Department of Pathology, Medical College of Ohio, Toledo, Ohio 43614-5806
Received September 8, 1999; accepted December 1, 1999
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ABSTRACT |
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Key Words: c-myc; c-jun; dichloroacetic acid; DNA methylation; trichloroacetic acid; trichloroethylene; mRNA expression; methionine; protooncogenes.
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INTRODUCTION |
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DCA, TCA, and TCE are peroxisome proliferators (Bruckner et al., 1989; Elcombe, 1985
; Goldsworthy and Popp, 1987
; Odum et al., 1988
) that induce foci of altered hepatocytes and hepatocellular adenomas and carcinomas in B6C3F1 mice (Bull et al., 1990
; Herren-Freund et al., 1987
; Latendresse and Pereira, 1997
; Pereira, 1996
; Pereira et al., 1997
; Pereira and Phelps, 1996
). Mouse liver tumors induced by DCA, TCA, and TCE, relative to spontaneous tumors, have been shown not to contain a unique mutation in either the H- or K-ras oncogenes (Anna et al., 1994
; Ferreira-Gonzalez et al., 1995
; Fox et al., 1996
; Leavitt et al., 1997
; Schroeder et al., 1997
). Due to their very weak genotoxicity in in vitro and in vivo assays (Chang et al., 1992
; Fahrig et al., 1995
; Fox et al., 1996
; Meier and Blazak, 1990
), an epigenetic mechanism has been proposed for their carcinogenic activity that is associated with increased cell proliferation (Butterworth et al., 1992
; Goodman et al., 1991
; Roberts et al. 1997
).
DCA, TCA, and TCE have been shown to induce cell proliferation in mouse liver (Bull et al., 1990; Channel et al., 1998
; Pereira, 1996
). The immediate-early protooncogenes, c-jun and c-myc, participate in the control of cell proliferation and apoptosis (Cole and McMahan, 1999; Fausto and Shank, 1983
; Fausto and Webber, 1993
; Sæter and Seglen, 1990
). Increased expression of these two protooncogenes has been observed in the liver after partial hepatectomy and during treatment with epigenetic carcinogens (Fausto and Shank, 1983
; Fausto and Webber, 1993
; Sæter and Seglen, 1990
). Increased levels of the mRNA and protein for c-jun and c-myc genes have been reported in liver and liver tumors from mice treated with DCA, TCA, or TCE (Latendresse and Pereira, 1997
; Nelson et al, 1990
; Tao et al., 1999
). The c-jun and c-fos proteins form the transcription factor, AP-1, that primes cells for proliferation during liver regeneration following partial hepatectomy (Brenner, 1998
; Fausto and Webber, 1993
). c-Myc protein is required for efficient progression through the cell cycle (Cole and McMahon, 1999
; Facchini and Penn, 1998
). It forms a heteromeric complex with the murine Myn protein (Prendergast et al., 1991
) and the human homolog, Max protein (Blackwood and Eisenman, 1991
) to activate transcription (Dang, 1999
; Steiner et al., 1996
). With respect to increased cell proliferation, target genes for up-regulation have been proposed to include cdc25A and cyclins A, D1, and E (Dang, 1999
). c-Myc also regulates cyclin-dependent kinases (cdk) including inhibition of cdk inhibitors (Steiner et al., 1996
).
Methylation of DNA as 5-methylcytosine (5-MeC) in the promoter region of genes regulates mRNA expression, including that of the protooncogenes, c-jun and c-myc (Bird, 1986; Garcea et al, 1989
; Herman et al., 1994
; Jones and Buckley, 1990
; Razin and Kafri, 1994
; Stoger et al., 1993
; Wainfan and Poirier, 1992
). Decreased levels of 5MeC in DNA and in specific genes including c-jun and c-myc are frequently identified as early events in both human and mouse neoplasias (Gama-Sosa et al., 1983
; Jones and Buckley, 1990
; Lapeyre et al., 1981
; Vogelstein et al., 1988
). The extent of DNA methylation has been reported to decrease further with the progression from benign to metastatic neoplasm (Gama-Sosa et al., 1983
; Lapeyre et al., 1981
; Tao, et al., 1998
). In mouse liver, non-genotoxic carcinogens including phenobarbital, a choline-devoid and methionine-deficient diet, DCA, TCA, and TCE decreased the methylation of DNA (Christman et al., 1993
; Counts and Goodman, 1995
; Counts et al., 1996
, 1997
; Tao et al., 1998
, 1999
). Hence, DNA hypomethylation has been proposed as a mechanism for non-genotoxic carcinogens to increase cell proliferation and to induce tumors (Counts and Goodman, 1995
).
