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Transmethylation is an important reaction that transfers a methyl group in S-adenosylmethionine (SAM) to substrates such as DNA, RNA, and proteins. It is known that transmethylation plays critical roles in various cellular responses. In this study, we examined the effects of transmethylation on tumorigenic responses and its regulatory mechanism using an upregulation strategy of adenosylhomocysteine (SAH) acting as a negative feedback inhibitor. Treatment with adenosine dialdehyde (AdOx), an inhibitor of transmethylation-suppressive adenosylhomocysteine (SAH) hydrolase (SAHH), enhanced the level of SAH and effectively blocked the proliferation, migration, and invasion of cancer cells; the treatment also induced the differentiation of C6 glioma cells and suppressed the neovascular genesis of eggs in a dose-dependent manner. Through immunoblotting analysis, it was found that AdOx was capable of indirectly diminishing the phosphorylation of oncogenic Src and its kinase activity. Interestingly, AdOx disrupted actin cytoskeleton structures, leading to morphological changes, and suppressed the formation of a signaling complex composed of Src and p85/PI3K, which is linked to various tumorigenic responses. In agreement with these data, the exogenous treatment of SAH or inhibition of SAHH by specific siRNA or another type of inhibitor, 3-deazaadenosine (DAZA), similarly resulted in antitumorigenic responses, suppressive activity on Src, the alteration of actin cytoskeleton, and a change of the colocalization pattern between actin and Src. Taken together, these results suggest that SAH/SAHH-mediated transmethylation could be linked to the tumorigenic processes through cross-regulation between the actin cytoskeleton and Src kinase activity.

Hepatocytes are resistant to tumor necrosis factor-α- (TNF) induced killing/apoptosis under normal circumstances, but primary hepatocytes from rats chronically fed alcohol have increased TNF cytotoxicity. Therefore, there must be mechanism(s) by which alcohol exposure “sensitizes” to TNF hepatotoxicity. Abnormal metabolism of methionine and S-adenosylmethionine (SAM) are well-documented acquired metabolic abnormalities in ALD. S-adenosylhomocysteine (SAH) is the product of SAM in hepatic transmethylation reactions, and SAH hydrolase (SAHH) is the only enzyme to metabolize SAH to homocysteine and adenosine. Our previous studies demonstrated that chronic intracellular accumulation of SAH sensitized hepatocytes to TNF cytotoxicity in vitro. In the current study, we extended our previous observations by further characterizing the effects of chronic alcohol intake on mitochondrial SAM levels in liver and examining its possible involvement in SAH sensitization to TNF hepatotoxicity. Chronic alcohol consumption in mice not only increased cytosolic SAH levels, but also decreased mitochondrial SAM concentration, leading to decreased mitochondrial SAM to SAH ratio. Moreover, accumulation of hepatic SAH induced by administration of 3-deaza-adenosine (DZA-a potent inhibitor of SAHH) enhanced lipopolysaccharide (LPS)/TNF hepatotoxicity in mice in vivo. Inhibition of SAHH by DZA resulted not only in accumulation of cytoplasmic SAH, but also in depletion of the mitochondrial SAM pool. Further studies using mitochondrial SAM transporter inhibitors showed that inhibition of SAM transport into mitochondria sensitized HepG2 cells to TNF cytotoxicity. In conclusion, our results demonstrate that depletion of the mitochondrial SAM pool by SAH, which is elevated during chronic alcohol consumption, plays a critical role in SAH induced sensitization to TNF hepatotoxicity.

