Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors
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.
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], [12], [13], [14], [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 non-genotoxic 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], [19], [20]. Increased cell proliferation in the liver is associated with increased expression of the immediate-early 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], [26], [27], [28], [29]. DNA is methylated by DNA methyltransferase (DNA MTase) with S-adenosyl methionine (SAM) as the methyl donor [27], [28], [29], [30], [31]. Hypomethylation of genes including c-jun and c-myc is a frequent early event of carcinogenesis in both humans and rodents [25], [26], [27], [28], [29], [31], [32], [33], [34]. Hypomethylation has been reported to decrease further with the progression from benign to metastatic neoplasm [31], [32], [33], [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], [28], [29], [30], [31].
In mouse liver, subchronic exposure to non-genotoxic carcinogens including phenobarbital, choline–methionine deficient diet, DCA, TCA and trichloroethylene have been reported to decrease the methylation DNA [24], [32], [33], [34], [35], [36], [37], [38], [39]. Liver tumors induced by a choline–methionine deficient diet in both mice and rats exhibited hypomethylation and overexpression of the H-ras, c-myc and c-fos genes [25], [36], [37], [38], [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.
Section snippets
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 c-jun 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+ nylon membranes [α-32P]dCTP
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 TCA-promoted 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
Discussion
DCA and TCA are important environmental contaminants and metabolites of trichloroethylene and tetrachloroethylene [1], [2], [3], [4] that in B6C3F1mice are carcinogenic in the liver [5], [6], [7], [8], [9], [10] and promote MNU-initiated liver tumors [7], [8]. It is likely that their carcinogenic mechanism is non-genotoxic and involves enhancement of cell proliferation and/or prevention of apoptosis [3], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. During cell proliferation
Acknowledgements
This research was supported in part by U.S. Environmental Protection Agency Grant No. R 825384-01-0.
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