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Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors
Cancer Letters 158 (2000) 185±193
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Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors Lianhui Tao*, Siming Yang, Mi Xie, Paula M. Kramer, Michael A. Pereira Department of Pathology, Medical College of Ohio, Health Education Building, 3055 Arlington Ave., Toledo, OH 43614-5806, USA Received 20 April 2000; received in revised form 12 June 2000; accepted 16 June 2000
Abstract Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are mouse liver carcinogens. Methylation of the c-jun and c-myc genes, expression of both genes and DNA methyltransferase (DNA MTase) activity were determined in liver tumors initiated by N-methyl-N-nitrosourea and promoted by DCA and TCA in female B6C3F1 mice. Hypomethylated and over-expression of c-jun and c-myc genes were found in DCA- and TCA-promoted liver tumors. DNA MTase activity was increased in tumors while decreased in non-involved liver. Thus, DCA- and TCA-promoted carcinogenesis appears to include decreased methylation and increased expression of c-jun and c-myc genes in the presence of increased DNA MTase activity. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dichloroacetic acid; Trichloroacetic acid; Mouse liver tumor; c-jun; c-myc; DNA methylation
1. Introduction Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are common organic contaminants of drinking water formed as by-products during chlorine disinfection [1,2]. They are also metabolites of trichloroethylene, a common industrial and commercial solvent and an environmental contaminant found in ground water and at hazardous waste sites [3]. TCA is also a major metabolite of tetrachloroethylene [4]. Hence, there exists human exposure to DCA and TCA either from chlorinated drinking water or from metabolism of trichloroethylene and tetrachloroethylene. * Corresponding author. Tel.: 11-419-383-4294; fax: 11-419383-3089. E-mail address: [email protected] (L. Tao).
DCA and TCA in B6C3F1 mice have been shown to induce hepatocellular adenomas and carcinomas [3,5,6,9,10] and to promote N-methyl-N-nitrosourea (MNU)-initiated foci of altered hepatocytes and liver tumors [7,8]. However, the mechanism for their carcinogenicity in mouse liver is unclear. Mouse liver tumors induced by DCA and TCA do not contain a unique mutation spectrum in the ras oncogene relative to spontaneous tumors [11±15]. Due to their very weak genotoxicity [16,17] and their ability to promote liver tumors in mice [7,8], they likely induce liver cancer by a nongenotoxic mechanism. DCA and TCA have been shown to increase cell proliferation in mouse liver [5,6] that is a proposed mechanism for non-genotoxic carcinogens [18±20]. Increased cell proliferation in the liver is associated with increased expression of the immediate-early
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00518-8
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protooncogenes, c-fos, c-jun and c-myc [21,22]. It has been reported that c-jun and c-myc proteins are increased in DCA and TCA-induced foci of altered hepatocytes and tumors in female mice [9,23]. Furthermore, in mouse liver 5 days of exposure to DCA and TCA also increased the level of the mRNA and protein of both protooncogenes [24]. Methylation of DNA as 5-methylcytosine (5-MeC) in the promoter regions of genes including c-jun and c-myc can regulate their expression [25±29]. DNA is methylated by DNA methyltransferase (DNA MTase) with S-adenosyl methionine (SAM) as the methyl donor [27±31]. Hypomethylation of genes including c-jun and c-myc is a frequent early event of carcinogenesis in both humans and rodents [25±29,31±34]. Hypomethylation has been reported to decrease further with the progression from benign to metastatic neoplasm [31±34]. Interestingly, the activity of DNA MTase has been reported to be increase in tumors of humans and laboratory animals even though the DNA is hypomethylated [27±31]. In mouse liver, subchronic exposure to non-genotoxic carcinogens including phenobarbital, choline± methionine de®cient diet, DCA, TCA and trichloroethylene have been reported to decrease the methylation DNA [24,32±39]. Liver tumors induced by a choline±methionine de®cient diet in both mice and rats exhibited hypomethylation and overexpression of the H-ras, c-myc and c-fos genes [25,36±39]. We have previously demonstrated that mouse liver tumors initiated by MNU and promoted by either DCA or TCA contained hypomethylated DNA [34]. The study reported here demonstrates that liver tumors from DCA and TCA-treated mice have decreased methylation in the promoter regions of the c-jun and c-myc protooncogenes and increased expression of their mRNA and proteins. We also reported that DNA MTase activity is increased in the liver tumors of mice initiated with MNU and promoted with DCA and TCA.
2. Materials and methods 2.1. Chemicals DCA and TCA were obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). Ribonuclease A type
III-A and proteinase K were from Sigma Chemical Co., Inc. (St. Louis, MO). TRIzol Reagent was purchased from Gibco BRL/Life Technologies, Inc. (Gaithersburg, MD). Oligonucleotide probes for cjun and c-myc were obtained from Oncogene Research Products (Cambridge, MA). Restriction endonucleases, HpaII, XbaI and EcoO109I were from New England BioLabs (Beverly, MA). Hybond-N 1 nylon membranes [a- 32P]dCTP (6000 Ci/mmol), [g- 32P]ATP (5000 Ci/mmol), enhanced chemiluminescence reagents, polydeoxyinosine± polydeoxycytidine (poly dI-dC) template and T4 polynucleotide kinase were obtained from Amersham Corp. (Arlington Heights, IL). Prime-a-Gene-Labeling System was from Promega Corp. (Madison, WI). Rabbit polyclonal antibodies of c-jun and cmyc, and anti-rabbit IgG-HRP and protein molecular weight standards were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Bio-Rad Protein assay was obtained from Bio-Rad Laboratories (Hercules, CA). [ 3H-methyl]SAM was from Moravek Biochemicals (Brea, CA). All other chemicals were molecular biology, electrophoresis, or HPLC grade as commercially available. 2.2. Mouse liver tumors The source of the liver tumors used in this study has been published [7]. Brie¯y, female B6C3F1 mice at 15 days of age were administered 25 mg/kg N-methylN-nitrosourea (MNU) by intraperitoneal injection. At 6 weeks of age, the pups started to receive in their drinking water 20 mmol/l of either DCA or TCA continuously until euthanized at 52 weeks of age. At necropsy, the liver was excised and examined for tumors. A portion of the tumors were ®xed in 10% phosphate-buffered formalin for histopathological analysis and the remainder rapidly frozen in liquid nitrogen and stored at 2708C. Pieces of non-involved liver tissue around the tumors that initiated with MNU and promoted with DCA or TCA were also harvested and rapidly frozen in liquid nitrogen, i.e. DCA-liver and TCA-liver. No liver tumors were found at necropsy in MNU-treated mice that did not receive a chloroacetic acid. However, the liver tissue from MNU-initiated mice without promotion treatment was also harvested and rapidly frozen in liquid nitrogen, i.e. MNU-liver.
