Reversion to a homocysteine-responsive phenotype in a human melanoma cell line is associated with diminished growth potential and increased methionine biosynthesis

Our aim was to determine if the isolation of cells capable of proliferating in methionine-free homocysteine-containing medium, from the human MeWo-LC1 melanoma tumor cell line which is unable to proliferate or survive under such conditions, was associated with altered growth properties. Cells which were able to proliferate in methionine-free homocysteine-containing medium (homocysteine-responsive cells) were obtained from the homocysteine-nonresponsive MeWo-LC1 cell line after 8 months of continuous exposure to methionine-free homocysteine-containing medium. Unlike the parental MeWo-LC1 cell line, these homocysteine-responsive cells were also able to proliferate normally in methionine-free medium containing 5'-deoxy-5'-methylthioadenosine. In vitro growth rate, methionine requirement, and capacity to form colonies on soft agarose of these homocysteine-responsive cells were reduced compared to those of the homocysteine-nonresponsive parental MeWo-LC1 cell line. Unlike MeWo-LC1, these homocysteine-responsive cells were able to synthesize [3H]S-adenosylmethionine from [3H]adenine and homocysteine. The failure of the MeWo-LC1 cell line to proliferate in methionine-free homocysteine-containing medium may be due to a deficiency in the synthesis of methionine from homocysteine and 5-methyltetrahydrofolic acid. These results indicate that acquisition of a homocysteine-responsive phenotype in homocysteine-nonresponsive malignant human tumor cells is associated with a reduction in the autonomous growth potential of such cells.     

Changes in cobalamin metabolism are associated with the altered methionine auxotrophy of highly growth autonomous human melanoma cells.

Our aim was to identify the biochemical defect responsible for the inability of highly growth autonomous human tumor cells to proliferate in culture medium devoid of methionine, but containing homocysteine and 5-methyletrahydrofolic acid. We have adopted the terms "homocysteine-responsive" and "homocysteine-nonresponsive" to describe cells which can or cannot proliferate in methionine-free homocysteine-supplemented medium. Using a panel of genetically related homocysteine-responsive and -nonresponsive human melanoma cell lines, the results from a number of experiments indicate that acquisition of the "homocysteine-nonresponsive phenotype" is associated with the reduced intracellular accumulation of methyl-cobalamin, a critical cofactor of the methionine synthase enzyme. When in vitro methionine synthase assays were performed in the presence of exogenously added methyl-cobalamin, specific methionine synthase activity in extracts obtained from homocysteine-responsive cells was only twofold greater than that observed with extracts prepared from homocysteine-nonresponsive cells. However, when exogenous methyl-cobalamin was omitted from the enzyme assays, methionine synthase activity in extracts derived from homocysteine-nonresponsive cells was dramatically reduced, compared with the small decrease observed with homocysteine-responsive cell extracts. Compared with their homocysteine-responsive counterparts, homocysteine-nonresponsive cells exhibited increased levels of cobalamin efflux and decreased intracellular accumulation of methyl-cobalamin. There was a clear relationship between the abilities of these related melanoma cell lines to proliferate in methionine-free homocysteine-supplemented medium, and the extent of cobalamin loss and capacity of exogenously added methyl-cobalamin to stimulate in vitro methionine synthase activity. These results indicate a link between alterations in the intracellular trafficking and/or metabolism of cobalamin and the increased growth autonomy of human melanoma cells.     

Lack of methionine biosynthesis de novo in Butyrivibrio fibrisolvens strain E14

Butyrivibrio fibrisolvens strain E14 has an absolute requirement for methionine. Metabolism of L-[ β-¹⁴C]-serine to methionine occurred in the methionine-independent B. fibrisolvens strain H17c but not in strain E14. The absolute requirement for methionine in strain E14 could be met by addition of S-adenosylmethionine to the medium, but incorporation was not due to the presence of free methionine in the S-adenosylmethionine preparation. The results show that B. fibrisolvens strain E14 is unable to synthesize methionine de novo , probably due to a lack of methionine synthase. Butyrivibrio fibrisolvens may also possess an alternative pathway of methionine biosynthesis from S-adenosylmethionine.     

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a Km for methionine of about 3-12 microM. In selected lines we have demonstrated a requirement for Mg2+ in addition to that needed to form the Mg X ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.     

Reversion to methionine independence by malignant rat and SV40-transformed human fibroblasts.

