Hypomethylation and overexpression of c-jun and c-myc protooncogenes and increased DNA methyltransferase activity in dichloroacetic and trichloroacetic acid-promoted mouse liver tumors

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.     

Detection of early gene expression changes by differential display in the livers of mice exposed to dichloroacetic acid

Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in mice when administered in drinking water. The mechanism of DCA carcinogenicity is not clear and we speculate that changes in gene expression may be important. In order to analyze early changes in gene expression induced by DCA treatment we used the differential display method. Mice were treated with 2 g/l DCA in drinking water for 4 weeks. Total RNAs were obtained from livers of both control and treated mice for analysis. Of approximately 48 000 bands on the differential display gels representing an estimated 96% of RNA species, 381 showed differences in intensity. After cloning and confirmation by both reverse-northern and northern analyses, six differentially expressed genes were found. The expression of five of these genes was suppressed in the DCA-treated mice while one was induced. After sequencing, four genes were identified and two were matched to expressed sequence tags through the BLAST program. These genes are alpha-1 protease inhibitor, cytochrome b5, stearoyl-CoA desaturase and carboxylesterase. Stearoyl-CoA desaturase was induced approximately 3-fold in the livers of DCA-treated mice and the other three genes were suppressed approximately 3-fold. Stearoyl-CoA desaturase, cytochrome b5 and carboxylesterase are endoplasmic reticulum membrane-bound enzymes involved in fatty acid metabolism. The expression pattern of four of these genes was similar in DCA-induced hepatocellular carcinomas and the 4 week DCA-treated mouse livers. The expression of stearoyl-CoA desaturase and one of the unidentified genes returned to control levels in the carcinomas. Understanding the roles and interactions between these genes may shed light on the mechanism of DCA carcinogenesis.     

Consideration of the evidence for mechanisms of 1,1,2-trichloroethylene metabolism,including new identification of its dichloroacetic acid and trichloroacetic acid metabolites in mice

Data derived from studies with vinylidene chloride (1,1-dichloroethylene) and 1,1,2-trichloroethylene suggest that similar mutagenic and tumorogenic properties in mice may be attributable to rearrangement of the 2 haloalkene-derived haloepoxides, respectively, into chloroacetyl chloride and dichloroacetyl chloride. On the other hand, the relative harmlessness of 1,1,2-trichloroethylene in rats and man is due to alternative rearrangement of 1,1,2-trichloroethylene oxide into chloral and the further products of its metabolism. The identification in mice of the new 1,1,2-trichloroethylene metabolite, dichloroacetic acid (in addition to trichloroacetic acid) strongly supports this supposition. The small proportion of dichloroacetic acid in relation to the large proportion of trichloroacetic acid in the urine of the treated mice is consistent with a spill-over model that is now tentatively proposed for 1,1,2-trichloroethylene metabolism in these animals.     

Effect of subsequent treatment of chloroform or phenobarbital on the incidence of liver and lung tumors initiated by ethylnitrosourea in 15 day old mice

The effect of subsequent administration of chloroform or phenobarbitalon the incidence of ethylnitrosourea (ENU) initiated liver andlung tumors was investigated. Fifteen day old Swiss mice wereadministered ENU, and at weaning they started to receive either1800 p.p.m. chloroform or 500 p.p.m. sodium phenobarbital intheir drinking water. The mice continued to receive either chloroformor phenobarbital until 51 weeks of age. They were sacrificedat 52 weeks of age. ENU at 5 and 20 mg/kg, caused a dose-dependentincrease in liver and lung tumors. The male mice were more sensitiveto the induction of liver tumors, while no sex preference wasobserved for the induction of lung tumors. In male mice chloroforminhibited, while in female and male mice phenobarbital promotedspontaneous and ENU-induced liver tumors. Subsequent treatmentwith either chloroform or phenobarbital did not affect the incidenceof ENU-induced lung tumors. In conclusion, when administeredin the drinking water, chloroform inhibited while phenobarbitalpromoted hepatocarcinogenesis in mice.     

ras proto-oncogene activation in dichloroacetic add-, trichloroethylene- and tetrachloroethylene-induced liver tumors in B6C3F1 mice

