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.     

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.     

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.     

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.     

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.     

Acetate transport and utilization in the rat brain

Acetate, a glial-specific substrate, is an attractive alternative to glucose for the study of neuronal-glial interactions. The present study investigates the kinetics of acetate uptake and utilization in the rat brain in vivo during infusion of [2-¹³C]acetate using NMR spectroscopy. When plasma acetate concentration was increased, the rate of brain acetate utilization (CMR_ace) increased progressively and reached close to saturation for plasma acetate concentration > 2–3 mM, whereas brain acetate concentration continued to increase. The Michaelis–Menten constant for brain acetate utilization (      = 0.01 ± 0.14 mM) was much smaller than for acetate transport through the blood–brain barrier (BBB) (      = 4.18 ± 0.83 mM). The maximum transport capacity of acetate through the BBB (      = 0.96 ± 0.18 μmol/g/min) was nearly twofold higher than the maximum rate of brain acetate utilization (      = 0.50 ± 0.08 μmol/g/min). We conclude that, under our experimental conditions, brain acetate utilization is saturated when plasma acetate concentrations increase above 2–3 mM. At such high plasma acetate concentration, the rate-limiting step for glial acetate metabolism is not the BBB, but occurs after entry of acetate into the brain.     

Detection of N-acetyl methionine in human and murine brain and neuronal and glial derived cell lines

Despite the fact that N-acetyl methionine (NAM) supplementation has long been reported as a bioavailable source of methionine in humans, and known to reduce liver toxicity after acetaminophen overdose, its cellular endogenous presence has never been investigated. We demonstrate for the first time that NAM is present in both human and mouse tissues and cells in culture. A wide variety of cultured cells, including a number of brain derived cell types, as well as mouse and human brain tissue all have clearly detectable levels of NAM. Methionine is rapidly acetylated to form NAM in cultured human oligodendroglioma cells with an initial rate of 0.44 ± 0.064 atom percent excess per minute. The presence of measurable quantities of NAM in brain cells in combination with its rapid formation point to a potential physiological role for N-acetylated methionine in the brain. Aminoacylase 1 is responsible for metabolism of NAM to methionine and acetate. Deficiencies in aminoacylase 1 have been linked to a variety of neurological disorders; however, it is unclear whether and how the brain is affected by this defect. The reported presence of NAM in the human brain may provide an invaluable key to discovering the link between aminoacylase 1 mutations and neurological problems.     

Expression of fatty acid binding proteins is altered in aged mouse brain

Brain membrane lipid fatty acid composition and consequently membrane fluidity change with increasing age. Intracellular fatty acid binding proteins (FABPs) such as heart H-FABP and the brain specific B-FABP, detected by immunoblotting of brain tissue, are thought to be involved in fatty acid uptake, metabolism, and differentiation in brain. Yet, almost nothing is known regarding the effect of age on the expression of the cytosolic fatty acid binding proteins (FABPs) or their content in brain subfractions. Electrophoresis and quantitative immunoblotting were used to examine the content of these FABPs in synaptosomes in brains from 4, 15, and 25 month old C57BL/6NNia male mice. Brain H-FABP and B-FABP were differentially expressed in mouse brain subcellular fractions. Brain H-FABP was highly concentrated in synaptosomal cytosol. The level of brain H-FABP in synaptosomes, synaptosomal cytosol, and intrasynaptosomal membranes was decreased 33, 35, and 43%, respectively, in 25 month old mice. B-FABP was detected in lower quantity than H-FABP. More important, B-FABP decreased in synaptosomes, synaptic plasma membranes, and synaptosomal cytosol from brains of 25 month old mice. In contrast to H-FABP, B-FABP was not detectable in the intrasynaptosomal membranes in any of the three age groups of mice. In conclusion, expression of both H-FABP and B-FABP was markedly reduced in aged mouse brain. Age differences in brain H-FABP and B-FABP levels in synaptosomal plasma membranes and synaptosomal cytosol may be important factors modulating neuronal differentiation and function.     

Radioactive methionine: determination, and distribution of radioactivity in the sulfur, methyl and 4-carbon moieties

A simple and inexpensive method is described for isolation and determination of [14C]methionine in the non-protein fraction of tissues extensively labeled with 14C. The effectiveness of the method was demonstrated by isolation of non-protein [14C]methionine (as the carboxymethylsulfonium salt) of proven radiopurity from the plant Lemna which had been grown for a number of generations on [U-14C]sucrose and contained a 2000-fold excess of 14C in undefined non-protein compounds. To our knowledge, this is the first reported assay for radioactive methionine under these demanding conditions. This method also offers an attractive alternative to the use of more expensive and sophisticated equipment for assay of radioactive methionine under less demanding conditions. An advantage is that the isolated methioninecarboxymethylsulfonium salt is readily degraded to permit separate determination of radioactivity in the 4-carbon, methyl and sulfur moieties of methionine. During this work, a facile labilization of 3H attached to the (carboxy)methylene carbon of methioninecarboxymethylsulfonium salt was observed. This labilization is ascribed to formation of a sulfur ylid.     

腺苷蛋氨酸预防酒精性肝病及其作用机制探讨

目的探讨腺苷蛋氨酸(SAM)防治大鼠酒精性肝病的作用机制。方法健康雄性wistar大鼠30只,随机分为对照组、模型组和SAM干预组。模型组大鼠采用逐渐增加浓度(30%-60%)和剂量(5-9g·kg-1·d-1)的方法酒精灌胃16周,干预组增加SAM(100mg/kg)灌胃,其它同模型组。16周末随机处死动物,检测血清总同型半胱氨酸(tHcy)的浓度和肝组织胱硫醚β合成酶(CBS)的活性;分别采用免疫组化方法和RT-PCR法检测肝组织中GRP-78、calpain 2及其caspase-12的表达;采用TUNEL染色法检测肝细胞凋亡。结果模型组大鼠16周末出现肝细胞弥漫小泡性脂肪变性,窦周纤维化,汇管区纤维组织增生并有纤维间隔形成。SAM组病理改变较模型组明显减轻。SAM组血清tHcy的浓度(7.00±0.79)较模型组(9.85±0.12)明显降低,而CBS的活性(511.60±57.44)较模型组(390.45±31.17)升高,F值分别为147.28和41.14,P值均<0.01;免疫组化和RT-PCR结果显示SAM组GRP-78、Calpain 2、caspase-12的表达较模型组减弱;SAM组的肝细胞凋亡指数(31.24±2.65)较模型组(65.71±9.78)降低,F值为301.79,P<0.01。结论在大鼠酒精性肝病中,腺苷蛋氨酸通过提高胱硫醚β合成酶活性,改善内质网应激,减少肝细胞凋亡,减轻肝脏损伤。