Although many lines of malignant and transformed cells are unable to grow in folate- and cobalamin-supplemented medium in which methionine is replaced by homocysteine its immediate metabolic precursor, rare cells from these lines regained the normal ability to grow under these conditions. Six revertant lines, one from Walker-256 rat breast carcinoma cells and five from SV40-transformed human fibroblasts, have been characterized with regard to growth and three measures of methionine biosynthetic capacity: methionine synthetase and methylenetetrahydrofolate reductase activities in cell extracts, and uptake of label from [5-¹⁴C]methyltetrahydrofolate by intact cells. When all three measures of methionine biosynthetic capacity were considered, two revertants isolated from SV40-transformed cells had regained the ability to grow like normal cells in homocysteine medium without substantial changes in these measures. Increased methionine biosynthesis thus is not a prerequisite to reversion of the methionine auxotrophy present in the transformed parental lines.     

Identification of minor tightly bound H1 histone subfractions which fail to cleave their initiator methionine

Groups of CBA mice were administered [³⁵S] methionine (1 mCi/mouse). Non-histone proteins, H1 and H1⁰ histones and nucleosomal core histones were isolated from different issues by selective extractions. The measurements of radioactivity of individual bands and autoradiography of dry gels were used to identify methionine-containing and methionine-free histone variants. H1A and H1B histone variants extracted with 5% perchloric acid were methionine-free. However, minor sub-fractions of these histones which are more tightly bound to DNA (and which can be extracted only with 0.25 N HC1) contained [³⁵S] methionine and did show a higher specific activity than methionine-containing nucleosomal hitones. Cyanogen Bromide reaction which destroys non-histone proteins and methionine-containing nucleosomal histones removes radioactivity but does not alter the position of methionine-containing H1 minor bands. This indicates that the radioactive methionine occupies only the N-terminus of the H1 molecules. It is suggested that this methionine is an uncleaved initiator methionine. The presence of these methionine-containing minor H1 subfractions varies in different tissues.     

Methanol production by Mycobacterium smegmatis.

Mycobacterium smegmatis cells produce [3H]methanol when incubated with [methyl-3H]methionine. The methanol is derived from S-adenosylmethionine rather than methyltetrahydrofolate. M. smegmatis cells carboxymethylate several proteins, and some of the methanol probably results from their demethylation, but most of the methanol may come from an unidentified component with a high gel mobility. Although methanol in the medium reached 19 microM, it was not incorporated into the methylated mannose polysaccharide, a lipid carrier in this organism.     

Pharmacokinetic studies of methionine in rats using deuterium-labeled methionine quantitative assessment of remethylation

The pharmacokinetics of methionine has been studied in rats by means of stable isotope methodology. After the i.v. bolus injection of [2H7]methionine (5 mg/kg body wt.), the plasma concentrations of [2H7]methionine, demethylated [2H4]homocysteine and remethylated [2H4]methionine were determined simultaneously with endogenous methionine and homocysteine by gas chromatography-mass spectrometry. The half-life for [2H7]methionine were 35.0 +/- 6.9 min. The appearance of the metabolites, [2H4]homocysteine and [2H4]methionine, in the plasma was very rapid. The fraction of [2H7]methionine that remethylated to [2H4]methionine through [2H4]homocysteine were 0.185 +/- 0.028. The administered [2H7]methionine did not influence the plasma levels of endogenous methionine and homocysteine. The present stable isotope methodology has made it possible to evaluate the pharmacokinetics of methionine, including the estimation of remethylation.     

Metabolism to methionine and growth stimulation by 5'-methylthioadenosine and 5'-methylthioinosine in mammalian cells

Viable human and murine lymphoblasts, and normal human tissue extracts, converted the thioether nucleosides 5'-methylthioadenosine (MeSAdo) and 5'-methylthioinosine (MeSIno) to methionine. Both MeSAdo and MeSIno, but not homocysteine, supported the short-term growth of human or murine lymphoblasts in methionine deficient medium. However, MeSAdo at concentrations greater than 25 microM inhibited cell growth. MeSIno was non-toxic at concentrations up to 200 microM, and supported the long-term growth of lymphoblasts in methionine-free medium.     

