Elsie Wainfan

Methyl deficiency, DNA methylation, and cancer: Studies on the reversibility of the effects of a lipotrope-deficient diet

Methylation of C residues in CpG sites in the regulatory regions of a wide variety of genes has been linked to silencing of their expression. During normal mammalian development, loss of methylation at specific sites accompanies tissue-specific activation of genes. Overall decreases in the level of DNA methylation and alterations in the pattern of methylation of specific genes are also closely linked to tumor development in humans and other mammals. Dietary methyl deficiency sufficient to cause hepatocarcinogenesis in male rats induces profound and rapid changes in the morphology and metabolic activity of liver cells. As we have previously reported, these changes include a decrease in the overall level of DNA methylation and alternations in the patterns of methylation and levels of transcripts of specific growth-related genes. These alterations persist as long as the rats are maintained on a methyl-deficient diet. The starting hypothesis for the studies summarized here is that methyl deficiency induced changes in liver cells that persist, even when dietary sources of methyl groups are restored, are more likely to be critical for establishment of neoplasia than those that are reversible. We find that loss of methylation at specific sites in liver DNA persists for at least 9 weeks after restoration of methionine, choline, folate, and vitamin B12 to the diet of rats previously deprived of these nutrients for 4 weeks. Other molecular changes are reversed in less than 3 weeks. This suggests that exposure of rats to alternating periods of dietary methyl deficiency and sufficiency may provide an experimental model for determining whether persistent alterations in methylation of growth regulatory genes allow affected hepatocytes to escape constraints on cell division because they respond to growth stimuli differently than cells in which the genes are normally methylated.

Cytokinins that inhibit transfer RNA methylating enzymes

Regulation of the tRNA methylases is, as yet, only poorly understood, but alteration in specificities and levels of activities of these enzymes has been observed after virus infection [ 1 ] , latent virus induction [2, 31, in transformed cells [4], and in neoplasms [5111. Naturally occurring inhibitors of the tRNA methylases have been observed after lambda bacteriophage induction [2] and in normal adult animal tissues [ 121. Adenosine [ 131, adenine, and several cytotoxic analogs of these compounds [ 141 were found to be inhibitors of tRNA methylating enzymes. These original findings have been confirmed and extended by other investigators [ 151. In this paper, we report that four adenosine analogs which are cytokinins [ 161 have been found to be inhibitors of transfer RNA methylating enzymes in vitro. These compounds include 6-furfurylamino purine riboside (Kinetin riboside), N6 -(A’-isopentenyl) adenosine, N6 -(trans-4-hydroxy-3-methylbut-2-enyl) adenosine (zeatin riboside), and 6-benzylamino purine riboside. Each of these cytokinins inhibited the rate at which 14CH3 transfer from 14CH, Sadenosyl methionine to methyl-deficient tRNA was catalyzed by enzyme extracts from animal and bacterial cells. Kinetin riboside was found to be a competitive inhibitor of the enzyme-catalyzed methyl transfer from S-adenosyl methionine to guanine in tRNA. Partial purification of calf spleen enzymes by precipitation with 60% saturated (NH4)

Inhibition of transfer ribonucleic acid methylating enzymes by cytotoxic analogs of adenosine

04 did not alter the response to inhibitors.

Tubercidin and isopropylureidopurine—Inhibitors of ribothymidine synthesis in vitro

Abstract Several purine derivatives have been found to inhibit tRNA methylases of either animal or bacterial origin. The experiments reported here show that arabinosyl adenine, 6-chloropurine ribonucleoside, 2,6-diaminopurine ribonucleoside, 6-methyl-aminopurine ribonucleoside, and 6,6′-dimethylaminopurine ribonucleoside are inhibitors of tRNA methylation. The ribonucleosides of methylaminopurine and dimethylaminopurine inhibit guanine tRNA methylation by competition with the methyl donor, S -adenosyl methionine, for the enzyme. 6-Chloropurine ribonucleoside inhibits the reaction non-competitively. All of these analogs, along with isopentenyladenosine, 6-furiurylamino purine ribonucleoside, and 7-deazaadenosine, which we showed earlier to be inhibitors of tRNA methylation, are cytotoxic and have shown some usefulness as antitumor agents.

