Reversibility of changes in nucleic acid methylation and gene expression induced in rat liver by severe dietary methyl deficiency.

As we have reported previously, both DNA and tRNA become hypomethylated in livers of rats fed a cancer promoting, methyl-deficient diet (MDD) for as short a period as one week. Within the same period, activities of tRNA and DNA methyltransferases (MTases) increase and levels of mRNAs for several genes believed to have roles in growth regulation are altered. These diet-induced changes in nucleic acid methylation and gene expression increased in extent when MDD was fed continuously for four weeks. We also observed hypomethylation of specific CCGG sites within several genes for which mRNA levels were increased. These included c-myc, c-fos and c-Ha-ras. To investigate the reversibility of such diet-induced alterations in methylation and gene expression, animals were fed MDD for four weeks, after which a diet supplemented with adequate sources of methyl groups (CSD) was fed for 1-3 weeks. One to two weeks after the restoration of an adequate diet, the overall extent of methylation of tRNA and DNA from livers of these rats did not differ from that of tRNA and DNA from livers of age matched animals continually maintained on CSD. At the same time, activities of MTases in the liver dropped to normal values. Levels of mRNAs for all genes studied returned to control levels within three weeks after ending MDD feeding, although at different rates. In contrast, MDD-induced hypomethylation of some HpaII sites in c-myc, c-fos and c-Ha-ras genes persisted after 3 weeks refeeding of an adequate diet. These results, which demonstrate that most of the effects of MDD on the parameters we have studied occur rapidly and are essentially reversible, are consistent with the role of MDDs as promoters of hepatocarcinogenesis. However, the finding that unmethylated sites persist in genes that play a role in growth regulation suggests a mechanism by which intermittent or long term exposure to MDDs could result in heritable phenotypic changes in some hepatocytes that lead to hyperplasia and tumorigenesis.

Alterations in expression and methylation of specific genes in livers of rats fed a cancer promoting methyl-deficient diet.

We have reported earlier that hypomethylated DNA is rapidly induced in the livers of male Fischer rats fed an extremely methyl-deficient diet (MDD). The early effects of dietary methyl deficiency on the expression of several genes in the livers of such animals have now been investigated. Poly(A)+ RNA was isolated from the livers of rats fed MDD or a similar diet supplemented with adequate supplies of choline, methionine, folic acid and vitamin B12 (CSD) for periods ranging from 1 to 4 weeks. The levels of mRNAs for the c-myc and c-fos protooncogenes in livers of rats given either MDD or the liver carcinogen, 2-acetylaminofluorene (AAF), were compared by Northern blot analysis with those in livers of animals given control diets. Both AAF and MDD induced significant elevations in levels of mRNAs specific for these two genes. After 1 week of MDD intake, large increases in the levels of c-myc and c-fos mRNAs and a smaller increase in the levels of c-Ha-ras mRNAs were observed. In contrast, there were marked decreases in the levels of mRNAs for epidermal growth factor receptor and for epidermal growth factor. These effects on mRNA accumulation persisted and were further enhanced during a 4 week period of MDD feeding. The appearance of hypomethylated DNA in the livers of these MDD-fed rats coincided with the observed changes in levels of mRNA for these genes associated with the regulation of cell growth. Increases in levels of mRNA for c-fos, c-Ha-ras and c-myc were correlated with loss of methylation at specific sites within these genes as early as 1 week after the start of MDD feeding. These combined observations are consistent with the hypothesis that methyl-deficient diets are cancer promoting and/or carcinogenic, at least in part, because they induce hypomethylation of DNA with concomitant alterations in the regulation of gene expression.

Isolation of cDNA clones for the interferon-induced 67,000-dalton protein: direct induction of a family of mRNAs by human interferon-alpha and interferon-gamma.

A partial cDNA clone for the interferon (IFN)-induced 67,000-dalton (67K) protein was isolated by immunological screening and used as a probe to study the expression of mRNAs encoding this protein. Northern blot analyses of RNA from IFN-treated GM2767 cells revealed the presence of two major 67K-specific RNA species, 2.7 and 4.3 kb in length, and two minor RNA species, 5.7 and 7.2 kb long. All of these 67K-specific RNAs were polyadenylated. Multiple 67K-specific mRNAs were observed to be induced in several cell lines. IFN-gamma was more effective at inducing these mRNAs than was IFN-alpha. In IFN-alpha-treated GM2767 cells, the 67K-specific mRNAs were detectable 6 h following IFN treatment, but not 12, 18, or 24 h following treatment. In IFN-gamma-treated cells, these mRNAs were detectable 6 h after treatment and continued to be present 24 h after treatment. The induction of the 67K-specific mRNAs in GM2767 cells did not require protein synthesis as the RNAs were induced by IFN-alpha or IFN-gamma in the presence of cycloheximide (CHX, 50 micrograms/ml). Treatment of cells with the combination of CHX and IFN-alpha mediated an enhanced accumulation of the 67K-specific mRNAs, suggesting that ongoing protein synthesis may downregulate the induction or accumulation of the IFN-alpha-induced 67K-specific mRNAs. Western blot analysis employing a monoclonal antibody to the 67K protein revealed that several distinctly sized but immunologically related proteins were induced in IFN-treated cells.

