Inhibition of DNA synthesis by methylglyoxal bis(guanylhydrazone) during lymphocyte transformation

The phytohemagglutinin induced DNA synthesis in guinea pig lymph node cells was inhibited remarkably by methylglyoxal bis(guanylhydrazone). This inhibitory effect was dependent on the time of its addition to the lymph node cell culture after stimulation with phytohemagglutinin. If methylglyoxal bis(guanylhydrazone) was added 48 hr after the stimulation, no inhibition of DNA synthesis was observed. Exogenous spermidine added at an early time of cell culture reversed the inhibitory effect of methylglyoxal bis(guanylhydrazone). However, no reversion occurred when spermidine was added at a late time of the cell culture.     

Polyamine replacement by magnesium ions in BHK-21/C13 cells

Cultures of BHK-21/C13 cells, whose growth was inhibited by deprivation of serum, were stimulated to grow by addition of serum to the culture medium. Addition of MgCl(2) to the medium, to increase the concentration of Mg(2+) ions by 15mm, 30min before addition of serum, had no effect on the stimulation of cell growth, but inhibited the accumulation of cellular spermidine, so that the spermidine/spermine molar ratio was lower in these cultures than in cultures that had received no additional cations. The increase in the activity of ornithine decarboxylase that occurs 4-5h after serum ;step-up' was substantially diminished by increasing the concentration of Mg(2+) ions, but not of Na(+) or K(+) ions, in the medium by 30mm, 30min before addition of serum, and this inhibition was maintained for at least 24h. Methylglyoxal bis(guanylhydrazone), added to serum-deprived cultures to a concentration of 20mum, 30min before addition of serum, severely inhibited the increase in cell growth. The inhibitory effects of the drug were prevented by simultaneous addition of spermidine to the medium (to 100mum), and were partly prevented by the simultaneous addition of Mg(2+) ions (to 30mm). Mg(2+) ions were particularly effective in overcoming the inhibitory effect of methylglyoxal bis(guanylhydrazone) on the synthesis of DNA. Thus although a certain lack of specificity for cations exists in BHK-21/C13 cells, in that Mg(2+) ions can be substituted for polyamines, particularly spermidine, to some extent, there are cellular processes for which the requirement for polyamines as cations is specific.     

Serum-induced expression of metallothionein isoforms in K-562 cells

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1-1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.     

Intracellular putrescine and spermidine deprivation induces increased uptake of the natural polyamines and methylglyoxal bis(guanylhydrazone).

Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium. In tumour cells exposed to the drug for 2 days, the intracellular concentration of spermidine was decreased to less than 10% of that found in untreated cells. However, the strikingly stimulated transport system brought the concentration of spermidine to the control values in less than 2h after supplementation of the cells with micromolar concentrations of the polyamine. In the absence of polyamine deprivation, tumour cells did not accumulate extracellular polyamines to any appreciable extent. Ascites-tumour cells deprived of putrescine and spermidine likewise concentrated methylglyoxal bis(guanylhydrazone) [1,1'-[methylethanedylidine)dinitrilo]diguanidine] at a greatly enhanced rate. A previous "priming of tumour cells with difluoromethylornithine followed by an exposure of the cells to methylglyoxal bis(guanylhydrazone) resulted in a marked and rapid anti-proliferative effect.     

Induction of ornithine decarboxylase activity in mouse tissues by phorbol ester is effectively blocked by methylglyoxal bis(butylamidinohydrazone)

Ornithine decarboxylase (ODC) was induced in the liver, lung and brain of the mouse injected intraperitoneally with 12-O-tetradecanoylphorbol 13-acetate (TPA), showing maximal enzyme activity four hours after the injection. The increase of ODC activity was due to the enhanced syntheses of mRNA and protein. The induction of ODC activity by TPA was specifically blocked by methylglyoxal bis(butylamidinohydrazone) (MGBB), a competitive inhibitor of ODC and S-adenosylmethionine decarboxylase, but not by the analog methylglyoxal bis(guanylhydrazone) (MGBG).     