INHIBITION OF METHYLATED NUCLEOSIDE SYNTHESIS In Vivo: ACCUMULATION OF INCOMPLETELY METHYLATED TRANSFER RNA IN ETHIONINE-TREATED CELLS OF Escherichia coli B*

Abstract Two purine analogs, 7-deazaadenosine (tubercidin) and isopropylureidopurine (IPUP), have been found to inhibit the uracil tRNA-methylating enzymes of Escherichia coli . Conversion of isopropylureidopurine to its ribonucleoside derivative rendered it ineffective as a methylation inhibitor. The uracil tRNA-methylating enzyme system, which specifically catalyzes the biosynthesis of ribothymidine in transfer RNA, is relatively resistant to inhibition by a number of adenine derivatives that were previously shown to interfere with the activity of enzymes that catalyze the methylation of guanine in tRNA. Included amongst the substances that inhibited the enzymatic transfer of methyl groups to tRNA guanine, but at comparable concentrations did not significantly affect uracil tRNA methylation, were kinetin riboside, 6-methylaminopurine riboside, isopentenyladenosine and 6-isoamylureidopurine riboside. Tubercidin and IPUP, however, inhibited both guanine and uracil tRNA-methylating enzymes. These results indicate that the structural requirements for inhibitors of the various base specific tRNA-methylating enzymes are not identical. It, therefore, seems possible that the relative activities of these enzymes can be selectively altered by drugs and that they may be separately regulated in vivo .

A simple specific assay for uracil tRNA methylases of enteric bacteria: Use of ribothymidine-deficient tRNA

tRNA prepared from cells of E. coli B that had been incubated with 0.5% DL-ethionine (Ethio sRNA) was found to accept methyl groups from 14CH3-S-adenosyl-methionine in the enzymatic reaction catalyzed in vitro by tRNA methyl transferases from untreated cells of the same organism. tRNA from cells that were not exposed to ethionine did not accept a significant level of methyl groups when incubated with the same enzyme system. Base ratio analysis of the product obtained after in vitro addition of methyl groups to Ethio sRNA by enzymes from normal E. coli B indicated that a high proportion of uracil sites in this tRNA were available for enzymatic methylation. These results indicated that tRNA from ethionine-treated organisms was recognized by the homologous enzymes to be incompletely methylated, while, as previously shown, all methyl-acceptor sites on tRNA from normal cells were already filled, and that Ethio sRNA was preferentially deficient in methyl groups on uracil moieties in the RNA molecules. Ethionine thus appears to interfere with normal tRNA modification in vivo.

Genetic control of tRNA methylation in inbred mice

Abstract A simple quantitative assay that is about 95% specific for uracil tRNA methylases of E. coli and A. aerogenes has been developed. tRNA was isolated from a strain of E. coli carrying the trm − mutation. These organisms have a low level of uracil methylase and consequently produce tRNA with a selective deficiency of ribothymidine. This RNA acted as a specific substrate for uracil tRNA methylases, when exposed to cell extracts from E. coli or A. aerogenes containing tRNA-methylating enzymes of multiple specificities. This assay can be used to screen organisms for trm − mutations and for studies with inhibitors.

Methyl groups in carcinogenesis: effects on DNA methylation and gene expression.

The tRNA methyltransferases from widely divergent species differ in their activity levels and base specificities (Borek and Kerr, 1972; Kerr and Borek, 1972; Srinivasan and Borek, 1964). However, very little is known about intra-species differences in activities of these enzymes except for those found in bacterial mutants which lack certain base-specific tRNA methylases (Bjork and Isaksson, 1970; Yang et al., 1973) and in rat liver tRNA methylases which exhibit sex specific activity differences ((Wainfan et al., 1980). In order to learn whether there are geneticallydetermined intra-species differences in mammalian tRNA methyltransferases, we are investigating various characteristics of these enzymes in inbred strains of mice. Since the tRNA methyltransferases are known to have altered activities in tumors and during chemically induced carcinogenesis (Borek and Kerr, 1972; Craddock, 1970; Hancock and Forrester, 1973; Kerr and Borek, 1972; Wainfan et al., 1975, 1977, 1978, 1979) it seemed of interest to compare the enzymes from inbred mice with either high (AKR/J) or low (C57BL.6J) incidence of spontaneous leukemia (Altman and Katz, 1979).