Prolonged survival of female AKR mice fed diets supplemented with methionine and choline.

Female mice of the AKR/J (AK) strain were fed a control diet (Purina Rodent Laboratory Chow) or a lipotrope-supplemented diet (Purina Rodent Chow plus 2% D,L-methionine and 1% choline chloride) beginning at 1 day after weaning. Food consumption and weight gain were found to be the same in both groups of animals. Mice of this inbred strain spontaneously develop thymic lymphoma, with close to 100% mortality expected by 12-13 months of age. Two separate experiments were carried out with 50 mice per group in one, and 40 mice per group in the other. The slopes of the survival curves for the animals in the control group and supplemented group of mice diverged after the animals reached 6.5 months of age. In both experiments, 20% of the mice receiving supplemented diet were still alive at 1 year, while 3% in one experiment and 8% in the other experiment survived in the control groups. Each experiment was terminated when the animals reached 13 months of age. At that time the survival rate of the controls was 2 and 4%, and survival in the groups of mice receiving supplemented diet was 14 and 18%. Necropsy revealed that the animals in both groups had advanced malignant lymphoma. Our results demonstrate that intake of a chow diet that is supplemented with moderate quantities of methionine and choline results in enhanced survival of spontaneously leukemic AK mice, in comparison with animals of this strain fed the same diet without supplements of choline and methionine.

Rapid appearance of hypomethylated DNA in livers of rats fed cancer-promoting, methyl-deficient diets.

Prolonged intake of diets deficient in sources of methyl groups leads to development of hepatomas in rats and promotes chemical carcinogenesis in both rats and certain strains of mice. Since methylation of cytosine residues in regulatory regions can affect gene activity, several investigators have postulated that the effects of methyl-deficient diets on tumorigenesis result from the inability of cells to maintain normal patterns of DNA methylation. However, significant decreases in the 5-methylcytosine content of liver DNA have not been reported to occur until rats have consumed methyl-deficient diets for several months. To determine whether methyl-deficient diets have immediate effects on nucleic acid methylation, we assessed the degree to which hepatocyte DNA and tRNA were methylated in vivo, by measuring their ability to act as methyl acceptors in vitro. Hypomethylation of DNA and tRNA was detected within 1 week after rats were started on a diet deficient In methionine, choline, folic acid, and vitamin B12 and it persisted throughout the 4 weeks of study. A significant elevation in liver DNA synthesis occurred in parallel with increased hypomethylation of DNA. Chronic failure to fully methylate DNA that is newly synthesized in response to liver damage induced by methyl-deficient diets provides a feasible mechanism for changing patterns of DNA methylation. Our results indicate that such changes could occur rapidly enough to play a causal role in the cancer-promoting and, in some instances, cancer-inducing properties of the diet.

Altered expression of retrovirus-like sequences and cellular oncogenes in mice fed methyl-deficient diets.

Methyl-deficient (lipotrope-deficient) diets enhance liver carcinogenesis in rodents. Although the mechanisms responsible for the cancer-promoting activity of such diets have not been identified, they have been observed to cause impaired immune response, alterations in methylation of liver RNA and DNA, and enhanced susceptibility to oxidative damage. Since alterations in gene expression may also play a critical role, the present studies examined the expression of the c-myc, c-H-ras, epidermal growth factor receptor, and ornithine decarboxylase genes, as well as endogenous retrovirus-like sequences, in C57BL/6J x C3H/HeJ F1 mouse liver during the first 2 weeks of feeding of a methyl-deficient diet. The kinetics of liver cell proliferation was investigated in parallel. Increased [3H]thymidine incorporation into liver DNA was found at day 4 and reached a maximum at days 7-11 after commencement of the methyl-deficient diet, when compared to age-matched mice fed a complete diet. Northern blot analysis of polyadenylated liver RNA samples indicated an increase in the levels of RNA homologous to Moloney murine leukemia virus and intracisternal A particle sequences but no significant change in the level of VL30 retrovirus-related RNA in the samples from mice fed methyl-deficient diets. A marked increase in the levels of c-myc and a slight increase in the levels of ornithine decarboxylase and c-H-ras transcripts were seen in the liver RNA samples from the treated mice. Of particular interest was a decrease in the abundance of epidermal growth factor receptor transcripts in the liver RNA samples from the treated mice. These changes in cellular levels of specific RNA resemble, in several respects, those we have previously described in rodent liver during regeneration and tumor promotion and also those seen in rodent hepatomas. They may reflect, therefore, a common profile of gene expression relevant to cell proliferation.

