 started prior to its hypomethylation of the c-myc gene. However, Wy-14,643 did not appear to prevent methylation by inhibiting of DNA MTase, decreasing the availability of SAM or increasing the concentration of SAH. Furthermore, Wy-14,643 prevented the methylation of only a few of the 12 CCGG sites in the region probed. We therefore propose that Wy-14,643 caused the hypomethylation of the c-myc gene by interacting with the chromatin surrounding a few of the CCGG sites to block their methylation by DNA MTase and that methionine prevents the hypomethylation by increasing the synthesis of polyamines of the methylation of histones.

Wy-14,643–Induced Hypomethylation of the c-myc Gene in Mouse Liver (3)

FIG. 1.

Effect of Wy-14,643 on the methylation of the c-myc gene. (A) Ethidium bromide fluorescence of gel prior to transfer. (B) Hybond™-+ nylon membranes probed for the promoter region of c-myc. DNA were digested with Hpa II, electrophoresed in a 1% agarose gel, transferred to the nylon membrane, and probed for the c-myc promoter region. The lanes labeled Oil and Wy contained DNA from mice administered either the corn oil vehicle or 50 mg/kg Wy-14,643, respectively. The indicated time is from the first dose of Wy-14,643. The arrows in the right margin indicate the size of the bands.

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Wy-14,643–Induced Hypomethylation of the c-myc Gene in Mouse Liver (4)

FIG. 2.

Effect of Wy-14,643 on the PCNA-Labeling Index in mouse liver. Liver sections were stained with monoclonal mouse anti-PCNA and approximately 4,000 hepatocytes were evaluated in order to determine the PCNA-Labeling Index. Wy indicates that the mice received 50 mg/kg Wy-14,643 daily and Wy + Methionine indicates that the mice received Wy-14,643 followed 30 min by 450 mg/kg methionine The methionine and corn oil groups received daily the corn oil vehicle followed by methionine or the corn oil vehicle followed by the saline vehicle for methionine. The results are mean ± SE for groups of six mice, each. The asterisk (*) indicates significant difference from the corn oil vehicle control group, p-value < 0.05.

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Wy-14,643–Induced Hypomethylation of the c-myc Gene in Mouse Liver (5)

FIG. 3.

Effect of methionine on Wy-14,643–induced hypomethylation of the c-myc gene. DNA was digested with Hpa II, electrophoresed in a 1% agarose gel, transferred to nylon, and probed for the c-myc promoter region. The DNA was from mice that received daily in lanes labeled Wy, 50 mg/kg Wy-14,643; lanes labeled Wy + M, Wy-14,643 followed 30 min with 450 mg/kg methionine; lanes labeled M, 450 mg/kg methionine; and lanes labeled Wy + SM, Wy-14,643 followed 30 min with saline for the first 2 doses and thereafter followed with 450 mg/kg methionine. The arrows in the right margin indicate the size of the bands.

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Wy-14,643–Induced Hypomethylation of the c-myc Gene in Mouse Liver (6)

FIG. 4.

Effect of Wy-14,643 and methionine on the concentration of SAM and SAH in mouse liver. (A) SAM. (B) SAH. Wy indicates the mice received 50 mg/kg Wy-14,643 daily, Wy + Methionine indicates they received 50 mg/kg Wy-14,643 followed 30 min with 450 mg/kg methionine, Methionine indicates they received the corn oil vehicle for Wy-14,643 followed by 450 mg/kg methionine, and Corn Oil indicates they received both the corn oil and the saline vehicles.

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Wy-14,643–Induced Hypomethylation of the c-myc Gene in Mouse Liver (7)

FIG. 5.

Effect of Wy-14,643 on DNA MTase activity in mouse liver. DNA MTase activity was determined in 2-h incubations of nuclei extracts from mice administered 50 mg/kg of Wy-14,643. The results are mean ± SE for groups of 6 animals, each. The asterisk (*) indicates significant difference from the corn oil control group, p-value < 0.05.

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Wy-14,643–Induced Hypomethylation of the c-myc Gene in Mouse Liver (8)

FIG. 6.

Effect of Wy-14,643 on DNA MTase activity in nuclear extracts. Wy-14,643 was added to nuclear extracts from livers of untreated mice and incubated for 2 h. The results are mean ± SE for 3 incubations, each. The asterisk (*) indicates significant difference from control incubations containing 0.01% DSMO, p-value < 0.05.

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1

To whom correspondence should be addressed at Department of Pathology, Health Education Bldg., Rm. 200F, Medical College of Ohio, 3055 Arlington Ave., Toledo, OH, 43614–5806. Fax: (419) 383-3089. E-mail: mpereira@mco.edu.

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