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2.3. Analysis of DNA methylation status in the promoter regions of the c-jun and c-myc protooncogenes Methylation status in the promoter regions for c-jun and c-myc protooncogenes was evaluated using the methylation-sensitive restriction endonuclease HpaII digestion followed by Southern blot analysis as described previously [24,32]. Brie¯y, DNA was isolated from liver tumors and non-involved liver by digestion with 400 mg/ml RNase A and 200 mg/ml proteinase K followed by organic extraction with phenol, chloroform, and isoamyl alcohol. The isolated DNA was digested at 378C with HpaII (2.5 units/mg DNA) followed by electrophoresis on 1% agarose gel and transferred to Hybond-N 1 nylon membranes. HindIII-produced DNA fragments of lambda-phage were included with each gel as molecular size markers. The 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. The probes for c-jun and c-myc contained the 1914±2422 and 1±1315 bp, respectively, in the promoter region of the genes as previously published [24]. Some DNA was also digested with the XbaI and EcoO109I restriction enzymes. 2.4. Analysis for mRNA expression of the c-jun and c-myc protooncogenes Expression of the mRNA for c-jun and c-myc was evaluated by Northern blot analysis as described previously [24,32]. Brie¯y, total RNA was extracted from liver tumors and non-involved liver using TRIzol Reagent. The yield, purity, and integrity of the RNA were assessed by absorbance at 260 nm, the A260/A280 ratio (1.7±1.9) and agarose/formaldehyde gel electrophoresis, respectively. The RNA was electrophoresed on denaturing formaldehyde gels and then transferred to Hybond-N 1 nylon membranes by downward alkaline capillary action. Oligonucleotide probes for mouse c-jun and c-myc were labeled with [g- 32P]ATP by the 5 0 -end labeling procedure to a minimum speci®c activity of 10 8 cpm/mg and separated from unincorporated [g- 32P]ATP using a Sephadex G-50 column. The Northern blots were prehybridized at 658C for 1 h in hybridization solution
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(1.0 M NaCl, 50 mM Tris±HCl (pH 7.5), 10% dextran sulfate, 1% SDS, and 100 mg/ml denatured nonhomologous DNA). The 5 0 -end labeled probe (65 ng) was then added and hybridization proceeded overnight at 658C. After hybridization, the membrane was stringently washed four times with 2 £ SSC containing 0.1% SDS at room temperature, once at 658C for 30 min and once again at room temperature for 5 min followed by a brief wash with 2 £ SSC. The membrane was dried and sealed in a plastic bag. Autoradiography was processed at 2708C using Kodak Biomax MR X-ray ®lm with an intensifying screen. 2.5. Western blot analyses for protein xpression of c-jun and c-myc genes Levels of c-jun and c-myc proteins were determined by Western blot analysis as described previously [24]. Liver tumors and non-involved liver were homogenized in a solution containing 20 mM Tris±HCl (pH 7.5), 10 mM EGTA (pH 7.5), 1 mM EDTA (pH 8.0), 10 mM b-mercaptoethanol, 1 mM phenylmethylsulfonyl ¯uoride, 0.02% leupeptin, 0.04% trypsin inhibitor, 0.25 M sucrose and 0.1% Triton X-100. The homogenates were sonicated and centrifuged at 12 000 £ g for 30 min. Protein concentration in the supernatant was determined using the Bio-Rad Protein assay. The supernatant (30 mg protein) was electrophoresed on 12% SDS±PAGE minigels under reducing conditions and then electrophoretically blotted to Immobilon-P membranes. Detection of c-jun and c-myc protein was performed using 1:200 dilution of rabbit polyclonal antibodies to the protooncogenes (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 1:1000 of anti-rabbit IgG-HRP. The blots were developed with enhanced chemiluminescence reagents. 2.6. Determination of DNA MTase activity DNA MTase activity was determined by a modi®cation of the DNA MTase assay developed by Adams [40]. Nuclei were isolated from liver tumors and noninvolved liver as well as liver from MNU-initiated mice that were not exposed to either chlorinated acetic acid. The isolated nuclei were incubated on ice with 0.8 M KCl, 50 mM Tris±HCl (pH 7.8) for 10 min followed by dilution to 0.3 M KCl with 10 mM Tris±HCl (pH 7.8) and incubated for 30 min. After centrifugation, the
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supernatant (12.5 mg protein) was incubated with 4.5 mCi [ 3H-methyl]SAM (14.5 Ci/mmol), 2 mg poly dI-dC template in a total volume of 100 ml. After 2 h of incubation at 378C, stop reaction reagent (1% SDS, 2 mM EDTA, 5% 2-propanol, 125 mM NaCl, 0.25 mg/kg salmon testes DNA and 1 mg/ml proteinase K) was added and the solution incubated for another hour at 378C. The poly dI-dC template plus carrier DNA was extracted with chloroform-phenol, precipitated with 70% ethanol, dissolved in water, and adsorbed onto ®lter paper discs. The ®lter discs were washed sequentially with ice cold 10% TCA, 5% TCA and 70% ethanol, dried and placed in scintillation vials containing scintillation cocktail. Radioactivity was determined by scintillation counting. Protein was determined using the Bio-Rad Protein assay. Results are expressed as dpm/mg protein.