The frequency and mutation spectra of proto-oncogene activationin hepatocellular neoplasms induced by tetrachloroethylene,trichloroethylene and dichloroacetic acid were examined to helpdefine the molecular basis for their carcinogenicity. H-rascodon 61 activation was not significantly different among dichloroaceticacid- and trichloroethylene-induced and combined historicaland concurrent control hepatocellular tumors (62%, 51% and 69%respectively). The mutation spectra of H-ras codon 61 mutationsshowed a significant decrease in AAA and increase in CTA mutationsfor dichloroacetic acid- and trichloroethylene-induced tumorswhen compared to combined controls. The H-ras codon 61 mutationfrequency for tetrachloroethylene-induced tumors was significantlylower (24%) than that of combined controls and also that ofthe two other chemicals. Mutations at codons 13 and 117 plusa second exon insert contributed 4% to the total H-ras frequenciesfor trichloroethylene and tetrachloroethylene. There was alsoa higher incidence of K-ras activation (13%) in tetrachloroethylene-inducedtumors than in the other chemically induced or control tumors.Four liver tumors were found to contain insertions of additionalbases within the second exon of K- or H-ras. These findingssuggest that exposure to dichloroacetic acid, trichloroethyleneand tetrachloroethylene provides a selective growth advantageto spontaneously occurring mutations in codon 61 of H-ras and,at the same time, is responsible for a small number of uniquemolecular lesions suggestive of either a random genotoxic modeof action or a non-specific result of secondary DNA damage.However, the absence of ras activation in many of the liverneoplasms suggests that alternative mechanisms are also importantin B6C3F1 mouse hepatocarcinogenesis.     

Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice

Genomewide DNA hypomethylation is a consistent finding in human tumors, but the importance of this change for human tumorigenesis remains an open question. We have previously reported that mice carrying a hypomorphic allele for the maintenance DNA methyltransferase (Dnmt1(chip/-)) are hypomethylated and develop thymic lymphomas, demonstrating that genomewide DNA hypomethylation can induce tumors. Hypomethylated cells exhibit inherent chromosomal instability, which is revealed in the lymphomas as a consistent trisomy of chromosome 15. We now report another aspect of the molecular basis for tumor development upon DNA hypomethylation. Seven out of 16 hypomethylation-induced lymphomas were found to contain an intracisternal A particle (IAP) somatic insertion in the middle of the Notch1 genomic locus, leading to generation of an oncogenic form of Notch1 in the tumors. This finding suggests that the molecular basis for hypomethylation-induced tumors in this model involves chromosomal instability events accompanied by activation of endogenous retroviral elements. Our findings validate the proposed role of DNA methylation in suppression of transposable elements in mammalian cells and demonstrate the importance of DNA methylation for normal cell function as well as the potential consequences of spontaneously occurring or chemically induced DNA hypomethylation.     

Effect of phenobarbital on the development of liver tumors in juvenile and adult mice

The effect of long-term exposure to phenobarbital (CAS: 50-06-6) subsequent to tumor initiation on the development of liver tumors in BALB/c and (C57BL/6 X C3H/Anf)F1 (B6C3F1) mice was determined. In male B6C3F1 mice that received either 15 or 45 ppm diethylnitrosamine [(DENA) CAS: 55-18-5] between 6 and 10 weeks of age, subsequent treatment with 500 ppm sodium phenobarbital in the drinking water resulted in the promotion of liver tumors. However, in male B6C3F1 mice initiated on day 15 of age with 25 mg DENA/kg, beginning long-term treatment of 500 ppm sodium phenobarbital at 4 weeks of age inhibited the development of liver tumors, whereas in male BALB/c mice initiated with 25 mg DENA/kg on day 15 of age, beginning the long-term treatment with 500 ppm sodium phenobarbital at 4 weeks of age promoted the development of liver tumors. Hence phenobarbital can either enhance or inhibit the formation of liver tumors, depending both on the mouse strain used and the animal's age at the start of exposure.     

The expression of c-myc and c-N-ras in human cirrhotic livers, hepatocellular carcinomas and liver tissue surrounding the tumors

To study the possible role of proto-oncogenes in the multistep process of human liver hepatocarcinogenesis, we have examined the expression of c-N-ras and c-myc in human hepatocellular carcinomas and liver tissue surrounding the tumors as well as cirrhotic livers which are generally considered to precede the formation of human hepatocellular carcinoma. One to four-fold higher expression of the c-N-ras proto-oncogene was observed in twelve hepatoma patients as compared to normal liver. Increased expression of c-N-ras was also observed in liver tissue surrounding these tumors. Eight patients exhibited an apparent higher expression of the c-N-ras oncogene in adjacent liver tissue than in their corresponding tumor tissues. Six human liver cirrhosis patients also exhibited a slight increase in c-N-ras expression. Southern blot analysis demonstrated an amplified c-N-ras sequence in these tissues surrounding the tumors. In the study of the c-myc gene, variable degrees of highly enhanced expression were found in all twelve hepatoma patients as compared to normal liver. The c-myc gene was also expressed in the adjacent liver tissue and in some of the human cirrhotic livers. Our studies give further evidence that the expression of c-N-ras and c-myc proto-oncogenes are involved in the process of human hepatocarcinogenesis.     