The effects of 5'-deoxy-5'-(hydroxyethylthio)adenosine (HETA), a trypanocidal analog of 5'-deoxy-5'-(methylthio)adenosine (MTA), on polyamine synthesis and S-adenosylmethionine (AdoMet) metabolism were examined in bloodstream forms of Trypanosoma brucei brucei. HETA was cleaved by trypanosome MTA phosphorylase at the same rate as the natural substrate, MTA, in a phosphate-dependent reaction. Fluorine substitution at the 2-position of the purine ring increased activity by approximately 50%, whereas substitution with an amino group reduced activity to about one-third of the control. HETA was accumulated by trypanosomes with internal concentrations of 100-250 microM and800 microM after a 15-min incubation with 1 and 10 microM, respectively. Trypanosomes preincubated with HETA metabolized it at a rate of 21.9 nmol/hr/mg protein. Preincubation of cells with HETA at 1 or 10 microM inhibited spermidine synthesis from [3H]ornithine by 22-37%, and increased the cytosolic levels of AdoMet by 2- to 5-fold and that of MTA by up to 8-fold. S-Adenosylhomocysteine (AdoHcy) levels also increased 1.5- to 7-fold in treated cells, whereas decarboxylated AdoMet decreased 65%. Preincubation of trypanosomes with HETA for 4 hr also reduced the incorporation of [35S]methionine in trichloroacetic acid-precipitable material by 50-60%, and reduced the methyl group incorporation into protein from [U-14C]methionine by 65-70%. Thus, HETA interferes with a series of biochemical events involving the participation of AdoMet and methionine in polyamine synthesis, protein synthesis, and transmethylation reactions.

The present investigation was undertaken to determine the effect of various ions on the characteristics of S-adenosylhomocysteine (SAH) hydrolase from bovine kidney. The binding sites of [3H]-adenosine to purified SAH hydrolase were not influenced by phosphate, magnesium, potassium, sodium, chloride or calcium ions at physiological cytosolic concentrations. To test whether NAD+ in the SAH hydrolase is essential for adenosine binding, we prepared the apoenzyme by removing NAD+ with ammonium sulfate. The resulting apoenzyme did not exhibit any [3H]-adenosine binding. Since the apoenzyme was enzymatically inactive, it is suggested that adenosine binds to the active site and not to an allosteric site of the intact enzyme. The kinetics of the hydrolysis and the synthesis of SAH catalyzed by the enzyme SAH hydrolase were measured in the presence and absence of phosphate and magnesium. Phosphate increased the Vmax for both synthesis and hydrolysis. However, only the affinity of adenosine for SAH synthesis was significantly enhanced from 10.1+/-1.3 microM to 5.4+/-0.5 microM by phosphate. This effect was already maximal at a phosphate concentration of 1 mM. All other tested ions were without effect on the enzyme activity. Our results show that phosphate at physiological concentrations shifts the thermodynamic equilibrium of SAH hydrolase in the direction of SAH synthesis. These findings imply that SAH-sensitive transmethylation reactions are inhibited during renal hypoxia when intracellular levels of phosphate, adenosine, and SAH are elevated.

To provide a background for future studies on neurodegenerative changes in the spinal cord, the present study analysed the distribution of the activity of methionine adenosyltransferase (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6, MAT), an enzyme that catalyses the synthesis of the biological methyl group donor S-adenosylmethionine (AdoMet), in spinal cords from bovine and pig, and compared the results with those from human spinal cord. The enzyme activity was estimated by a radiochemical method measuring the rate of formation of [(3)H]AdoMet from L-[methyl-(3)H]methionine and ATP. The MAT activity (V(max)) was quite homogeneously distributed between spinal regions and species investigated (19-50 pmol [(3)H]AdoMet/mg protein/minute), with the highest level found in the male bovine group. The bovine group (both males and females) also presented a 20% higher enzymatic activity in the dorsal horn as compared to the ventral horn and white matter areas. In the pig spinal cord, the highest level of activity was found in the white matter. The lowest affinity for methionine (highest K(m)) was found in the human spinal cord. Whole spinal cords of one cat and one rhesus monkey were also analysed and the levels of MAT activity were similar to that of humans and bovine females, respectively. Studies of MAT stability in the rat spinal cord (post-mortem time 0-72 hr) showed a significant decrease in enzyme activity during the interval of 0-8 hr (23 degrees ). From this time point on and up to 72 hr (4 degrees ), the significant decrease in the activity remained at 60% of the initial value.