The promoter region of the c-myc gene contains 1540 bp and has ten CCGG sites; however, HpaIIdigested DNA from DCA- and TCA-promoted tumors when probed for the c-myc gene promoter contained a 2.2 kb band (Fig. 1B). The 2.2 kb band is larger than
3. Results 3.1. Methylation status in the promoter region of the c-jun and c-myc protooncogenes The methylation status in the promoter regions of the c-jun and c-myc genes in liver tumors and non-involved liver from MNU-initiated and DCA- and TCApromoted mice was evaluated by HpaII restriction enzyme digestion followed by Southern blot analysis (Fig. 1A,B). HpaII does not cut CCGG sites when the internal cytosine is methylated. Therefore, the ability to cut DNA indicates that the internal cytosine is not methylated while the inability of HpaII to cut DNA indicates that the cytosine is methylated. HpaII-digested DNA from DCA-and TCA-promoted tumors when probed for the c-jun gene contained bands of 1.5 and 3.2 kb (Fig. 1A) and when probed for the c-myc gene contained bands of 0.5, 1.0 and 2.2 kb (Fig. 1B). These bands were absent in tumor DNA not digested with HpaII and in HpaII-digested DNA from the liver of MNU-initiated but not promoted mice and from noninvolved liver of DCA- and TCA-promoted mice (Fig. 1A,B). Thus, liver DNA from DCA- and TCApromoted mice contained 5-MeC at the internal cytosine of CCGG sites in the promoter regions of the two genes. In contrast, tumor DNA from these mice no longer contained 5-MeC at CCGG sites, making it susceptible to digestion by HpaII.
Fig. 1. Methylation analysis of the promoter region for the c-jun (A) and c-myc (B) protooncogenes in DCA and TCA-promoted liver tumors and in non-involved liver. DNA (30 mg) except for lanes 1, 5 and 10 was digested with the HpaII restriction enzyme.The DNA was then electrophoresed in a 1% agarose gel, transferred to a Hybond-N 1 membrane, and hybridized to a 32P-labeled probe for the promoter region of the c-jun or c-myc genes. Lane 1 is from an MNU-initiated mouse that was not promoted by either chlorinated acetic acid. Lanes 2±4 are from non-involved liver of MNUinitiated mice followed by exposure to DCA, TCA, or neither chlorinated acetic acid (C), respectively. Lanes 5±9 and 10±14 are from liver tumors initiated with MNU and promoted with either DCA or TCA, respectively. The arrows in the right margin indicate the size of the bands.
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the distance between CCGG sites in the promoter region. To demonstrate that this band contains the promoter sequence of the c-myc gene, two ¯anking restriction enzymes, XbaI and EcoO109I were used to cut the 5 0 - and 3 0 -ends of the promoter region at bases 2 and 1490, respectively. Fig. 2 demonstrates that XbaI and EcoO109I-digested DNA from both non-involved liver and DCA-promoted tumors yielded only the expected 1.4 kb band between the sensitive site of each ¯anking restriction enzymes. DNA from non-involved liver digested by HpaII and the two ¯anking restriction enzymes also contained only the 1.4 kb band. However, HpaII digestion of DCA-promoted tumors either before or after treatment with the two ¯anking restriction enzymes resulted in bands of 0.5, 0.7 and 1.0 kb instead of the 1.4 kb band. The 0.7 kb band was not present in HpaII-digested tumor DNA without treat-
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ment with the two ¯anking restriction enzymes. The 2.2 kb band present when tumor DNA was digested with only HpaII is proposed to result from a cut at a CCGG site in the probed promoter region and another site downstream from the probe. Hence, when the DNA was digested with both HpaII and the two ¯anking restriction enzymes, the 2.2 kb band was greatly decreased with the appearance of the 0.7 kb band. The 0.7 kb band corresponded to the size between a CCGG site and the EcoO109I site in the promoter region. Thus 0.7 kb of the 2.2 kb band were demonstrated to be in the promoter region of the c-myc gene. Furthermore, it con®rmed that some of the CCGG sites in DCA-promoted tumors contained unmethylated cytosine that were methylated in DNA from non-involved liver of mice treated with DCA and TCA and from liver of mice not treated with either chlorinated acetic acids. 3.2. Levels of mRNA expression for the c-jun and c-myc protooncogenes The levels of mRNA for c-jun and c-myc genes were virtually undetectable in liver from MNUinitiated mice that did not receive either chlorinated acetic acid and in non-involved liver from DCA- or TCA-treated mice (Fig. 3). Messenger RNA levels of both genes were increased in DCA- and TCApromoted liver tumors. Furthermore, DCA- and TCA-promoted tumors did not differ with respect to the mRNA levels of either gene. However, the level of the mRNA for c-jun appeared to be increased to a greater extent than c-myc. The expression of the mRNA for the two protooncogenes was also increased in the liver of mice administered a single 2-ml/kg dose of carbon tetrachloride (Fig. 3, lane 1: positive control). 3.3. Protein expression for the c-jun and c-myc genes
Fig. 2. Southern analysis of mouse c-myc gene promoter. DNA (30 mg) was digested with two ¯anking restriction enzymes, XbaI and EcoO109I, before and after HpaII digestion. Lanes 1±4 are from non-involved liver and lanes 5±8 are from a liver tumor of an MNUinitiated mice promoted by DCA. Lanes 1 and 5 were digested with both XbaI and EcoO109I (X 1 E); lanes 2 and 6 were digested with HpaII; lanes 3 and 7 were digested with XbaI and EcoO109I before HpaII digestion (X 1 E 1 HpaII); lanes 4 and 8 were digested with XbaI and EcoO109I after HpaII digestion (HpaII 1 X 1 E). The arrows in the right margin indicate the size of the bands.
Western blot analysis of the protein for c-jun and for c-myc migrated as 39 and 65 kDa bands, respectively (Fig. 4). In liver from MNU-initiated mice and in non-involved liver from DCA- and TCA-promoted mice, the c-jun and c-myc proteins were present at very low levels. The protein level of both protooncogenes was increased in DCA- and TCA-promoted liver tumors (Fig. 4).
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Fig. 4. Protein level of the c-jun and c-myc protooncogenes in the livers and tumors promoted by DCA and TCA. Western blot analysis of the protein (30 mg) was performed using rabbit polyclonal antibodies (1:200 dilution) and anti-rabbit IgG-HRP. Blots were developed with enhanced chemiluminescence reagents. Lanes 1±3 contain protein from non-involved liver of MNU-initiated mice followed by exposure to DCA, TCA, or neither chlorinated acetic acid (C), respectively. Lanes 4±6 and 7±9 are from liver tumors promoted by DCA and TCA, respectively.