Effect on the expression of c-met, c-myc and PPAR-{alpha} in liver and liver tumors from rats chronically exposed to the hepatocarcinogenic peroxisome proliferator WY-14, 643

The induction of rodent hepatic tumors by peroxisome proliferators(PP) appears to depend on focal growth of hepatocytes. Expressionof the oncogenes c-met and c-mycis altered following regenerativestimuli in rat liver, suggesting involvement of their proteinproducts in hepatocyte replication. In addition, increases inc-myc and c-met mRNA expression are observed in multiple typesof human and rodent tumors, including hepatocellular carcinoma.A study was designed to test the hypothesis that developmentof PP-induced hepatic neoplasms occurs as a result of overexpressionof c-met or c-myc. Male F344 rats were exposed to WY-14, 643for 22 or 78 weeks (1000 p.p.m. in the diet). Messenger RNAwas extracted from liver tumors (78 weeks) and surrounding non-lesionliver of exposed rats and non-lesion liver from age-matchedcontrol rats. Levels of mRNA expression were compared usingNorthern analysis. Significant increases in c-met (     

Effect of verapamil on cell cycle transit and c-myc gene expression in normal and malignant murine cells

Verapamil, the prototype calcium channel blocker, reversibly inhibits cell proliferation in many normal and tumour cell lines (Schmidt et al., Cancer Res., 48, 3617, 1988). We have found that two closely related cell lines - B16 murine melanoma cells and B10.BR normal murine melanocytes growing in culture - behave differently in the presence of verapamil, and we are now utilising these two related cell lines to help elucidate the molecular basis of verapamil's antiproliferative effect. In this study, we studied cell cycle phase distribution and c-myc gene expression in both cell lines in the absence of verapamil, during incubation with verapamil and after the cells were washed free of verapamil. Our studies show that 100 microM verapamil rapidly blocks DNA synthesis in melanocytes but not in B16 cells. Similarly, incubation with verapamil for 6-24 h results in a decreased c-myc signal in melanocytes, but a transient increase in c-myc expression in B16 cells. After verapamil is washed from the cells following a 24-h incubation with drug, c-myc expression increases in melanocytes as they begin again to proliferate, but decreases in B16 cells as they begin to die. Our disparate results with these cell lines suggest that c-myc gene expression, regardless of its known involvement in growth control, is not the immediate target for verapamil's inhibitory action.     

Effect of budesonide on the methylation and mRNA expression of the insulin-like growth factor 2 and c-myc genes in mouse lung tumors

The use of surrogate end-point biomarkers could help in the development of chemopreventive agents. To define potential surrogate end-point biomarkers, the ability of budesonide to decrease mRNA expression of the insulin-like growth factor-2 (Igf-II) and c-myc genes and to cause the remethylation of the genes was investigated in lung tumors. Lung tumors were induced in female strain A mice by administering i.p. 16 mg/kg vinyl carbamate for 2 consecutive wk or by a single dose of 100 mg/kg benzo[a]pyrene (B[a]P). Thirty-four weeks later, the mice given vinyl carbamate received budesonide (0.6 or 2.4 mg/kg diet) for 7 d and then were killed. Mice were killed 24 wk after administration of B[a]P. The mRNA expression of the Igf-II and c-myc genes was increased in lung tumors relative to normal lung tissue. Budesonide decreased mRNA expression of both genes in tumors. The methylation status of 27 CpG sites in the differentially methylated region 2 in the Igf-II gene was determined with the bisulfite-treated DNA-sequencing procedure. The numbers of methylated CpG sites were 17-21 in normal lung (70.4 +/- 2.6%); 0-2, and 1-2 in lung tumors induced by vinyl carbamate and B[a]P (4.9 +/- 1.2% and 4.6 +/- 1.2%, respectively); and 4-5 or 7-16 in tumors after treatment with 0.6 or 2.4 mg/kg budesonide (16.0 +/- 1.2% and 46.2 +/- 5.1%, respectively). Thus, lung tumors had strikingly less methylated CpG sites than normal lung tissue, while even limited treatment with budesonide resulted in remethylation of the CpG sites in tumors. With HpaII digestion followed by Southern blot analysis, the internal cytosine of CCGG sites in the c-myc gene was found to be methylated in normal lung tissue, whereas some of the sites were unmethylated in lung tumors. Treatment for 7 d with budesonide resulted in the remethylation of these sites. In conclusion, mouse lung tumors showed decreased methylation of the Igf-II and c-myc genes that was associated with increased expression of these genes. Budesonide treatment caused remethylation and decreased expression of both genes. The results support the possibility of using decreased mRNA expression and remethylation of the Igf-II and c-myc genes as biomarkers for the efficacy of budesonide.     