Methionine is required for the synthesis of S-adenosylmethionine (SAM), the cofactor for DNA methylation. Choline-devoid and methionine-deficient diet has been shown to cause hepatocellular carcinomas in both rats and mice (Copeland and Salmon, 1946; Ghoshal and Farber, 1984
; Mikol et al., 1983
; Newberne et al., 1982
) and to induce hypomethylation of H-ras, c-myc, and c-fos genes (Henning and Swendseid, 1996
; Wainfan and Poirier, 1992
). Methionine has been reported to prevent aflatoxin B1-induced liver tumorigenesis (Newberne et al., 1990
) and diethylnitrosamine-initiated and phenobarbital-promoted liver carcinogenesis (Fullerton et al., 1990
). Thus, methionine deficiency appears to enhance liver carcinogenesis while supplemental methionine appears to prevent liver cancer. Furthermore, it appears that the cancer-enhancing activity of methionine deficiency is associated with DNA hypomethylation, including the hypomethylation of protooncogenes. Therefore, we evaluated the ability of methionine to prevent the decrease in the methylation of the c-jun and c-myc genes and the increase in the expression of their mRNA and proteins that are induced by DCA, TCA, and TCE.
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MATERIALS AND METHODS |
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Animals and treatments.
VAF (viral antibody-free) B6C3F1 female mice were purchased from Charles River Breeding Laboratories (Portage, MI). Female mice were used, since the carcinogenic and tumor-promoting activities of DCA and TCA were first observed in this sex (Herren-Freund et al., 1987; Pereira and Phelps, 1996
). Six-week-old mice arrived at the AAALAC-accredited facility of the Medical College of Ohio. They were housed in accordance with the U.S. Public Health Service "Guide for the Care and Use of Laboratory Animals." Deionized and filtered tap water and Laboratory Rodent Diet 5001 (J & B Feed, Toledo, OH) were provided ad libitum to the mice.
At 8 weeks-of-age, the mice were dosed by gavage 500 mg/kg DCA or TCA in water neutralized with sodium hydroxide to pH 6.57.5, or 1000 mg/kg TCE in corn oil. The doses of DCA, TCA, and TCE were chosen because they have been reported to increase liver growth, cell proliferation, and lipid peroxidation in mice (Channel et al., 1998; Dees and Travis, 1994
; Larson and Bull, 1992
; Styles et al., 1991
). DCA, TCA, and TCE were administered once a day for 5 days. Vehicle-control mice received the same volume of water or corn oil. At 30 min after each dose of carcinogen or vehicle, the mice received 0, 30, 100, 300, or 450 mg/kg methionine by ip injection. The high dose level of 450 mg/kg was selected because it has been reported to prevent bromobenzene-induced decrease in hepatic SAM and alterations in DNA methylation (Lertratanangkoon et al., 1996
, 1997
). The mice were euthanized by carbon dioxide asphyxiation at 100 min after the fifth dose of carcinogen or vehicle. Sacrifice of the animals at 100 min after the last dose was chosen because it resulted in the peak increase in the level of the mRNA of the two protooncogenes after administering TCE (Tao et al., 1999
). As a positive control for enhancement of mRNA expression of the protooncogenes, mice were administered by gavage a single 2 ml/kg dose of carbon tetrachloride (1:1 by weight in corn oil) and killed 100 min later. At necropsy, the liver was rapidly excised, weighed, frozen in liquid nitrogen and stored at 70°C.
Analysis of DNA methylation status in the promoter region for the c-jun and c-myc protooncogenes.
