Elevated overall rates of transmethylation in cell lines from diverse human tumors

Summary In a study of a diverse set of human tumor cell lines previously shown to all have a defect in methionine metabolism (Stern, P. H., Wallace, C. D. and Hoffman, R. M. J. Cellular Physiology119, 29–34, 1984), we demonstrate in this report that all have enhanced overall rates of transmethylation compared to normal human fibroblasts. Transmethylation rates were measured by blocking S-adenosylhomocysteine hydrolase and measuring the AdoHcy which accumulates as a result of transmethylation. The enhanced transmethylation rates may be the basis of the above-mentioned defects in methionine metabolism previously reported in human tumor cells, including the basis of the inability of the majority of the tumor cells to grow when methionine is replaced by homocysteine. The excess and unbalanced tRNA methylation observed for the last 25 years in many types of cancer may be at least in part explained by our results of elevated rates of overall transmethylation in cancer cells. The alteration of such a fundamental process as transmethylation in cancer may be indicative of its importance in the oncogenic process. This study was supported by grants 1348A and 1496R1 from the Council for Tobacco Research-USA, Inc., grant CA27564 from the National Cancer Institute, and Research Career Development Award CA00804 from the National Cancer Institute, all to Robert M. Hoffman, and by the George A. Jacobs Memorial Fund for Cancer Research. Editor's Statement This report describes increased rates of transmethylation in a large number of human tumor cell lines in culture, compared to transmethylation rates of several strains of untransformed human fibroblasts. All studies of this kind, using tumor cell lines of epithelial origin and employing as controls “normal” (untransformed) cell strains that are solely of fibroblastic origin, are difficult to interpret and remain open to question. However, the authors' observations that cell lines derived from both sarcomas and carcinomas exhibit enhanced transmethylation rates may strengthen, the case somewhat. More importantly, the potential relationship discussed by the authors of enhanced transmethylation rates to the phenomena of methionine dependence and unbalanced tRNA methylation make the data presented worthy of note. Gordon H. Sato     

The role of methionine recycling for ethylene synthesis in Arabidopsis

The methionine (Met) cycle contributes to sulfur metabolism through the conversion of methylthioadenosine (MTA) to Met at the expense of ATP. MTA is released as a by-product of ethylene synthesis from S-adenosylmethionine (AdoMet). Disruption of the Met cycle in the Arabidopsis mtk mutant resulted in an imbalance of AdoMet homeostasis at sulfur-limiting conditions, irrespective of the sulfur source supplied to the plants. At a low concentration of 100 mum sulfate, the mtk mutant had reduced AdoMet levels and growth was retarded as compared with wild type. An elevated production of ethylene was measured in seedlings of the ethylene-overproducing eto3 mutant. When Met cycle knockout and ethylene overproduction were combined in the mtk/eto3 double mutant, a reduced capacity for ethylene synthesis was observed in seedlings. Even though mature eto3 plants did not produce elevated ethylene levels, and AdoMet homeostasis in eto3 plants did not differ from that in wild type, shoot growth was severely retarded. The mtk/eto3 double mutant displayed a metabolic plant phenotype that was similar to mtk with reduced AdoMet levels at sulfur-limiting conditions. We conclude from our data that the Met cycle contributes to the maintenance of AdoMet homeostasis, especially when de novo AdoMet synthesis is limited. Our data further showed that the Met cycle is required to sustain high rates of ethylene synthesis. Expression of the Met cycle genes AtMTN1, AtMTN2, AtMTK, AtARD1, AtARD2, AtARD3 and AtARD4 was not regulated by ethylene. This result is in contrast to that found in rice where OsARD1 and OsMTK are induced in response to ethylene. We hypothesize that the regulation of the Met cycle by ethylene may be restricted to plants that naturally produce high quantities of ethylene for a prolonged period of time.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Effect of oral methionine on blood lipid peroxidation and antioxidants in alloxan-induced diabetic rats

Supplementation of thiol compounds has been suggested to protect against the toxic effects of reduced oxygen species by contributing to the thiol pool of the cell. The present study was designed to determine whether supplementation of methionine in the diet of diabetic animals protected against the oxidative stress in diabetic pathology. Oral methionine was administered at a dosage of 330 mg/100 g feed to diabetic rats. The effect was compared with the effect of insulin administration. Levels of lipid peroxides were measured in plasma, erythrocytes, and erythrocyte membrane. Anti-oxidants were measured in plasma. Diabetic condition was associated with increased lipid peroxidation and depletion in antioxidant levels. Although methionine did not affect the level of blood glucose and some of the antioxidants, it lowered the lipid peroxide content in blood. Erythrocyte lipid peroxidation activity was unaffected by methionine treatment. Administration of insulin lowered both plasma and erythrocyte lipid peroxide levels.     