4. Discussion Fig. 3. Expression of the mRNA for c-jun and c-myc protooncogenes in the livers and tumors from DCA- and TCA-treated female B6C3F1 mice. Northern blot analysis of the RNA (30 mg) was performed using oligonucleotide probes for c-jun and c-myc. Lane 1 is the carbon tetrachloride (2 ml/kg) positive control for enhanced expression of the protooncogenes. Lanes 2±4 contain RNA from non-involved liver of MNU-initiated mice followed by exposure to DCA, TCA, or neither chlorinated acetic acid (C), respectively. Lanes 5±7 and 8±10 are from DCA- and TCA-promoted tumors, respectively. Ethidium bromide staining of the c-myc gel depicting the 18S and 28Ss RNA is presented to demonstrate equal loading of the lanes (bottom). Similar evidence of equal loading was obtained for the c-jun gel.
DCA and TCA are important environmental contaminants and metabolites of trichloroethylene and tetrachloroethylene [1±4] that in B6C3F1mice are carcinogenic in the liver [5±10] and promote MNU-initiated liver tumors [7,8]. It is likely that their carcinogenic mechanism is non-genotoxic and
3.4. DNA MTase activity DNA MTase activity was increased in liver tumors from both DCA- and TCA-promoted mice relative to non-involved liver from the same mouse (Fig. 5). DNA MTase activity in DCA- but not TCA-promoted tumors was also increased when compared with the liver from MNU-initiated mice that did not receive either chlorinated acetic acid. Interestingly, DNA MTase activity was decreased in non-involved liver from both DCA- and TCA-promoted mice relative to MNU-initiated mice that did not receive them (P , 0:05). Thus, long-term exposure to DCA and TCA decreased DNA MTase activity in non-involved mouse liver while promoting liver tumors with increased activity.
Fig. 5. DNA MTase activity in non-involved liver and tumors from MNU-initiated mice exposed to DCA or TCA. The results are mean ^ SE. The asterisk indicates signi®cant difference from non-involved liver of MNU-initiated mice that were administered neither chlorinated acetic acid (MNU-Liver). (a) and (b) indicate signi®cant difference between tumor and non-involved liver from mice administered the same chlorinated acetic acid (P , 0:05).
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involves enhancement of cell proliferation and/or prevention of apoptosis [3,11±20]. During cell proliferation induced in the liver by chemicals or partial hepatectomy, the mRNA and protein levels of immediate-early protooncogenes including c-jun and c-myc are increased [21,22]. Liver tumor promoters and carcinogens including DCA, TCA, and trichloroethylene, as well as carbon tetrachloride, cyproterone acetate, ethylene dibromide, furan, lead nitrate, nafenopin, and phenobarbital have been shown to increase the expression of the mRNA and/or proteins of the two protooncogenes [22±24,32,41±44]. Methylation of CpG sites in the promoter region of genes that are near, or directly within, transcription factorbinding motifs can decrease transcription [25±28]. Increased expression of c-jun and c-myc have been demonstrated in human tumors in the absence of gene mutation and ampli®cation [45,46], suggesting an epigenetic mechanism for the regulation of their expression. Hence, decreased methylation of the c-jun and c-myc genes in liver tumors has been associated with increased expression of their gene [22± 28,47]. The current study demonstrated that the promoter regions of the c-jun and c-myc genes were hypomethylated and the expression of the mRNA and proteins of the two protooncogenes were increased in DCA- and TCA-promoted liver tumors. Liver tumors in humans and laboratory animals including those promoted by DCA and TCA have been shown to contain decreased levels of DNA methylation [25± 28,34±39]. Liver tumors from mice treated with phenobarbital or with a choline±methionine de®cient diet also possess decreased methylation of the c-myc gene and other protooncogenes including H-ras, raf, and c-fos [25,35±39]. In contrast, the non-involved liver from DCA- and TCA-treated mice did not contain a decrease in the methylation of the c-jun and c-myc genes. This is consistent with the noninvolved liver containing normal level of DNA methylation after similar chronic exposure to the chlorinated acetic acids [34]. In contrast, after subchronic exposure of up to 32 days both DNA and the c-jun and c-myc genes were hypomethylated [24,34]. Thus, DCA- and TCA-promoted liver tumors similar to other laboratory animal and human tumors contain hypomethylated genes, while non-involved liver appears to contain hypomethylated genes only
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early during the exposure to the chlorinated acetic acids. Liver tumors from DCA- and TCA-treated mice also possessed increased expression of the mRNA and protein of the two protooncogenes relative to the non-involved liver. Consistent with this observation, increased protein levels of the two protooncogenes have been detected by immunocytochemistry in DCA- and TCA-induced liver tumors without signi®cant expression in the surrounding noninvolved liver [9,23]. We have also previously reported that subchronic exposure to DCA and TCA for 5±32 days results in an increased expression of the mRNA and protein levels of c-jun and c-myc[24]. Thus, both hypomethylation and increased expression of the two protooncogenes in non-involved liver appear to occur only for a limited time after the start of exposure to DCA and TCA and then to return to pre-exposure levels even though exposure is continued. Furthermore, increased expression of c-jun and c-myc has been associated with increased cell proliferation [21,22]. Hence, increased expression and decreased methylation of the c-jun and c-myc genes could be involved in the carcinogenic activity of DCA and TCA by facilitating cell proliferation. Increased DNA MTase activity has been found in liver and other tumors of mice and humans [27±31]. We observed that DNA MTase activity was increased in liver tumors while decreased in non-involved liver from mice exposed to DCA or TCA. This corresponds to the level of cell proliferation being increased in the tumors while decreased in non-involved liver after long-term exposure to the chlorinated acetic acids [5,6]. Furthermore, although in our previous study methylation of c-jun and c-myc in the liver was decreased by short-term exposure of 5±32 days to DCA and TCA [24], in the present study methylation of the two protooncogenes in non-involved liver was unaffected by long-term exposure. Thus, while DNA MTase activity was increased in tumors with decreased methylation of DNA, it was decreased in non-involved liver with unaffected levels of DNA methylation. A later event than DNA hypomethylation and increased DNA MTase activity in mouse and human tumors is the hypermethylation of tumor suppressor genes that results in the down regulation of their mRNA [27±31,47]. The lost of activity of tumor
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suppressor genes results in growth advantage enhancing the progression to cancer. Increased activity of DNA MTase has been associated with the hypermethylation of these genes [27±31,47]. Therefore, the increased DNA MTase activity in DCA- and TCA-promoted tumors could increase the silencing of tumor suppressor genes by increasing their susceptibility to hypermethylation. This should result in the promotion of the neoplastic progression to cancer.