Methylation status in the promoter region for c-jun and c-myc protooncogenes was evaluated using Hpa II restriction-enzyme digestion followed by Southern blot analysis, as described previously (Tao et al, 1999). Briefly, DNA was isolated from the liver tissues by digestion with 400 µg/ml RNase A and 200 µg/ml proteinase K followed by organic extraction with phenol, chloroform, and isoamyl alcohol. The isolated DNA was digested at 37°C with the methyl-sensitive restriction enzyme, Hpa II (10 U/µg DNA). Hpa II does not cut CCGG sites when the internal cytosine is methylated. The digested DNA was electrophoresed on 1% agarose gel and then transferred to HybondTM-N+ nylon membranes. Hind III produced DNA fragments of Lambda-phage were included with each gel as molecular size markers.
DNA was cross-linked by ultraviolet irradiation with an UV Stratalinker 2400 (Strategene, La Jolla, CA) and hybridized with 32P-labeled probes by random priming procedure. Probes for the promoter regions of the protooncogenes were produced by PCR amplification of normal mouse-liver genomic DNA using the following primers for c-jun, sense 5'-GTTGTAGTGGACCTCATTCGCTCTA-3' and antisense 5'-AGGGGTGTTCGCTTTGCCTCCGAGG-3' and for c-myc, sense 5'-TCTAGAACCAATGCACAGAGCAAAAG-3' and antisense 5'-GCCTCAGCCCGCAGTCCAGTACTCC-3'. The probes were designed from the GenBank database (Accession no. U60582 for c-jun and M12345 for c-myc genes) and contained, for c-jun and c-myc, the 19142422 and 11315 bp, respectively, in the promoter region of the genes.
Analysis for mRNA expression of protooncogenes.
Expression of mRNA for c-jun and c-myc protooncogenes was evaluated by Northern blot analysis, as described previously (Tao et al, 1999). Briefly, total RNA was isolated from the liver tissue by using TRIZOL Reagent (Chomczynski, 1993
). The yield, purity, and integrity of the RNA were assessed by absorbance at 260 nm, the A260/A280 ratio (1.71.9), and agarose/formaldehyde gel electrophoresis, respectively. The RNA was electrophoresed on the denaturing formaldehyde gels and then transferred to HybondTM-N+ nylon membranes by downward alkaline capillary action (Ming et al., 1994
). Oligonucleotide probes for mouse c-jun and c-myc were labeled with (
-32P)-ATP by the 5'-end labeling procedure to a minimum specific activity of 108 cpm/µg and separated from unincorporated ATP using a Sephadex G-50 column. Using a Biometra Mini Hybridization Oven, OV3, (Biometra, Inc., Germany), the Northern blots were prehybridized at 65°C for 1 h in hybridization solution (1.0 M NaCl, 50 mM Tris-HCl, pH 7.5, 10% dextran sulfate, 1% SDS, and 100 µg/ml denatured nonhomologous DNA). The 5' end-labeled probe (65 ng) was then added and hybridization proceeded overnight at 65°C. After hybridization, the membrane was washed 4x with 2 x SSC containing 0.1% SDS at room temperature, once for 30 min at hybridization temperature, and once again at room temperature for 5 min, followed by a brief wash with 2 x SSC. The membrane was dried and sealed in a plastic bag. Autoradiography was processed at 70°C using Kodak Biomax MR X-ray film with an intensifying screen.
Western blot analyses for c-jun and c-myc proteins.
Liver tissue was homogenized in a buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM EGTA (pH 7.5), 1 mM EDTA (pH 8.0), 10 mM ß-mercaptoethanol, 1 mM phenylmethyl-sulfonyl fluoride, 0.02% leupeptin, 0.04% trypsin inhibitor, 0.25M sucrose and 0.1% triton X-100, sonicated and centrifuged at 12,000g for 30 min at 40°C. Protein concentration in the supernatant was determined using the Bio-Rad Protein Assay (BioRad Corp. Richmond, CA). The supernatant (30 µg protein) was electrophoresed on 12% SDS-PAGE minigels under reducing conditions and blotted electrophoretically to Immobilon-P membranes. Detection of c-jun and c-myc was performed using 1:500 dilution of the mouse monoclonal antibodies, Ab-3 and Ab-2, respectively, and anti-mouse IgG-HRP. The blot was developed with enhanced chemiluminescence reagents.