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

Formation and utilization of methionine by rat liver cells in culture

Summary The enzymeN ⁵-methyltetrahydrofolate: homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells. Presented in part at the Conference on Differentiation and Carcinogenesis in Liver Cell Cultures sponsored by the New York Academy of Sciences, October 11, 1979 (see reference 1).     

Somatic embryo development in carrot is associated with an increase in levels of S-adenosylmethionine,S-adenosylhomocysteine and DNA methylation

Almost homogeneous populations representing different developmental stages of somatic embryos (globular, torpedo-shaped, plantlets) and vacuolated cells were obtained from a cell suspension culture of carrot. The concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and methylated DNA were determined in embryos at different developmental stages and were found to increase during somatic embryogenesis. The highest increase during embryogenesis was a 5-fold increase in the level of SAM. A considerable increase in the methylation index (SAM/SAH ratio) was also found. We propose that the levels of SAM and SAH may be involved in the control of somatic embryogenesis by affecting the level of DNA methylation, which in turn might cause differential changes in gene activation. An increase in the level of SAM may be a prerequisite for progression of embryogenesis and the development of complete embryos.     

Inhibition of juvenile hormone III biosynthesis in cockroach corpora allata by interference with the S-adenosylmethionine-dependent transmethylation

The synthesis of juvenile hormone-III by corpora allata of the cockroach Diploptera punctata is dependent under in vitro conditions upon a supply of exogenous methionine. Radiolabelled S-adenosylmethionine was identified by HPLC in extracts of corpora allata incubated with either [methyl-³H]methionine or [³⁵S]methionine. Juvenile hormone (JH) synthesis by intact glands in vitro was inhibited by cycloleucine and selenomethionine, but this inhibition could be relieved by increasing the concentration of methionine. S-adenosylhomocysteine or sinefungin had little or no inhibitory effect on JH synthesis by intact glands, but 5′-deoxy-5′-methylthioadenosine was inhibitory. Adenosine and homocysteine synergistically inhibited JH synthesis. These results show that JH-III synthesis by intact glands can be inhibited by interfering with the S-adenosylmethionine-dependent transmethylation, and suggest that the product and inhibitor of that reaction, S-adenosyl-homocysteine, is rapidly hydrolyzed to adenosine and homocysteine in the corpora allata.     

Methylenetetrahydrofolate reductase C677T and methionine synthase A2756G polymorphisms influence on leukocyte genomic DNA methylation level

Methionine synthase (MTR) and methylenetetrahydrofolate reductase (MTHFR) enzymes are involved in the metabolism of methyl groups, and thus have an important role in the maintenance of proper DNA methylation level. In our study we aimed to evaluate the effect of the polymorphism A2756G (rs1805087) in the MTR gene on the level of human leukocyte genomic DNA methylation. Since the well-studied polymorphism C677T (rs1801133) in the MTHFR gene has already been shown to affect DNA methylation, we aimed to analyze the effect of MTR A2756G independently of the MTHFR C677T polymorphism. For this purpose, we collected the groups of 80 subjects with the MTR 2756AA genotype and 80 subjects with the MTR 2756GG genotype, having equal numbers of individuals with the MTHFR 677CC and the MTHFR 677TT genotypes, and determined the level of DNA methylation in each group. Individuals homozygous for the mutant MTR 2756G allele showed higher DNA methylation level than those harboring the MTR 2756AA genotype (5.061 ± 1.761% vs. 4.501 ± 1.621%, P = 0.0391). Individuals with wild-type MTHFR 677СC genotype displayed higher DNA methylation level than the subjects with mutant MTHFR 677TT genotype (5.103 ± 1.767% vs. 4.323 ± 1.525%, P = 0.0034). Our data provide evidence that the MTR A2756G polymorphism increases the level of DNA methylation and confirm the previous reports that the MTHFR C677T polymorphism is associated with DNA hypomethylation.     

Lysine enhances methionine content by modulating the expression of S-adenosylmethionine synthase

Lysine and methionine are two essential amino acids whose levels affect the nutritional quality of cereals and legume plants. Both amino acids are synthesized through the aspartate family biosynthesis pathway. Within this family, lysine and methionine are produced by two different branches, the lysine branch and the threonine-methionine branch, which compete for the same carbon/amino substrate. To elucidate the relationship between these biosynthetic branches, we crossed two lines of transgenic tobacco plants: one that overexpresses the feedback-insensitive bacterial enzyme dihydrodipicolinate synthase (DHPS) and contains a significantly higher level of lysine, and a second that overexpresses Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Significantly higher levels of methionine and its metabolite, S-methylmethionine (SMM), accumulated in the newly produced plants compared with plants overexpressing AtCGS alone, while the level of lysine remained the same as in those overexpressing DHPS alone. The increased levels of methionine and SMM were correlated with increases in the mRNA and protein levels of AtCGS and a reduced mRNA level for the genes encoding S-adnosylmethionine (SAM) synthase, which converts methionine to SAM. Reduction in SAMS expression level leads most probably to the reduction of SAM found in plants that feed with lysine. As SAM is a negative regulator of CGS, this reduction leads to higher expression of CGS and consequently to an increased level of methionine. Elucidating the relationship between lysine and methionine synthesis may lead to new ways of producing transgenic crop plants containing increased methionine and lysine levels, thus improving their nutritional quality.     