Growth-inhibition effects of oleic acid, linoleic acid, and their methyl esters on transplanted tumors in mice

We investigated the effects of oleic acid and linoleic acid on transplanted Ehrlich ascites carcinoma and Ehrlich solid carcinoma in ACR mice. Both acids significantly prolonged the life spans of Ehrlich ascites carcinoma-bearing mice and inhibited the growth of Ehrlich solid carcinoma in mice compared with the findings in untreated control mice. Methyl esters of these acids also prolonged the survival of Ehrlich ascites carcinoma-bearing mice, but they were less effective in lengthening the survival of mice given transplants of Ehrlich ascites carcinoma. In addition, gas-chromatography analysis of tumor cell lipids showed that appreciable changes occurred in the fatty acid composition of the tumor cell grown in mice treated with oleic acid or linoleic acid. Linoleic acid caused more pronounced alterations in fatty acid composition of tumor cell lipids than did oleic acid, a feature that parallels the intensity of the cytotoxicity potential of the two free fatty acids. These results suggest that (a) the free carboxyl group of free fatty acids plays a role in killing tumor cells and (b) the modification of the fatty acid composition of tumor cells also correlates with the antitumor effects of oleic and linoleic acids. In addition, these results indicate that free fatty acids may be of tumor-oriented distribution; as a consequence, free fatty acids selectively inhibit the growth of tumor cells.     

Combined DNA methylation and gene expression profiling in gastrointestinal stromal tumors reveals hypomethylation of SPP1 as an independent prognostic factor

Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to localization, genotype and aggressiveness. DNA methylation at CpG dinucleotides is an important mechanism for regulation of gene expression. We performed targeted DNA methylation analysis of 1.505 CpG loci in 807 cancer‐related genes in a cohort of 76 GISTs, combined with genome‐wide mRNA expression analysis in 22 GISTs, to identify signatures associated with clinicopathological parameters and prognosis. Principal component analysis revealed distinct DNA methylation patterns associated with anatomical localization, genotype, mitotic counts and clinical follow‐up. Methylation of a single CpG dinucleotide in the non‐CpG island promoter of SPP1 was significantly correlated with shorter disease‐free survival. Hypomethylation of this CpG was an independent prognostic parameter in a multivariate analysis compared to anatomical localization, genotype, tumor size and mitotic counts in a cohort of 141 GISTs with clinical follow‐up. The epigenetic regulation of SPP1 was confirmed in vitro, and the functional impact of SPP1 protein on tumorigenesis‐related signaling pathways was demonstrated. In summary, SPP1 promoter methylation is a novel and independent prognostic parameter in GISTs, and might be helpful in estimating the aggressiveness of GISTs from the intermediate‐risk category.     

肝细胞肝癌及癌周组织中P~（53）、C－myc基因产物表达的研究

应用免疫组化ＡＢＣ法，对肝癌高发区６２例肝细胞癌组织及癌周肝组织进行了Ｐ５３和Ｃ－ｍｙｃ基因产物的标记。结果显示，肝细胞肝癌组织中Ｐ５３与Ｃ－ｍｙｃ基因蛋白均过量表达，阳性率分别为７４．１９％与６７．７４％，均高于有关文献报道的检测率。癌周肝组织中Ｐ５３与Ｃ－ｍｙｃ阳性检出率均低于癌组织。ＨＢｓＡｇ阳性病例、癌周伴肝硬变病例Ｐ５３与Ｃ－ｍｙｃ基因异常表达率均显著高于相应的阴性病例。本文随访病例中，术后肿瘤复发组Ｐ５３与Ｃ－ｍｙｃ基因表达的阳性率极明显地高于未复发尚生存组。文中还讨论了Ｐ５３和Ｃ－ｍｙｃ基因产物的表达在肝细胞癌发生中的可能作用