Effects of the transmethylation inhibitors 3-deazaadenosine (c 3 Ado) and 3-deaza-(±)-aristeromycin (c 3 Ari) on cell cycle and cyclic AMP (cAMP) concentrations in human promyelocytic leukemia cells (HL-60) were studied by flow cytometry and radioimmunoassay techniques. Previously described cell cycle accumulations, after incubation with drugs (25 μM) for two cell doublings (36 hr), were localized to G 1 and G 2 after incubation with c 3 Ado and c 3 Ari, respectively. cAMP levels were elevated in cells treated with c 3 Ado (35%) and c 3 Ari (92%) for 36 hr. Addition of the phosphodiesterase (PDE) inhibitor theophylline, increased cAMP levels further, while cAMP responsiveness to the β-adrenergic stimulator isoproterenol was attenuated after c 3 Ado and c 3 Ari incubation. Homocysteine thiolactone (Hcy) alone reduced cell growth slightly (5%) and increased cAMP levels (17%). Hcy increased the growth inhibitory effects of c 3 Ado, while no modulating effect was seen in combination with c 3 Ari, nor did Hcy counteract the effects on the cell cycle perturbations. The results suggest that c 3 Ado- and c 3 Ari-induced cell cycle accumulation is, at least in part, mediated through cAMP elevation, possibly due to PDE inhibition secondary to S-adenosyl-homocysteine hydrolase inhibition and 5-adenosyl-homocysteine build-up.

Homocysteine has been shown to increase glutathione levels in C3H/10T1/2 Cl 8 cells. The present paper confirms that this increase was specific for non-dividing cells. Several other thiols and disulfides, including cysteamine, mercaptoethanol and dithioerythritol, also increased glutathione, but the specificity for quiescent non-dividing cells was confined to homocysteine only. Cysteamine was most efficient, increasing glutathione 5-fold in confluent, non-dividing cells, and 3.2-fold in exponentially growing Cl 8 cells. The results indicate that the increase in glutathione was not specific for homocysteine or other cysteine generating agents, but rather related to the presence of potential thiol, either in free form, as thiolactone or in its oxidized, disulfide form. The effect of the glutathione synthesis inhibitor BSO was investigated in detail in both C3H/10T1/2 Cl 8 cells and in Rl.1. mouse lymphoma cells. Twenty-four hours after addition of 20 μM BSO to exponential growing Cl 8 cells the glutathione content was reduced to 5.5%, with minimal toxic effect. To achieve the same GSH depleting effect on exponential growing Rl.l. cells, the BSO concentration had to be increased to 50 μM, which had a slight, but distinct growth inhibitory effect on the lymphoma cells. Based on these data, the possibility that glutathione mediated homocysteine production was investigated in part by depleting the cells of glutathione and determining the homocysteine export rate as a measure of the intracellular production of the metabolite. The results showed that glutathione depletion by BSO had no effect on the homocysteine export rate in Cl 8 cells, while in R1.1. cells a moderate decrease in homocysteine export rate accompanied by a slight, but distinct decrease in growth rate, was observed when the cells were depleted of glutathione. In addition, these data indicate that BSO did not interfere with the overall transmethylation rate, and this observation supports the view of BSO as a specific inhibitor of GSH synthesis. A general difference between the homocysteine export rate in Cl 8 and R1.1. cells was observed. The former demonstrated a decreasing export rate during exponential growth, while the latter showed an initial decrease and then a slight increase in homocysteine export rate.