The logic of the hepatic methionine metabolic cycle

This review describes our current understanding of the “traffic lights” that regulate sulfur flow through the methionine bionetwork in liver, which supplies two major homeostatic systems governing cellular methylation and antioxidant potential. Theoretical concepts derived from mathematical modeling of this metabolic nexus provide insights into the properties of this system, some of which seem to be paradoxical at first glance. Cellular needs supported by this network are met by use of parallel metabolic tracks that are differentially controlled by intermediates in the pathway. A major task, i.e. providing cellular methylases with the methylating substrate, S-adenosylmethionine, is met by flux through the methionine adenosyltransferase I isoform. On the other hand, a second important function, i.e., stabilization of the blood methionine concentration in the face of high dietary intake of this amino acid, is achieved by switching to an alternative isoform, methionine adenosyltransferase III, and to glycine N-methyl transferase, which together bypass the first two reactions in the methionine cycle. This regulatory strategy leads to two metabolic modes that differ in metabolite concentrations and metabolic rates almost by an order of magnitude. Switching between these modes occurs in a narrow trigger zone of methionine concentration. Complementary experimental and theoretical analyses of hepatic methionine metabolism have been richly informative and have the potential to illuminate its response to oxidative challenge, to methionine restriction and lifespan extension studies and to diseases resulting from deficiencies at specific loci in this pathway.     

Alpha-lipoic acid does not alter stress protein response to acute exercise in diabetic brain

Heat shock proteins (HSPs) are molecular chaperones which may act protective in cerebrovascular insults and peripheral diabetic neuropathy. We hypothesized that alpha-lipoic acid (LA), a natural thiol antioxidant, may enhance brain HSP response in diabetes. Rats with or without streptozotocin-induced diabetes were treated with LA or saline for 8 weeks. Half of the rats were subjected to exhaustive exercise to investigate HSP induction, and the brain tissue was analyzed. Diabetes increased constitutive HSC70 mRNA, and decreased HSP90 and glucose-regulated protein 75 (GRP75) mRNA without affecting protein levels. Exercise increased HSP90 protein and mRNA, and also GRP75 and heme oxygenase-1 (HO-1) mRNA only in non-diabetic animals. LA had no significant effect on brain HSPs, although LA increased HSC70 and HO-1 mRNA in diabetic animals and decreased HSC70 mRNA in non-diabetic animals. Eukaryotic translation elongation factor-2, essential for protein synthesis, was decreased by diabetes and suggesting a mechanism for the impaired HSP response related to translocation of the nascent chain during protein synthesis. LA supplementation does not offset the adverse effects of diabetes on brain HSP mRNA expression. Diabetes may impair HSP translation through elongation factors related to nascent chain translocation and subsequent responses to acute stress.     

HPLC/RIA analysis of bioactive methionine enkephalin content in the seizure-susceptible El mouse brain

We previously reported a deficit of methionine enkephalin-like immunoreactivity (ME-LI), in the cerebral cortex, septal area, hippocampus, and striatum and the abnormal metabolism of opioid peptides in the hippocampus and striatum of seizure-susceptible El mice, which are involved in the pathogenesis of seizures. However, these findings suggest that the ME-LI does not necessarily reflect the bioactive methionine enkephalin (ME). Herein, we measured the biologically active peptide, ME excluding cross-reactive substances by using HPLC coupled with radioimmunoassay to clarify the abnormal function of enkephalinergic neurons in the El mouse brain. The ME content in 25-day-old El mice that had no seizures was significantly decreased in the hippocampus and septal area, as compared with corresponding regions in ddY mice (seizure-nonsusceptible; the mother strain of El). At the age of 50 days when El mice displayed abortive seizures, this content in both stimulated El[s] and nonstimulated El[ns] was significantly reduced in the septal area and cerebral cortex. At the age of 150 days when El mice exhibit tonic-clonic seizures, this content in both El[s] and El[ns] was significantly reduced in the septal area, cerebral cortex and striatum. These findings were generally compatible with our previous findings. This study further supports our hypothesis that a deficit of anticonvulsant endogenous ME, in the cerebral cortex, septal area, and hippocampus of seizuresusceptible El mice play an important role in the pathogenesis of seizures.     