[12]
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Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors Lianhui Tao*, Siming Yang, Mi Xie, Paula M. Kramer, Michael A. Pereira Department of Pathology, Medical College of Ohio, Health Education Building, 3055 Arlington Ave., Toledo, OH 43614-5806, USA Received 20 April 2000; received in revised form 12 June 2000; accepted 16 June 2000
Abstract Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are mouse liver carcinogens. Methylation of the c-jun and c-myc genes, expression of both genes and DNA methyltransferase (DNA MTase) activity were determined in liver tumors initiated by N-methyl-N-nitrosourea and promoted by DCA and TCA in female B6C3F1 mice. Hypomethylated and over-expression of c-jun and c-myc genes were found in DCA- and TCA-promoted liver tumors. DNA MTase activity was increased in tumors while decreased in non-involved liver. Thus, DCA- and TCA-promoted carcinogenesis appears to include decreased methylation and increased expression of c-jun and c-myc genes in the presence of increased DNA MTase activity. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dichloroacetic acid; Trichloroacetic acid; Mouse liver tumor; c-jun; c-myc; DNA methylation
1. Introduction Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are common organic contaminants of drinking water formed as by-products during chlorine disinfection [1,2]. They are also metabolites of trichloroethylene, a common industrial and commercial solvent and an environmental contaminant found in ground water and at hazardous waste sites [3]. TCA is also a major metabolite of tetrachloroethylene [4]. Hence, there exists human exposure to DCA and TCA either from chlorinated drinking water or from metabolism of trichloroethylene and tetrachloroethylene. * Corresponding author. Tel.: 11-419-383-4294; fax: 11-419383-3089. E-mail address: [email protected] (L. Tao).
DCA and TCA in B6C3F1 mice have been shown to induce hepatocellular adenomas and carcinomas [3,5,6,9,10] and to promote N-methyl-N-nitrosourea (MNU)-initiated foci of altered hepatocytes and liver tumors [7,8]. However, the mechanism for their carcinogenicity in mouse liver is unclear. Mouse liver tumors induced by DCA and TCA do not contain a unique mutation spectrum in the ras oncogene relative to spontaneous tumors [11±15]. Due to their very weak genotoxicity [16,17] and their ability to promote liver tumors in mice [7,8], they likely induce liver cancer by a nongenotoxic mechanism. DCA and TCA have been shown to increase cell proliferation in mouse liver [5,6] that is a proposed mechanism for non-genotoxic carcinogens [18±20]. Increased cell proliferation in the liver is associated with increased expression of the immediate-early
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00518-8
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protooncogenes, c-fos, c-jun and c-myc [21,22]. It has been reported that c-jun and c-myc proteins are increased in DCA and TCA-induced foci of altered hepatocytes and tumors in female mice [9,23]. Furthermore, in mouse liver 5 days of exposure to DCA and TCA also increased the level of the mRNA and protein of both protooncogenes [24]. Methylation of DNA as 5-methylcytosine (5-MeC) in the promoter regions of genes including c-jun and c-myc can regulate their expression [25±29]. DNA is methylated by DNA methyltransferase (DNA MTase) with S-adenosyl methionine (SAM) as the methyl donor [27±31]. Hypomethylation of genes including c-jun and c-myc is a frequent early event of carcinogenesis in both humans and rodents [25±29,31±34]. Hypomethylation has been reported to decrease further with the progression from benign to metastatic neoplasm [31±34]. Interestingly, the activity of DNA MTase has been reported to be increase in tumors of humans and laboratory animals even though the DNA is hypomethylated [27±31]. In mouse liver, subchronic exposure to non-genotoxic carcinogens including phenobarbital, choline± methionine de®cient diet, DCA, TCA and trichloroethylene have been reported to decrease the methylation DNA [24,32±39]. Liver tumors induced by a choline±methionine de®cient diet in both mice and rats exhibited hypomethylation and overexpression of the H-ras, c-myc and c-fos genes [25,36±39]. We have previously demonstrated that mouse liver tumors initiated by MNU and promoted by either DCA or TCA contained hypomethylated DNA [34]. The study reported here demonstrates that liver tumors from DCA and TCA-treated mice have decreased methylation in the promoter regions of the c-jun and c-myc protooncogenes and increased expression of their mRNA and proteins. We also reported that DNA MTase activity is increased in the liver tumors of mice initiated with MNU and promoted with DCA and TCA.
2. Materials and methods 2.1. Chemicals DCA and TCA were obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). Ribonuclease A type
III-A and proteinase K were from Sigma Chemical Co., Inc. (St. Louis, MO). TRIzol Reagent was purchased from Gibco BRL/Life Technologies, Inc. (Gaithersburg, MD). Oligonucleotide probes for cjun and c-myc were obtained from Oncogene Research Products (Cambridge, MA). Restriction endonucleases, HpaII, XbaI and EcoO109I were from New England BioLabs (Beverly, MA). Hybond-N 1 nylon membranes [a- 32P]dCTP (6000 Ci/mmol), [g- 32P]ATP (5000 Ci/mmol), enhanced chemiluminescence reagents, polydeoxyinosine± polydeoxycytidine (poly dI-dC) template and T4 polynucleotide kinase were obtained from Amersham Corp. (Arlington Heights, IL). Prime-a-Gene-Labeling System was from Promega Corp. (Madison, WI). Rabbit polyclonal antibodies of c-jun and cmyc, and anti-rabbit IgG-HRP and protein molecular weight standards were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Bio-Rad Protein assay was obtained from Bio-Rad Laboratories (Hercules, CA). [ 3H-methyl]SAM was from Moravek Biochemicals (Brea, CA). All other chemicals were molecular biology, electrophoresis, or HPLC grade as commercially available. 2.2. Mouse liver tumors The source of the liver tumors used in this study has been published [7]. Brie¯y, female B6C3F1 mice at 15 days of age were administered 25 mg/kg N-methylN-nitrosourea (MNU) by intraperitoneal injection. At 6 weeks of age, the pups started to receive in their drinking water 20 mmol/l of either DCA or TCA continuously until euthanized at 52 weeks of age. At necropsy, the liver was excised and examined for tumors. A portion of the tumors were ®xed in 10% phosphate-buffered formalin for histopathological analysis and the remainder rapidly frozen in liquid nitrogen and stored at 2708C. Pieces of non-involved liver tissue around the tumors that initiated with MNU and promoted with DCA or TCA were also harvested and rapidly frozen in liquid nitrogen, i.e. DCA-liver and TCA-liver. No liver tumors were found at necropsy in MNU-treated mice that did not receive a chloroacetic acid. However, the liver tissue from MNU-initiated mice without promotion treatment was also harvested and rapidly frozen in liquid nitrogen, i.e. MNU-liver.