Statistical evaluation.
SigmaStat software, version 2.03, (Jandel Corp., San Rafeal, CA) was used to perform the statistical analysis. The results were analyzed for statistical significance by a 1-way analysis of variance followed by a Tukey test with a p-value < 0.05.
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RESULTS |
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Dose-Response for the Prevention by Methionine of TCE-, DCA- and TCA-Induced Hypomethylation of c-Jun and c-Myc
The dose-response relationship for the prevention by methionine of DCA, TCA, and TCE-induced hypomethylation in the promoter regions of c-jun and c-myc genes is presented in Figure 3. Methionine at doses of 0, 30, 100, and 300 mg/kg was administered 30 min after each treatment of DCA, TCA, and TCE. Similar to the experiment presented in Figure 2
, Hpa II digested DNA from DCA-, TCA- and TCE-treated mice contained 2 bands of 3.2 and 1.5 kb and 3 bands at 2.2, 1.0, and 0.5 kb after probing for c-jun and c-myc, respectively. Furthermore, the results confirm that TCE induces 2 and 3 bands in Hpa II-digested DNA probed for the promoter regions of the c-jun and c-myc genes, respectively (Tao et al., 1999
). These bands were absent in DNA from vehicle-treated control mice with/without treatment with methionine. When the mice received 300-mg/kg methionine after each dose of DCA, TCA, or TCE, the 2 and 3 bands after probing for c-jun and c-myc, respectively, were absent. When each dose of the DCA, TCA, and TCE was followed with 100 mg/kg methionine but not with 30 mg/kg methionine, the density of the bands after Hpa II digestion and probing for c-jun and c-myc was reduced. Hence, methionine exhibited a dose-dependent prevention of DCA-, TCA-, and TCE-induced demethylation of the promoter regions of the c-jun and c-myc genes, with 30 mg/kg being an ineffective dose.
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DISCUSSION |
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Elevated levels of c-jun and c-myc mRNA and protein have been demonstrated in human tumors in the absence of gene mutation and amplification (Erisman et al., 1985; Vogt and Bos, 1990
), which suggests that there exist some epigenetic mechanisms regulating gene expression. Methylation of CpG sites in the promoter region of genes, which are near, or directly within, transcription factor-binding motifs, can modulate the transcription (Baylin et al., 1998
). Hypomethylation of the immediate-early protooncogenes c-jun and c-myc has been associated with increased expression (Garcea et al., 1989
; Tao et al., 1999
; Wainfan and Poirier, 1992
). This includes our previous demonstration that TCE decreased the methylation of the promoter regions of the c-jun and c-myc genes (Tao et al., 1999
). In the study presented here, the promoter regions of both protooncogenes were demonstrated to be hypomethylated in the livers of mice in response to DCA, TCA, or TCE exposure for 5 days, which was associated with increased populations of the mRNA and proteins of both genes. Increased mRNA and proteins of the 2 protooncogenes has been reported in DCA- and TCA-induced foci of altered hepatocytes and liver tumors (Latendresse and Pereira, 1997
; Nelson et al., 1990
). Furthermore, these tumors also contained decreased methylation in the promoter regions of the c-jun and c-myc genes (unpublished results).
Other non-genotoxic carcinogens have been reported to cause hypomethylation and increased mRNA levels of protooncogenes in mouse liver. Phenobarbital decreased the methylation of the raf oncogene in B6C3F1 mouse liver (Ray et al., 1994). In phenobarbital-induced and spontaneous liver tumors, raf and H-ras were hypomethylated (Counts et al., 1997
; Ray et al., 1994
; Vorce and Goodman, 1991
). The H-ras gene was also hypomethylated in chloroform-induced liver tumors (Vorce and Goodman, 1991
). Increased levels of H-ras and c-myc mRNA in response to chloroform have been reported (Sprankle et al., 1996
). Choline-devoid and methionine-deficient diet induced hypomethylation and increased expression of H-ras, c-myc and c-fos genes in mouse liver and tumors (Henning and Swendseid, 1996
; Wainfan and Poirier, 1992
). Hence, demethylation of protooncogenes and increased expression of their mRNA and proteins appears to be associated with treatment of non-genotoxic carcinogens that results in liver tumors. This has lead to the hypothesis that decreased methylation and increased expression of the protooncogenes plays a role in the mechanism of non-genotoxic carcinogens (Counts and Goodman, 1995
). Our results indicate that DCA, TCA, and TCE can be added to the list of non-genotoxic carcinogens operating by this proposed mechanism.