In a previous report (De Clercq E, Cools M and Balzarini J, Biochem Pharmacol 38 : 1771–1778, 1989) we showed that homocysteine (Hcy) enhanced the antiviral and cytostatic activity of S -adenosylhomocysteine (AdoHcy) hydrolase inhibitors. The mechanism of synergistic action between Hcy and the isobutyl ester of ( RS )-3-(adenin-9-yl)-2-hydroxypropanoic acid [( RS )-AHPA] has been the subject of the present study. The selectivity index of ( RS )-AHPA against vaccinia virus in murine L929 cells was significantly increased if the drug was combined with 1 or 3 mM Hcy. Even if Hcy was added as late as 12 hr after ( RS )-AHPA, a synergistic antiviral activity was noted. Treatment of the L929 cells with ( RS )-AHPA caused a significant increase in AdoHcy levels, and these levels were further increased if, in addition to ( RS )-AHPA, Hcy (1mM) was added to the cell cultures. Double-pulse label experiments showed that the additional AdoHcy built up after the combined treatment of ( RS )-AHPA with Hcy did not originate from S -adenosylmethionine (via transmethylation reactions), but resulted from residual AdoHcy hydrolase activity (in the synthetic direction). To maintain sufficient levels of AdoHcy, AdoHcy hydrolase activity must be inhibited in the hydrolytic direction.

Elevated levels of adenosylmethionine (AdoMet) and adenosylhomocysteine (AdoHcy) were observed in livers of rats injected with 3-deazaadenosine, an inhibitor of adenosylhomocysteine hydrolase. A marked appearance of 3-deazaadenosylhomocysteine was observed. The ratio of AdoMet/AdoHcy in the liver was reduced from a normal value of 4.5 to 1.6 after two injections, and to 1.3 after three injections. Perturbation of biochemical transmethylations was achieved in rats injected with 3-deazaadenosine, as evidenced by: (a) a reduction in the level of creatine in the liver; (b) a decrease in the urinary excretion of 3-methoxy-4-hydroxymandelic acid; and (c) a drastic reduction in the methylation of lipids, as measured by methyl-3H-incorporation from [methyl-3H]AdoMet. These observations support the hypothesis that the ratio AdoMet/AdoHcy may be of critical importance in the regulation of biological methylations. The enzyme, adenosylhomocysteine hydrolase, therefore, may be considered a prime biological target in terms of in vivo regulation of transmethylation reactions.

Methylation reactions mediated by S- adenosyl- l -methionine (AdoMet) play an important role in a number of biological reactions including bacterial and human monocyte chemotaxis. This study evaluated the role of methylation reactions in histamine release from human basophils. Methylation was blocked by several methods. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an inhibitor of adenosine deaminase (EC 3.5.4.4), blocked the IgE-mediated histamine release with an ic 50 (concentration of drug required to produce 50 per cent inhibition) of 0.33 mM. Preincubation of leukocytes with adenosine caused some inhibition of IgE-mediated histamine release. This inhibition by adenosine was potentiated by the addition of 1 × 10−5 M EHNA which alone did not affect histamine release. Further addition of l -homocysteine thiolactone at 1 × 10−4 M potentiated the inhibitory effect of EHNA plus adenosine. The effect of l -homocysteine thiolactone was dose dependent. 3-Deazaadenosine (DZA), an inhibitor of S- adenosyl- l -homocysteine hydrolase (EC 3.3.1.1) inhibited IgE-initiated histamine release from human basophils with an ic 50 of ~1 mM. This inhibition was potentiated by l -homocycsteine thiolactone. The inhibition by DZA was not potentiated by two different phospho-diesterase inhibitors suggesting that the action of DZA was not through changes in intracellular levels of cyclic AMP. DZA inhibited during the Ca2+-independent activation step of IgE-mediated basophil histamine release. In contrast, when histamine release was induced by the calcium ionophore A23187, f'Met-Leu-Phe or zymosan-activated serum, there was either no inhibition or enhancement of histamine release by these inhibitors of transmethylation. These results suggest that AdoMet-mediated methylation plays an important role in IgE-initiated histamine secretion from human basophils and that release induced by A23187, fMet-Leu-Phe, or C5A, bypasses this reaction step.