Induction of spermidine/spermine N1-acetyltransferase by methylglyoxal bis(guanylhydrazone)

The anti-tumor agent methylglyoxal bis(guanylhydrazone) was found to be a competitive inhibitor of spermidine/spermine N1-acetyltransferase with a Ki of about 8 microM. Treatment of rats with this drug lead to a very large increase in the total amount of spermidine/spermine N1-acetyltransferase in liver, kidney and spleen. The total increase as measured using a specific antiserum amounted to 700-fold in liver and 100-fold in kidney within 18 h of treatment with 80 mg/kg doses. At least part of this induction was due to a pronounced increase in the half-life of the acetyltransferase which increased from 15 min to more than 12 h. The very large increase in the amount of the enzyme is likely to overwhelm the direct inhibition, and a net increase in the acetylation of polyamines by this enzyme would be expected to occur after treatment with methylglyoxal bis(guanylhydrazone). The acetylated polyamines are known to be rapidly degraded by polyamine oxidase producing putrescine. Direct evidence that a substantial part of the increase in the content of putrescine in the liver of rats treated with methylglyoxal bis(guanylhydrazone) occurs via the induction of this acetylase/oxidase pathway was obtained. These results indicate that methylglyoxal bis(guanylhydrazone) affects cellular polyamine levels not only by means of its inhibitory effect on S-adenosylmethionine decarboxylase and diamine oxidase but also by the induction of spermidine/spermine N1-acetyltransferase. They also raise the possibility that the enormous increase in this enzyme which occurs with higher doses may contribute to the very severe toxicity of methylglyoxal bis(guanylhydrazone).     

Ethylglyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in L1210 leukemia cells

Ethylglyoxal bis(guanylhydrazone), a close derivative of the known anti-cancer drug methylglyoxal bis(guanylhydrazone), is also a powerful inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), the enzyme needed for the synthesis of spermidine and spermine. There were, however, marked differences between the ethyl and methyl derivatives of glyoxal bis(guanylhydrazone) when tested in cultured L1210 cells. The cellular accumulation of ethylglyoxal bis(guanylhydrazone) represented only a fraction (20-25%) of that of the methyl derivative. Moreover, polyamine depletion, which is known to strikingly stimulate the uptake of methylglyoxal bis(guanylhydrazone), decreased, if anything, the uptake of ethylglyoxal bis(guanylhydrazone) by L1210 cells. The compound produced spermidine and spermine depletion fully comparable to that achieved with methylglyoxal bis(guanylhydrazone) at micromolar concentrations. Ethylglyoxal bis(guanylhydrazone) was growth-inhibitory to L1210 cells and produced an additive antiproliferative action when used together with 2-difluoromethylornithine. Ethylglyoxal bis(guanylhydrazone) was distinctly less effective than methylglyoxal bis(guanylhydrazone) in releasing bound polyamines from isolated cell organelles in vitro. Ethylglyoxal bis(guanylhydrazone) was also devoid of the early and profound mitochondrial toxicity typical to methylglyoxal bis(guanylhydrazone). These findings may indicate that this compound is a more specific inhibitor of polyamine biosynthesis with less intracellular polyamine 'receptor-site' activity than methylglyoxal bis(guanylhydrazone).     

Growth stimulation by serum in Entamoeba histolytica is associated with protein tyrosine dephosphorylation

Very little protein tyrosine phosphorylation was observed in growing (exponential-phase) Entamoeba histolytica cells by immunoblotting and quantitative immunofluorescence. After 1 h of serum deprivation, two proteins (42 and 38 kDa in SDS-PAGE) were tyrosine phosphorylated and two more proteins (96 and 63 kDa) also showed tyrosine phosphorylation when examined after 4 h of serum deprivation. Intense enhancements of anti-phosphotyrosine immunofluorescence levels were observed during this period of serum withdrawal. Membrane-associated tyrosine kinase activity reached a peak (3.5-fold increase) 1 h after serum deprivation and decreased thereafter reaching a basal level by 2 h of serum deprivation. Interestingly, tyrosine kinase activities remained unaffected by serum stimulation (2-60 min) of serum-deprived cells. Also, during this period of serum stimulation tyrosine phosphorylated proteins of serum-deprived cells were dephosphorylated. Tyrosine phosphatase activities were suppressed during serum deprivation and on serum addition to serum-deprived cells tyrosine phosphatase activities increased significantly. Our data attest that protein tyrosine phosphorylation was associated with growth inhibition of E. histolytica and serum stimulation of E. histolytica produced tyrosine phosphatase activation and protein tyrosine dephosphorylation.     

Insensitivity of cardiac phosphofructokinase to adrenergic activation in Zucker rats. A post-receptor defect.