DNA hypomethylation or demethylation can occur by three different mechanisms, which include (1) DNA demethylase activity, (2) removal of 5-MeC from DNA by 5-MeC-DNA glycosylase, and (3) prevention of the formation of 5-MeC in nascent DNA form during replication (Schmutte and Jones 1998; Wolffe et al., 1999
). Recently a DNA demethylase has been identified that hydrolyzes 5MeC to cytosine and methanol (Bhattacharya et al., 1999
; Ramchandani et al., 1999
). The regulation of this enzymatic activity is unknown. However up-regulation by DCA, TCA, and TCE could be involved in the DNA hypomethylation induced by them. Two 5-MeC-DNA glycosylases have been described (Schmutte and Jones 1998
). One is replication dependent and preferentially removes 5-MeC from hemi-methylated sites formed during DNA replication. Demethylation by this glycosylase is in competition with DNA methyltransferase (DNA MTase) for the hemi-methylated sites (Schmutte and Jones 1998
). The up-regulation of DNA MTase that occurs concurrently with DNA hypomethylation should prevent demethylation of DNA by this glycosylase (Belinsky et al., 1998; Lopatina et al., 1998
; Sun et al., 1997
). The other 5-MeC-DNA glycosylase is DNA replication-independent and appears to cause gene-specific demethylation. Since, DCA and TCA caused large decreases (3550%) in global DNA methylation (Tao et al., 1998
) it is not likely that induction of gene specific 5-MeC-DNA glycosylases would be responsible for such extensive decrease in DNA methylation.
Prevention of the methylation of nascent DNA during replication could result from a down-regulation/inhibition of DNA MTase or from a decrease in the availability of SAM to act as substrate. However, elevated expression and activity of DNA MTase has been found along with DNA hypomethylation in many human and mouse tumors including liver tumors (Belinsky et al., 1998; Lopatina et al., 1998; Sun et al., 1997
). Thus, decreased DNA MTase activity is not the likely cause of DNA hypomethylation. Another way to decrease the formation of 5-MeC in DNA is to decrease the concentration of SAM and/or increase the concentration of SAH, an inhibitor of DNA MTase. Administration of a choline-devoid and methionine-deficient diet leads to the development of hepatocellular carcinomas in both rats and mice, which has been associated with hypomethylation and overexpression of H-ras and c-myc genes (Henning and Swendseid, 199; Wainfan and Poirier, 1992
). Supplementation with both choline and methionine has prevented diethylnitrosamine-initiated and phenobarbital-promoted liver carcinoma formation in C3H/He mice (Fullerton et al., 1990
). This suggests that supplementation with methionine might prevent DNA hypomethylation. Hence, methionine prevented in a dose-dependent manner DCA-, TCA-, and TCE-induced hypomethylation of c-jun and c-myc protooncogenes. Methionine also prevented DCA-, TCA-, and TCE-induced increase in the expression of the mRNA and protein for the 2 protooncogenes, further indicating an association between hypomethylation of the genes and increased levels of their mRNA and protein. Since, hypomethylation and increased expression of the protooncogenes is associated with hepatocarcinogenesis, our results suggest that methionine would prevent liver cancer induced by DCA, TCA, and TCE.
In conclusion, DCA, TCA, and TCE decreased the methylation in the promoter regions for the c-jun and c-myc genes and increased the expression of their mRNA and proteins. Methionine, in a dose-dependent manner, prevented the decreased methylation and increased expression of the protooncogenes. Hence, the results are supportive of DNA hypomethylation as a mechanism for the carcinogenic activity of DCA, TCA, and TCE. Furthermore, the results suggest that the dose of DCA, TCA, and TCE must be sufficient to decrease the level of SAM in order for these carcinogens to be active.
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ACKNOWLEDGMENTS |
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NOTES |
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