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2.3. Analysis of DNA methylation status in the promoter regions of the c-jun and c-myc protooncogenes Methylation status in the promoter regions for c-jun and c-myc protooncogenes was evaluated using the methylation-sensitive restriction endonuclease HpaII digestion followed by Southern blot analysis as described previously [24,32]. Brie¯y, DNA was isolated from liver tumors and non-involved liver by digestion with 400 mg/ml RNase A and 200 mg/ml proteinase K followed by organic extraction with phenol, chloroform, and isoamyl alcohol. The isolated DNA was digested at 378C with HpaII (2.5 units/mg DNA) followed by electrophoresis on 1% agarose gel and transferred to Hybond-N 1 nylon membranes. HindIII-produced DNA fragments of lambda-phage were included with each gel as molecular size markers. The 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. The probes for c-jun and c-myc contained the 1914±2422 and 1±1315 bp, respectively, in the promoter region of the genes as previously published [24]. Some DNA was also digested with the XbaI and EcoO109I restriction enzymes. 2.4. Analysis for mRNA expression of the c-jun and c-myc protooncogenes Expression of the mRNA for c-jun and c-myc was evaluated by Northern blot analysis as described previously [24,32]. Brie¯y, total RNA was extracted from liver tumors and non-involved liver using TRIzol Reagent. The yield, purity, and integrity of the RNA were assessed by absorbance at 260 nm, the A260/A280 ratio (1.7±1.9) and agarose/formaldehyde gel electrophoresis, respectively. The RNA was electrophoresed on denaturing formaldehyde gels and then transferred to Hybond-N 1 nylon membranes by downward alkaline capillary action. Oligonucleotide probes for mouse c-jun and c-myc were labeled with [g- 32P]ATP by the 5 0 -end labeling procedure to a minimum speci®c activity of 10 8 cpm/mg and separated from unincorporated [g- 32P]ATP using a Sephadex G-50 column. The Northern blots were prehybridized at 658C for 1 h in hybridization solution
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(1.0 M NaCl, 50 mM Tris±HCl (pH 7.5), 10% dextran sulfate, 1% SDS, and 100 mg/ml denatured nonhomologous DNA). The 5 0 -end labeled probe (65 ng) was then added and hybridization proceeded overnight at 658C. After hybridization, the membrane was stringently washed four times with 2 £ SSC containing 0.1% SDS at room temperature, once at 658C for 30 min and once again at room temperature for 5 min followed by a brief wash with 2 £ SSC. The membrane was dried and sealed in a plastic bag. Autoradiography was processed at 2708C using Kodak Biomax MR X-ray ®lm with an intensifying screen. 2.5. Western blot analyses for protein xpression of c-jun and c-myc genes Levels of c-jun and c-myc proteins were determined by Western blot analysis as described previously [24]. Liver tumors and non-involved liver were homogenized in a solution containing 20 mM Tris±HCl (pH 7.5), 10 mM EGTA (pH 7.5), 1 mM EDTA (pH 8.0), 10 mM b-mercaptoethanol, 1 mM phenylmethylsulfonyl ¯uoride, 0.02% leupeptin, 0.04% trypsin inhibitor, 0.25 M sucrose and 0.1% Triton X-100. The homogenates were sonicated and centrifuged at 12 000 £ g for 30 min. Protein concentration in the supernatant was determined using the Bio-Rad Protein assay. The supernatant (30 mg protein) was electrophoresed on 12% SDS±PAGE minigels under reducing conditions and then electrophoretically blotted to Immobilon-P membranes. Detection of c-jun and c-myc protein was performed using 1:200 dilution of rabbit polyclonal antibodies to the protooncogenes (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 1:1000 of anti-rabbit IgG-HRP. The blots were developed with enhanced chemiluminescence reagents. 2.6. Determination of DNA MTase activity DNA MTase activity was determined by a modi®cation of the DNA MTase assay developed by Adams [40]. Nuclei were isolated from liver tumors and noninvolved liver as well as liver from MNU-initiated mice that were not exposed to either chlorinated acetic acid. The isolated nuclei were incubated on ice with 0.8 M KCl, 50 mM Tris±HCl (pH 7.8) for 10 min followed by dilution to 0.3 M KCl with 10 mM Tris±HCl (pH 7.8) and incubated for 30 min. After centrifugation, the
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supernatant (12.5 mg protein) was incubated with 4.5 mCi [ 3H-methyl]SAM (14.5 Ci/mmol), 2 mg poly dI-dC template in a total volume of 100 ml. After 2 h of incubation at 378C, stop reaction reagent (1% SDS, 2 mM EDTA, 5% 2-propanol, 125 mM NaCl, 0.25 mg/kg salmon testes DNA and 1 mg/ml proteinase K) was added and the solution incubated for another hour at 378C. The poly dI-dC template plus carrier DNA was extracted with chloroform-phenol, precipitated with 70% ethanol, dissolved in water, and adsorbed onto ®lter paper discs. The ®lter discs were washed sequentially with ice cold 10% TCA, 5% TCA and 70% ethanol, dried and placed in scintillation vials containing scintillation cocktail. Radioactivity was determined by scintillation counting. Protein was determined using the Bio-Rad Protein assay. Results are expressed as dpm/mg protein.