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1'-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in combination with difluoromethylornithine or related inhibitors of ornithine decarboxylase.     

Stimulation of melanotic expression in murine melanoma cells exposed to polyamine antimetabolites

An exposure of cultured Cloudman S91 melanoma cells to inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG), distinctly promoted the expression of differentiated biochemical functions of the tumor cells. Slight to moderate growth inhibition produced by the compounds was associated with a stimulation of melanogenesis, as reflected by a striking enhancement of tyrosinase (EC 1.10.3.1) activity and an increase in cellular melanin content. Both antimetabolites acted synergistically with α-melanotropin (MSH), as regards the stimulation of melanogenesis. Exposure of the melanoma cells to MSH resulted in most experiments in a marked decrease of the intracellular polyamine pools, usually involving all three polyamines (putrescine, spermidine and spermine). The DFMO-induced stimulation of melanogenesis was totally suppressed by the administration of putrescine, whereas the MSH-stimulated tyrosinase activity was not influenced by the diamine. Although many recent reports indicate that terminal differentiation is accompanied by a distinct stimulation of polyamine biosynthesis, our results suggest that in certain cells polyamine deprivation may lead to an enhanced expression of differentiated phenotype.     

Effect of 1,25-dihydroxyvitamin D on S-adenosylmethionine decarboxylase in chick intestine

The effect of 1,25-dihydroxyvitamin D on the activity of S-adenosylmethionine decarboxylase in the duodenal mucosa of vitamin D-deficient chicks was investigated. Enzyme activity increased dose-dependently in a biphasic manner with maximal responses at 1 and 6 h, due to an increase in Vmax in both cases. A second dose of 1,25-dihydroxyvitamin D, administered 6 h after the first, resulted in a significant increase in activity 1 h later, confirming the rapidity of the response. This early response was not seen with ornithine decarboxylase. The increase in S-adenosylmethionine decarboxylase activity particularly at 6 h may be due to a rise in cytosolic calcium, since hydrocortisone, an inhibitor of 1,25-dihydroxyvitamin D-stimulated calcium absorption, attenuates this enzyme's activity. Inhibitors of polyamine biosynthesis such as DL-alpha-difluoromethyl ornithine and methylglyoxal bis(guanylhydrazone) had no effect on calcium absorption, but the significance of this in evaluating the importance of 1,25-dihydroxyvitamin D stimulation of S-adenosylmethionine decarboxylase activity needs further investigation.     

Inhibition of polyamine synthesis blocks urinary secretion of beta-glucuronidase from mouse kidney

The effect of inhibition of polyamine synthesis on castrated male mouse kidney beta-glucuronidase induction and secretion by testosterone was studied. Inhibition of the activities of polyamine synthesis key-enzymes, L-ornithine and S-adenosyl-L-methionine decarboxylases, was performed with the combined treatment of 2-difluoromethylornithine and methylglyoxal' bis(guanylhydrazone). Blockage of polyamine synthesis did not affect testosterone-induced increase in renal beta-glucuronidase but blocked its secretion into the urine. After withdrawal of inhibitor-treatment beta-glucuronidase secretion normalized, and repeated testosterone administration produced undisturbed beta-glucuronidase secretion peak in urine suggesting that blockage of beta-glucuronidase secretion was not due to the tissue damage produced by inhibitors. These results indicate that the stimulation of renal polyamine synthesis by testosterone is not necessary for the induction of beta-glucuronidase but is required for the urinary secretion of this protein.     

Characterization of the ATP-G-actin aggregates formed at low potassium chloride concentration.

Three bis(guanylhydrazones) (those of methylglyoxal, glyoxal and ethylglyoxal) were compared for their affinity for the putative polyamine carrier and for their cellular retention in L1210 mouse leukaemia cells. All the bis(guanylhydrazones) inhibited equally effectively the uptake of spermidine by the tumour cells, indicating that the compounds had roughly equal affinity for the polyamine carrier. The fact that methylglyoxal bis(guanylhydrazone) and glyoxal bis(guanylhydrazone) were much more effectively concentrated in the animal cells than was ethylglyoxal bis(guanylhydrazone) was obviously attributable to the finding that the efflux rate of ethylglyoxal bis(guanylhydrazone) greatly exceeded that of the other bis(guanylhydrazones). The rate of efflux of the drugs was slowed down if the tumour cells were treated with 2-difluoromethylornithine before exposure to the bis(guanylhydrazones). These results suggest that intracellular binding of the bis(guanylhydrazones) determines their cellular accumulation.     