Comparison of methyltransferase activities of pair-fed rats given adequate or methyl-deficient diets.

The short-term effects of a lipotrope-deficient (methyl-deficient) diet on tRNA and protein methyltransferase activities have been studied using pair-fed male Fischer rats. The activity of liver N2-guanine tRNA methyltransferase II (NMG2) of animals receiving the methyl-deficient diet (MDD) for 2 weeks was found to be elevated more than 2-fold. This is in agreement with the results of earlier experiments in which the animals were fed ad libitum. These data indicate that the effects of lipotrope-deficient diets on NMG2 activity observed in the earlier studies can be attributed to the nature of the diet, and not to differences in caloric intake. In the same pair-fed animals, very little effect of MDD on the activity of NMG2 of either brain or spleen was observed. In liver, the activity of one of the enzymes that catalyze protein methylation--protein methylase I (S-adenosyl-methionine: protein-arginine N-methyltransferase)--was significantly elevated in response to the lipotrope-deficient diet. In contrast, the activities of protein methylase II (S-adenosylmethionine: protein-carboxy-O-methyltransferase), from control and experimental animals did not differ significantly. Lipotrope-deficient diets are thus seen to induce, within a short period of time, selective changes in the activities of some, but not all, of the liver enzymes that catalyze the methylation of tRNA and protein.

Infection and transformation of Fv-2rr erythroprogenitor cells with Friend virus.

Friend virus was used to infect and transform Fv-2rr erythroprogenitor cells in vivo. The RB (Fv-2rr) cell line was characteristic of Friend virus-induced cell lines in Fv-2ss mice, i.e., it produced infectious Friend virus and synthesized hemoglogin. The RB (Fv-2rr) cell line expressed the envelope protein of the spleen focus-forming virus (gp52) and a novel, related envelope protein (gp48). The results demonstrate that Fv-2rr erythroprogenitor cells can be infected and transformed in vivo.

Suppression by methionine and choline of onco-fetal patterns of liver tRNA methyltransferase activities in carcinogen-treated rats.

When male Fischer rats were fed Purina chow supplemented with 2% D,L-methionine and 1% choline chloride, the rapid increase in N2-guanine tRNA methyltransferase II (NMG2) activity otherwise seen in response to cancer-promoting doses (0.02% in the diet) of 2-acetylaminofluorene (AAF) was prevented, and the increase in NMG2 activity otherwise caused by carcinogenic doses of AAF (0.06% in the diet) was decreased by 50%. In addition, the return of NMG2 activity to a normal level after completion of a 3-week regimen of 0.06% AAF was accelerated in animals fed the methionine plus choline supplemented diet. As shown earlier in this laboratory, liver tRNA methylating enzyme activities are shifted rapidly to an onco-fetal pattern in rats receiving methyl-deficient diets. This pattern is characterized by selectively elevated NMG2 activity while the activities of other base-specific tRNA methylating enzymes are relatively unchanged. Our combined results indicate that the exogenous supply of methyl groups is a factor in regulating NMG2 activity and can modulate at least one response of animals to carcinogens.

Altered tRNA methylation in rats and mice fed lipotrope-deficient diets.

A diet that is deficient in methionine, choline, folic acid and vitamin B12 has been found to induce alterations rapidly in liver tRNA methylation in male Fischer rats. In vitro assays indicated that activity of N2-guanine tRNA methyltransferase II (NMG2) was increased to 150% of controls levels in 1 week and 300% of control levels after 2 weeks or longer on this diet. Incompletely methylated tRNA was isolated from livers of these same animals, indicating that there was impairment of methylation in vivo. The effects on liver tRNA methylation of this methyl-deficient diet were thus seen to mimic those of the liver carcinogen, ethionine, which also causes production of hypomethylated tRNA and increased activity of NMG2. The effect of the same diet on liver tRNA methyltransferase activity of C57BL/6J and C3H/HeJ inbred mice were also studied. Intake of the lipotrope-deficient diet induced elevation in activity of liver N2-guanine tRNA methyltransferase II activity in C57BL/6J mice, similar to that seen in rats. In contrast, the methyl-deficient diet had very little effect on the same enzyme activity in C3H/HeJ animals.