The promoter region of the c-myc gene contains 1540 bp and has ten CCGG sites; however, HpaIIdigested DNA from DCA- and TCA-promoted tumors when probed for the c-myc gene promoter contained a 2.2 kb band (Fig. 1B). The 2.2 kb band is larger than
3. Results 3.1. Methylation status in the promoter region of the c-jun and c-myc protooncogenes The methylation status in the promoter regions of the c-jun and c-myc genes in liver tumors and non-involved liver from MNU-initiated and DCA- and TCApromoted mice was evaluated by HpaII restriction enzyme digestion followed by Southern blot analysis (Fig. 1A,B). HpaII does not cut CCGG sites when the internal cytosine is methylated. Therefore, the ability to cut DNA indicates that the internal cytosine is not methylated while the inability of HpaII to cut DNA indicates that the cytosine is methylated. HpaII-digested DNA from DCA-and TCA-promoted tumors when probed for the c-jun gene contained bands of 1.5 and 3.2 kb (Fig. 1A) and when probed for the c-myc gene contained bands of 0.5, 1.0 and 2.2 kb (Fig. 1B). These bands were absent in tumor DNA not digested with HpaII and in HpaII-digested DNA from the liver of MNU-initiated but not promoted mice and from noninvolved liver of DCA- and TCA-promoted mice (Fig. 1A,B). Thus, liver DNA from DCA- and TCApromoted mice contained 5-MeC at the internal cytosine of CCGG sites in the promoter regions of the two genes. In contrast, tumor DNA from these mice no longer contained 5-MeC at CCGG sites, making it susceptible to digestion by HpaII.
Fig. 1. Methylation analysis of the promoter region for the c-jun (A) and c-myc (B) protooncogenes in DCA and TCA-promoted liver tumors and in non-involved liver. DNA (30 mg) except for lanes 1, 5 and 10 was digested with the HpaII restriction enzyme.The DNA was then electrophoresed in a 1% agarose gel, transferred to a Hybond-N 1 membrane, and hybridized to a 32P-labeled probe for the promoter region of the c-jun or c-myc genes. Lane 1 is from an MNU-initiated mouse that was not promoted by either chlorinated acetic acid. Lanes 2±4 are from non-involved liver of MNUinitiated mice followed by exposure to DCA, TCA, or neither chlorinated acetic acid (C), respectively. Lanes 5±9 and 10±14 are from liver tumors initiated with MNU and promoted with either DCA or TCA, respectively. The arrows in the right margin indicate the size of the bands.
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the distance between CCGG sites in the promoter region. To demonstrate that this band contains the promoter sequence of the c-myc gene, two ¯anking restriction enzymes, XbaI and EcoO109I were used to cut the 5 0 - and 3 0 -ends of the promoter region at bases 2 and 1490, respectively. Fig. 2 demonstrates that XbaI and EcoO109I-digested DNA from both non-involved liver and DCA-promoted tumors yielded only the expected 1.4 kb band between the sensitive site of each ¯anking restriction enzymes. DNA from non-involved liver digested by HpaII and the two ¯anking restriction enzymes also contained only the 1.4 kb band. However, HpaII digestion of DCA-promoted tumors either before or after treatment with the two ¯anking restriction enzymes resulted in bands of 0.5, 0.7 and 1.0 kb instead of the 1.4 kb band. The 0.7 kb band was not present in HpaII-digested tumor DNA without treat-
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ment with the two ¯anking restriction enzymes. The 2.2 kb band present when tumor DNA was digested with only HpaII is proposed to result from a cut at a CCGG site in the probed promoter region and another site downstream from the probe. Hence, when the DNA was digested with both HpaII and the two ¯anking restriction enzymes, the 2.2 kb band was greatly decreased with the appearance of the 0.7 kb band. The 0.7 kb band corresponded to the size between a CCGG site and the EcoO109I site in the promoter region. Thus 0.7 kb of the 2.2 kb band were demonstrated to be in the promoter region of the c-myc gene. Furthermore, it con®rmed that some of the CCGG sites in DCA-promoted tumors contained unmethylated cytosine that were methylated in DNA from non-involved liver of mice treated with DCA and TCA and from liver of mice not treated with either chlorinated acetic acids. 3.2. Levels of mRNA expression for the c-jun and c-myc protooncogenes The levels of mRNA for c-jun and c-myc genes were virtually undetectable in liver from MNUinitiated mice that did not receive either chlorinated acetic acid and in non-involved liver from DCA- or TCA-treated mice (Fig. 3). Messenger RNA levels of both genes were increased in DCA- and TCApromoted liver tumors. Furthermore, DCA- and TCA-promoted tumors did not differ with respect to the mRNA levels of either gene. However, the level of the mRNA for c-jun appeared to be increased to a greater extent than c-myc. The expression of the mRNA for the two protooncogenes was also increased in the liver of mice administered a single 2-ml/kg dose of carbon tetrachloride (Fig. 3, lane 1: positive control). 3.3. Protein expression for the c-jun and c-myc genes
Fig. 2. Southern analysis of mouse c-myc gene promoter. DNA (30 mg) was digested with two ¯anking restriction enzymes, XbaI and EcoO109I, before and after HpaII digestion. Lanes 1±4 are from non-involved liver and lanes 5±8 are from a liver tumor of an MNUinitiated mice promoted by DCA. Lanes 1 and 5 were digested with both XbaI and EcoO109I (X 1 E); lanes 2 and 6 were digested with HpaII; lanes 3 and 7 were digested with XbaI and EcoO109I before HpaII digestion (X 1 E 1 HpaII); lanes 4 and 8 were digested with XbaI and EcoO109I after HpaII digestion (HpaII 1 X 1 E). The arrows in the right margin indicate the size of the bands.
Western blot analysis of the protein for c-jun and for c-myc migrated as 39 and 65 kDa bands, respectively (Fig. 4). In liver from MNU-initiated mice and in non-involved liver from DCA- and TCA-promoted mice, the c-jun and c-myc proteins were present at very low levels. The protein level of both protooncogenes was increased in DCA- and TCA-promoted liver tumors (Fig. 4).
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Fig. 4. Protein level of the c-jun and c-myc protooncogenes in the livers and tumors promoted by DCA and TCA. Western blot analysis of the protein (30 mg) was performed using rabbit polyclonal antibodies (1:200 dilution) and anti-rabbit IgG-HRP. Blots were developed with enhanced chemiluminescence reagents. Lanes 1±3 contain protein from non-involved liver of MNU-initiated mice followed by exposure to DCA, TCA, or neither chlorinated acetic acid (C), respectively. Lanes 4±6 and 7±9 are from liver tumors promoted by DCA and TCA, respectively.