Measurement of intracellular sulfur amino acid metabolism in humans

Methionine metabolism forms homocysteine via transmethylation. Homocysteine is either 1) condensed to form cystathionine, which is cleaved to form cysteine, or 2) remethylated back to methionine. Measuring this cycle with the use of isotopically labeled methionine tracers is problematic, because the tracer is infused into and measured from blood, whereas methionine metabolism occurs inside cells. Because plasma homocysteine and cystathionine arise from intracellular metabolism of methionine, plasma homocysteine and cystathionine enrichments can be used to define intracellular methionine enrichment during an infusion of labeled methionine. Eight healthy, postabsorptive volunteers were given a primed continuous infusion of [1-13C]methionine and [methyl-2H(3)]methionine for 8 h. Enrichments in plasma methionine, [13C]homocysteine and [13C]cystathionine were measured. In contrast to plasma methionine enrichments, the plasma [13C]homocysteine and [13C]cystathionine enrichments rose to plateau slowly (rate constant: 0.40 +/- 0.03 and 0.49 +/- 0.09 h(-1), respectively). The enrichment ratios of plasma [13C]homocysteine to [13C]methionine and [13C]cystathionine to [13C]methionine were 58 +/- 3 and 54 +/- 3%, respectively, demonstrating a large intracellular/extracellular partitioning of methionine. These values were used to correct methionine kinetics. The corrections increase previously reported rates of methionine kinetics by approximately 40%.     

Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry

Total homocysteine, total cysteine, and methionine have been extracted and partially purified from serum and urine using reduction with 2-mercaptoethanol followed by cation-exchange chromatography and anion-exchange chromatography. The t-butyldimethylsilyl derivatives were prepared and analyzed using capillary gas chromatography-mass spectrometry with selected ion monitoring. The addition of DL-[3,3,3',3',4,4,4',4'-2H8]homocystine, DL-[3,3,3',3'-2H4]cystine, and L-[methyl-2H3]methionine to the starting samples prior to the reduction of all disulfides, including the deuterated internal standards, with 2-mercaptoethanol makes it possible to quantitate all three amino acids. Normal ranges for total homocysteine, total cysteine, and methionine have been determined in human and rat serum and in human urine.     

Assay and regulation of S-adenosylmethionine synthetase in Saccharomyces cerevisiae and Candida utilis.

A simple and sensitive assay for S-adenosylmethionine (SAM) synthetase is described which depends on the quantitative separation of the product, [14CH3]S-adenosylmethionine, from the substrate, L-[14CH3]methionine, on a Bio-Rex 70 column. L-Methionine protects the enzyme during preparation of cell extracts by sonic treatment but causes repression of enzyme activity during growth of Candida utilis. The presence of 5 mM methionine in the growth medium repressed SAM synthetase specific activity threefold compared to the specific acitivity of the enzyme isolated from cells grown in unsupplemented medium. Conversely, the presence of methionine in the growth medium resulted in an 80-fold increase in the intracellular concentration of SAM as compared to the Sam accumulated intracellularly in unsupplemented cultures.     

Correlation between the presence of T-antigen and the reinitiation of host DNA synthesis in senescent human diploid fibroblasts after SV40 infection

Senescent human diploid fibroblasts, TIG-1, had labelling indices of about 0.5-3% when labelled with [3H]thymidine for 3 days in fresh medium containing 10% fetal bovine serum. When these cells were infected with SV40, the percentage of nuclei incorporating [3H]thymidine increased by about 10-fold. The frequency of T-antigen-positive cells and that of [3H]thymidine-incorporating cells were almost the same. About 80% of T-antigen-positive cells were also positive to incorporation of [3H]thymidine, and the same result was obtained in infected young cells. These results indicated that senescent human diploid cells which are brought to synthesize T-antigen always initiate DNA synthesis as young cells do. The characteristics of senescent cells as compared with younger cells was low incidence of T-antigen-positive cells after infection. The basis of low susceptibility of senescent cells to initiate DNA synthesis by SV40 infection thus seems to be concerned with an event after the adsorption of virus, but before the synthesis of a detectable amount of T-antigen.     

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.     