Developmental and regional alteration of methionine enkephalin-like immunoreactivity in seizure-susceptible E1 mouse brain

Methionine enkephalin-like immunoreactivity (ME-LI) in the brain of El mice (seizure-susceptible strain) was measured by radioimmunoassay (RIA) to elucidate the relation between seizures and the opioid system. The lyophilized supernatants of tissue extracts were subjected to ME RIA. The concentration of ME-LI in 25-day-old El mice that had no seizures was significantly decreased in the hippocampus. At the age of 50 days when El mice displayed abortive seizures, the levels of ME-LI in both El(+) and nonstimulated El(o) mice were also significantly reduced in the hippocampus and septal area. It was further shown that the ME-LI concentrations in both 150-day-old adult El(+) during interictal periods and El(o) mice were markedly decreased in the cerebral cortex, septal area, and striatum, as compared with the corresponding regions in ddY mice (seizurenonsusceptible strain; the mother strain of El). The decrease of ME-LI in the El mouse brain was generally compatible with our previous findings concerning the up-regulation of opioid delta receptors in this species. These results suggest that the reduction of ME-LI in the El mouse brain is not due to convulsions, but could be associated with the pathogenesis of seizure diathesis and seizure manifestations in the El mouse.     

金属离子选择性的甲硫氨酰氨肽酶抑制剂研究进展

甲硫氨酰氨肽酶(MetAP)是潜在的抗细菌、抗真菌和肿瘤治疗的分子靶点。MetAP是一类两价金属离子依赖的蛋白水解酶。然而,生理状态下,MetAP在细胞内结合并利用的金属离子类型目前还没有定论。因而,研究和发展不同金属离子选择性的甲硫氨酰氨肽酶抑制剂对细胞内源性金属离子的解析以及新型抗肿瘤及抗感染药物的研发具有重要意义。     

High free-methionine and decreased lignin content result from a mutation in the Arabidopsis S-adenosyl-L-methionine synthetase 3 gene

As an approach to understand the regulation of methionine (Met) metabolism, Arabidopsis Met over-accumulating mutants were isolated based on their resistance to selection by ethionine. One mutant, mto3, accumulated remarkably high levels of free Met - more than 200-fold that observed for wild type - yet showed little or no difference in the concentrations of other protein amino-acids, such as aspartate, threonine and lysine. Mutant plants did not show any visible growth differences compared with wild type, except a slight delay in germination. Genetic analysis indicated that the mto3 phenotype was caused by a single, recessive mutation. Positional cloning of this gene revealed that it was a novel S-adenosylmethionine synthetase, SAMS3. A point mutation resulting in a single amino-acid change in the ATP binding domain of SAMS3 was determined to be responsible for the mto3 phenotype. SAMS3 gene expression and total SAMS protein were not changed in mto3; however, both total SAMS activity and S-adenosylmethionine (SAM) concentration were decreased in mto3 compared with wild type. Lignin, a major metabolic sink for SAM, was decreased by 22% in mto3 compared with wild type, presumably due to the reduced supply of SAM. These results suggest that SAMS3 has a different function(s) in one carbon metabolism relative to the other members of the SAMS gene family.     

Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae

Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged. In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strain's reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2. Replacement of the conserved His(174) with alanine within the C-terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro. Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth. Deletion mutants further revealed that the N-terminal region of MetAP2 (residues 2-57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth. Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N-terminal region to interfere with wild-type MetAP2 function.     

Immunogold study of altered expression of some interendothelial junctional molecules in the brain blood microvessels of diabetic scrapie-infected mice

Quantitative immunogold procedure was used to study the distribution of molecular components of interendothelial junctions in blood–brain barrier (BBB) microvessels of scrapie infected SJL/J hyperglycemic mice showing obesity and reduced glucose tolerance. Samples of brain (fronto-parietal cerebral cortex and thalamo-hypothalamic region) obtained from hyperglycemic (diabetic) mice and from non- infected, normoglycemic (non-diabetic) SJL/J mice, were processed for immunocytochemical examination. The localization of the following tight junction (TJ)-associated proteins was studied: occludin as an integral membrane (transmembrane) protein, and zonula occludens one (ZO-1) as a peripheral protein. The localization of β-catenin as a representative of the cadherin/catenin complex that is typical for adherens junctions (AJs) also was studied. Morphometric analysis revealed that the density of immunosignals for occludin, represented by colloidal gold particles (GPs), was significantly lower in the brain microvessels of diabetic than in non-diabetic mice. No significant differences in the density of immunosignals for ZO-1 and β-catenin between both experimental mouse groups were observed. It indicates that abnormal glucose metabolism affects mostly occludin which is believed to play a fundamental role in the maintenance of the tightness of endothelial lining in brain microvascular network and thereby in the preservation of its barrier function. These results also support the previously expressed opinion that occludin, detected with the applied morphological method, can be considered a sensitive indicator of altered molecular architecture of the interendothelial junctions due to the action of some metabolic or pathological insults.