The transmethylation catalysed by HMT (EC 2.1.1.8) ∗ has been considered as absolutely specific for histamine as acceptor substrate. In this investigation N α -MH, N α , N α DMH, spinaceamine and synthetically prepared 4-[(2-amino-ethylmercapto)-methyl]-imidazole could be identified as further methyl-group accepting substrates (optimum substrate concentration ~ 1 mM), but the yield of extractable 14 C-labelled methylation products was never greater than 21 per cent of that of histamine. The 3 per cent methylation of N α , N α -DMH was considerably smaller than that of 33 per cent reported in the literature. This discrepancy was resolved and found to be ascribable to an inappropriate product extraction procedure used in the former experiments. When N α -MH and N α , N α -DMH were the substrates, the corresponding products were isolated by t.1.c. in four different solvent systems and identified to be N τ , N α -DMH and N τ , N α , N α -TMH. Thus HMT catalysed in all cases a uniform methyl of the N α -nitrogen atom of the imidazole nucleus. The investigation of a series of various substituted imidazole compounds revealed that a methylation of the ring system had to be considered, if it was not substituted in the N τ -, 2- or N π -position and if it carried a 4-substituent with a strong basic aminogroup, whereas substitution in the ring 5-position seemed to be of minor importance. Furthermore H 1 -receptor antagonists, H 2 -receptor antagonists, the non-imidazole H 2 -receptor agonist dimaprit, as well as the enzyme inhibitors aminoguanidine, tranylcypromine, pargyline and nicolinamide, were not methylated under the catalysis of HMT. The evidence for a less high substrate specificity of HMT may influence the relevance of histamine determinations using this enzyme: caution seems necessary.

Confluent monolayers of human umbilical vein endothelial cells in culture responded with a 5-fold increase in thromboplastin (tissue factor) synthesis when exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (50 ng/ml) or endotoxin (ETX) (25 micrograms/ml) for 16 hr. This induced thromboplastin synthesis was markedly inhibited by exposure of the cells to two different phosphodiesterase inhibitors, methylisobutylxanthine (MIX) and rac-4(3-butoxy-4-methoxybenzyl)-2-imidazole idinone (RO-20-1724) and to the transmethylation inhibitors 3-deazaadenosine (DZA) and 1-homocysteine thiolactone (Hcy) in combination. It was slightly (TPA) or not at all (ETX) inhibited upon exposure of the cells to the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4, 5-trimethoxybenzoate hydrochloride (TMB-8). However, in the presence of MIX TMB-8 had a moderate additional inhibitory effect on TPA-induced thromboplastin response. The thromboplastin response of endothelial cells in vitro thus probably depends on transmethylation events for its full expression. It is also strongly modulated by the intracellular level of cAMP.

Evidence was obtained of the formation of diethyldithiocarbamic acid methyl ester (DDC-Me) from diethyldithiocarbamate in vitro and from disulfiram in vivo . The metabolite was identified by two independent methods, that is: (1) by chromatography in three solvent systems, and (2) by isotopic dilution with purification to constant count. Highly active S-adenosyi methionine transmethylase catalyzing methylation of DDC was found in both kidney and liver; the most active transmethylation was associated with the 100,000 g microsomal fraction. DDC-[ 35 S]Me was synthesized. Urinary metabolites of DDC-[ 35 S]Me and of DDC-[ 35 S] administered to rats were compared: over 60 per cent of a dose of DDC-[ 35 S]Me was excreted as urinary sulfate in the first 2 days; by contrast, only 16 per cent of a dose of DDC-[ 35 S] was eliminated as sulfate. No diethyldithiocarbamic acid glucuronide was detectable in the urine of DDC-[ 35 S]Me-treated animals; again, by contrast, the glucuronide was the major metabolite of DDC-[ 35 S] (30 per cent of dose). These findings suggest that conversion of DDC-Me to inorganic sulfate occurs without intermediate cleavage to DDC, and therefore point to a pathway to sulfate formation from DDC via its methylated metabolite, DDC-Me.