Differences in activity of N2-guanine tRNA methyltransferase II among several inbred strains of mice.

The tRNA methyltransferase activities of C57BL/6J, C57L/J, C58/J, AKR/J, and C3H/HeJ inbred mice were studied with the use of various amino acid-specific Escherichia coli tRNA's as substrates. Mice from two strains with high incidence of spontaneous leukemia (AKR/J and C58/J) exhibited levels of liver N2-guanine tRNA methyltransferase II (N2-MeGII) activity that were double those of two strains of mice with low incidence of spontaneous leukemia (C57BL/6J and C57L/J). Activities of liver and kidney N2-MeGII of the high spontaneous hepatoma strain C3H/HeJ were also found to be twice as high as those of C57BL/6J mice. The activities of other tRNA base-specific liver tRNA methyltransferases were very similar in all strains studied. The N2-MeGII activity of the F1 progeny of a cross between C57BL/6J and C3H/HeJ showed levels of activity intermediate to those of the parental strains. Activities of liver N2-MeGII of two inbred strains of mice that differ in their H-2 haplotype (C57BL/10SnJ and the congenic strain B10.BR/SgSnJ) were also compared. Both C57BL/10SnJ and B10.BR/SgSnJ strains exhibited low levels of liver N2-MeGII activity, indicating that H-2 does not directly control the activity of this enzyme.

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage.

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5' end of the molecule. This group of tRNAs includes E. coli tRNANfmet, tRNAAla1, tRNALeu1, or Leu2, and tRNASer3 (Group 1). In each case N2-methylguanine and N2,N2-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N2-guanine methyltransferase II (S-adenosyl-L-methionine : tRNA (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50 degrees C for min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preparations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N2-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.

Construction of a D2.B6-Fv-2r congenic mouse strain: nonlethality of the homozygous Fv-2' genotype.

Starting from the (C57BL/6 x DBA/2)F1 generation (Fv-2s/Fv-2r), 16 serial backcrosses to mice of the parental DBA/2 strain (Fv-2s/Fv-2s) were bred. In each generation, heterozygous Fv-2s/Fv-2r segregants were selected and backcrossed with a DBA/2 parent. In the 16th generation, Fv-2s/Fv-2r heterozygotes were intercrossed, and Fv-2r/Fv-2r homozygotes were selected for interbreeding to establish a congenic strain, D2.B6-Fv-2r. The successful establishment of this congenic strain is in contrast with previous findings suggesting that homozygosity for the Fv-2r allele might be a lethal genotype on the DBA/2 genetic background. At least 10 centimorgans of Fv-2r-linked chromosomal material originating from the C57BL/6 ancestor remains in the D2.B6-Fv-2r genome, as shown by the continued presence of C57BL/6 alleles at the flanking Kfo-1 and Bgl-s loci. The observed recombination frequency for the intervals between Fv-2 and Bgl-s was 6.8%.

A second gene affecting the sialylation of lysosomal alpha-mannosidase in mouse liver.

We have previously reported on a mouse liver-specific genetic polymorphism associated with altered sialylation of lysosomal alpha-mannosidase. A second electrophoretic polymorphism for liver lysosomal alpha-mannosidase has now been found and characterized. This variation, between SWR/J and SM/JCv inbred mice, is determined by a single genetic locus (Map-2) on chromosome 17 and appears to be the result of further differences in sialylation of the lysosomal enzyme. The Map-2 gene appears to affect the processing of liver, spleen, and lung lysosomal alpha-mannosidase, whereas the Map-1 gene appears to be specific to the processing of liver lysosomal alpha-mannosidase (Dizik and Elliott, 1977). The more negatively charged electrophoretic liver phenotype (MA-A) characteristic of the SM/JCv strain is recessive to the phenotype (MA-B) characteristic of the SWR/J strain. In contrast, at the Map-1 locus, the more negatively charged phenotype is dominant. The electrophoretic pattern of development of the liver enzyme from SM/JCv mice is described.

A gene apparently determining the extent of sialylation of lysosomal alpha-mannosidase in mouse liver.

Organ-specific electrophoretic heterogeneity of lysosomal alpha-mannosidase has been observed within individual strains of inbred mice. Polymorphism between C57BL/6J and CBA/J for liver lysosomal alpha-mannosidase is determined by a single genetic locus on chromosome 5 and appears to be the result of differences in sialylation of the lysosomal enzyme. Two different patterns of expression of development of the liver electrophoretic forms have been observed.