Biosynthesis of methylphosphomannosyl residues in the oligosaccharides of Dictyostelium discoideum glycoproteins. Evidence that the methyl group is derived from methionine

The phosphorylated oligosaccharides of Dictyostelium discoideum contain methylphosphomannosyl residues which are stable to mild-acid and base hydrolysis (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we present evidence that these methyl groups are derived from [methyl-3H]methionine, in vivo and [methyl-3H]S-adenosylmethionine in vitro. About 18% of the macromolecules secreted from vegetative cells labeled with [methyl-3H]methionine are released by digestion with preparations of endoglycosidase/peptide N-glycosidase F. The majority of the released molecules are sulfated, anionic high mannose-type oligosaccharides. Strong acid hydrolysis of the [3H]methyl-labeled molecules yields [3H]methanol with kinetics of release similar to those found for the generation of Man-6-P from chemically synthesized methylphosphomannose methylglycoside. Treatment of the [3H]methyl-labeled molecules with a phosphodiesterase from Aspergillus niger which is known to cleave this phosphodiester also releases [3H]methanol from a portion of the oligosaccharides. In vitro incorporation of [methyl-3H]S-adenosylmethionine into endogenous acceptors found in membrane preparations shows that the [3H]methyl group of the methylphosphomannose residues can be derived from this molecule.     

Formation and utilization of methionine by rat liver cells in culture

The enzyme N5-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.     

The metabolic defect of methionine dependence occurs frequently in human tumor cell lines

Methionine dependence is the inability of cells to grow when methionine (Met) is replaced by its immediate precursor homocysteine (Hcy) in the culture medium (Met^?Hcy⁺ medium). All normal unestablished cell strains tested to date have been shown to be methionine-independent and thus grow almost as well in Met^?Hcy⁺ medium as they do in Met⁺Hcy^? medium. Results presented here indicate that out of 23 cell lines derived from diverse types of human tumors, 11 do not grow at all in Met^?Hcy⁺ medium and are absolutely methionine-dependent and 3 grow only slightly in this medium. Many of the tumor cell lines tested have little else in common other than the fact that they are methionine-dependent. The high frequency of occurrence of methionine dependence in diverse types of human tumor cells indicates that methionine dependence may be an important aspect of oncogenic transformation and therapeutically exploitable.     

Altered methionine metabolism occurs in all members of a set of diverse human tumor cell lines

Methionine dependence is a metabolic defect found thus far only in transformed and malignant cells. The defect is manifested as the inability of cells to grow in media in which methionine (Met) is replaced by its immediate precursor homocysteine (Hcy). We have termed this Met ^? Hcy ⁺ media. We demonstrate here that methionine-dependent cells derived from human tumors, compared to normal methionine-independent cells, have low levels of free Met, low levels of S-adenosylmethionine (AdoMet) and elevated levels of S-adenosylhomocysteine (AdoHcy) when incubated in Met ^? Hcy ⁺ medium. Methionine-independent human tumor cells also have very low levels of free Met compared to normal cells but generally have levels of AdoMet and AdoHcy comparable to normal cells in Met ^? Hcy⁺ medium. All tumor cell types incorporate amounts of Met into protein similar to normal methionine-pindependent human fibroblasts when incubated in Met ^? Hcy⁺ medium, thereby indicating apparently normal levels of Met synthesis in the tumor cells. The methionine-independent tumor cell lines in Met ^? Hcy⁺ medium seem able to regulate their AdoMet/AdoHcy ratios normally despite this defect in having very low levels of free Met. Thus, in a diverse set of human tumor cell lines, all are defective in at least one aspect of Met metabolism, giving rise to the possibility of a general metabolic defect in cancer.     

Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous area 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of [methyl-3H]-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells.     

5-Azacytidine induction of thymidine kinase in a spontaneously enzyme-deficient murine tumor line

During the course of our studies on murine tumor cell metastases, one of our variant lines (called L61-M) was found to be unable to incorporate [methyl-3H]thymidine into DNA, due to a spontaneous deficiency in thymidine kinase (TK) activity. L61-M cells are unable to proliferate in HAT selection medium and are resistant to bromodeoxyuridine (BrdU). TK activity in L61-M cells is 4.2% of that found in the wild-type parental MDAY-D2 cell line. Treatment of L61-M with 5-azacytidine, a known inducer of DNA hypomethylation, resulted in the expression of TK activity. These observations suggest that the TK deficiency in the L61-M cell line was due in part to an alteration in the methylation pattern of DNA, resulting in the diminished expression of the TK gene. These results demonstrate the ability of 5-azacytidine to induce TK activity in a spontaneously enzyme-deficient murine tumor cell line.