4. Discussion Fig. 3. Expression of the mRNA for c-jun and c-myc protooncogenes in the livers and tumors from DCA- and TCA-treated female B6C3F1 mice. Northern blot analysis of the RNA (30 mg) was performed using oligonucleotide probes for c-jun and c-myc. Lane 1 is the carbon tetrachloride (2 ml/kg) positive control for enhanced expression of the protooncogenes. Lanes 2±4 contain RNA from non-involved liver of MNU-initiated mice followed by exposure to DCA, TCA, or neither chlorinated acetic acid (C), respectively. Lanes 5±7 and 8±10 are from DCA- and TCA-promoted tumors, respectively. Ethidium bromide staining of the c-myc gel depicting the 18S and 28Ss RNA is presented to demonstrate equal loading of the lanes (bottom). Similar evidence of equal loading was obtained for the c-jun gel.
DCA and TCA are important environmental contaminants and metabolites of trichloroethylene and tetrachloroethylene [1±4] that in B6C3F1mice are carcinogenic in the liver [5±10] and promote MNU-initiated liver tumors [7,8]. It is likely that their carcinogenic mechanism is non-genotoxic and
3.4. DNA MTase activity DNA MTase activity was increased in liver tumors from both DCA- and TCA-promoted mice relative to non-involved liver from the same mouse (Fig. 5). DNA MTase activity in DCA- but not TCA-promoted tumors was also increased when compared with the liver from MNU-initiated mice that did not receive either chlorinated acetic acid. Interestingly, DNA MTase activity was decreased in non-involved liver from both DCA- and TCA-promoted mice relative to MNU-initiated mice that did not receive them (P , 0:05). Thus, long-term exposure to DCA and TCA decreased DNA MTase activity in non-involved mouse liver while promoting liver tumors with increased activity.
Fig. 5. DNA MTase activity in non-involved liver and tumors from MNU-initiated mice exposed to DCA or TCA. The results are mean ^ SE. The asterisk indicates signi®cant difference from non-involved liver of MNU-initiated mice that were administered neither chlorinated acetic acid (MNU-Liver). (a) and (b) indicate signi®cant difference between tumor and non-involved liver from mice administered the same chlorinated acetic acid (P , 0:05).
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involves enhancement of cell proliferation and/or prevention of apoptosis [3,11±20]. During cell proliferation induced in the liver by chemicals or partial hepatectomy, the mRNA and protein levels of immediate-early protooncogenes including c-jun and c-myc are increased [21,22]. Liver tumor promoters and carcinogens including DCA, TCA, and trichloroethylene, as well as carbon tetrachloride, cyproterone acetate, ethylene dibromide, furan, lead nitrate, nafenopin, and phenobarbital have been shown to increase the expression of the mRNA and/or proteins of the two protooncogenes [22±24,32,41±44]. Methylation of CpG sites in the promoter region of genes that are near, or directly within, transcription factorbinding motifs can decrease transcription [25±28]. Increased expression of c-jun and c-myc have been demonstrated in human tumors in the absence of gene mutation and ampli®cation [45,46], suggesting an epigenetic mechanism for the regulation of their expression. Hence, decreased methylation of the c-jun and c-myc genes in liver tumors has been associated with increased expression of their gene [22± 28,47]. The current study demonstrated that the promoter regions of the c-jun and c-myc genes were hypomethylated and the expression of the mRNA and proteins of the two protooncogenes were increased in DCA- and TCA-promoted liver tumors. Liver tumors in humans and laboratory animals including those promoted by DCA and TCA have been shown to contain decreased levels of DNA methylation [25± 28,34±39]. Liver tumors from mice treated with phenobarbital or with a choline±methionine de®cient diet also possess decreased methylation of the c-myc gene and other protooncogenes including H-ras, raf, and c-fos [25,35±39]. In contrast, the non-involved liver from DCA- and TCA-treated mice did not contain a decrease in the methylation of the c-jun and c-myc genes. This is consistent with the noninvolved liver containing normal level of DNA methylation after similar chronic exposure to the chlorinated acetic acids [34]. In contrast, after subchronic exposure of up to 32 days both DNA and the c-jun and c-myc genes were hypomethylated [24,34]. Thus, DCA- and TCA-promoted liver tumors similar to other laboratory animal and human tumors contain hypomethylated genes, while non-involved liver appears to contain hypomethylated genes only
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early during the exposure to the chlorinated acetic acids. Liver tumors from DCA- and TCA-treated mice also possessed increased expression of the mRNA and protein of the two protooncogenes relative to the non-involved liver. Consistent with this observation, increased protein levels of the two protooncogenes have been detected by immunocytochemistry in DCA- and TCA-induced liver tumors without signi®cant expression in the surrounding noninvolved liver [9,23]. We have also previously reported that subchronic exposure to DCA and TCA for 5±32 days results in an increased expression of the mRNA and protein levels of c-jun and c-myc[24]. Thus, both hypomethylation and increased expression of the two protooncogenes in non-involved liver appear to occur only for a limited time after the start of exposure to DCA and TCA and then to return to pre-exposure levels even though exposure is continued. Furthermore, increased expression of c-jun and c-myc has been associated with increased cell proliferation [21,22]. Hence, increased expression and decreased methylation of the c-jun and c-myc genes could be involved in the carcinogenic activity of DCA and TCA by facilitating cell proliferation. Increased DNA MTase activity has been found in liver and other tumors of mice and humans [27±31]. We observed that DNA MTase activity was increased in liver tumors while decreased in non-involved liver from mice exposed to DCA or TCA. This corresponds to the level of cell proliferation being increased in the tumors while decreased in non-involved liver after long-term exposure to the chlorinated acetic acids [5,6]. Furthermore, although in our previous study methylation of c-jun and c-myc in the liver was decreased by short-term exposure of 5±32 days to DCA and TCA [24], in the present study methylation of the two protooncogenes in non-involved liver was unaffected by long-term exposure. Thus, while DNA MTase activity was increased in tumors with decreased methylation of DNA, it was decreased in non-involved liver with unaffected levels of DNA methylation. A later event than DNA hypomethylation and increased DNA MTase activity in mouse and human tumors is the hypermethylation of tumor suppressor genes that results in the down regulation of their mRNA [27±31,47]. The lost of activity of tumor
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suppressor genes results in growth advantage enhancing the progression to cancer. Increased activity of DNA MTase has been associated with the hypermethylation of these genes [27±31,47]. Therefore, the increased DNA MTase activity in DCA- and TCA-promoted tumors could increase the silencing of tumor suppressor genes by increasing their susceptibility to hypermethylation. This should result in the promotion of the neoplastic progression to cancer.
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