Effect of methylglyoxal bis(guanylhydrazone) on polyamine metabolism in normal and regenerating rat liver and rat thymus

1. Injections of sublethal doses of methylglyoxal bis(guanylhydrazone), a potent inhibitor of putrescine-activated S-adenosylmethionine decarboxylase in vitro, resulted after a few days in an immense increase in the activity of S-adenosylmethionine decarboxylase in normal and regenerating rat liver and in rat thymus. The increase in the activity of S-adenosylmethionine decarboxylase was at least partly due to a marked lengthening of the half-life of the enzyme. 2. In regenerating liver and thymus there was also a moderate stimulation of the activity of ornithine decarboxylase (EC 4.1.1.17) and a marked accumulation of tissue putrescine. 3. Injection of methylglyoxal bis(guanylhydrazone) into the rat also markedly decreased the activity of diamine oxidase (EC 1.4.3.6) in thymus. 4. In no cases where doses of methylglyoxal bis(guanylhydrazone) close to the LD(50) dose for the rat were used was it possible to lower tissue spermidine content to any significant extent. 5. Methylglyoxal bis(guanylhydrazone) seemed to act as a competitive inhibitor for the substrate S-adenosylmethionine and as an uncompetitive inhibitor for the activator putrescine in the decarboxylation of S-adenosylmethionine in vitro. 6. In the diamine oxidase reaction, with putrescine as the substrate, methylglyoxal bis(guanylhydrazone) was a non-competitive inhibitor for putrescine.     

A biochemical investigation of the adenovirus-induced G1 to S phase progression: Thymidine kinase,ornithine decarboxylase,and inhibitors of polyamine biosynthesis

Biochemical events were investigated in the G1 to S phase progression induced in quiescent rodent cells by human adenovirus type 5 (Ad5) and by serum. Thymidine kinase activity increased after infection of cells with Ad5 or addition of 10% serum. These stimulations were additive. An early viral gene was respnsible for induction by Ad5, but the early mutants ts36, ts37, and ts125 induced thymidine kinase at the permissive and nonpermissive temperatures. Several differences were found between cells stimulated by serum compared with Ad5. Induction of thymidine kinase was delayed in Ad5- infected cells, insensitive to 0.01 μ/ml actinomycin D and relatively resistant to reduced Ca²⁺ compared with induction by serum. Ornithine decarboxylase was induced by serum, but not by Ad5. α-Methylornithine had little effect on the induction of thymidine kinase by Ad5, but reduced the induction of thymidine kinase by serum, suggesting that Ad5-induced entry into S phase is uncoupled from polyamine biosynthesis. Methylglyoxal bis(guanylhydrazone), however, prevented the induction of thymidine kinase by both serum and Ad5. Adenovirus infection appears to induce cellular DNA synthesis and thymidine kinase in G1-arrested cells by a mechanism different from serum, and by passes events in the normal G1 to S phase progression.     

Inhibition of spermidine formation in rat liver and kidney by methylglyoxal bis(guanylhydrazone)

The effect of methylglyoxal bis(guanylhydrazone), a substance known to inhibit putrescine-dependent S-adenosyl-l-methionine decarboxylase, on polyamine metabolism in liver and kidney was investigated. Almost complete inhibition of the incorporation of putrescine into spermidine was obtained up to 8h after administration of 80mg of methylglyoxal bis(guanylhydrazone)/kg body wt. by intraperitoneal injection. However, by 20h after administration of the inhibitor spermidine synthesis was resumed. Considerable accumulation of putrescine occurred during this period (up to 3 times control concentrations in both tissues), but there was only a slight fall in the spermidine content. These results suggest that the putrescine-activated S-adenosyl-l-methionine decarboxylase plays an essential role in spermidine biosynthesis in rat liver and kidney, and the possibility of using methylglyoxal bis(guanylhydrazone) to study the role of polyamine synthesis in growth is discussed.     

Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation.     

Lack of a correlation between polyamine synthesis and DNA synthesis by cultured rat liver cells and fibroblasts

Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely α-hydrazino ornithine and methylglyoxal bis(guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth-stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G₁) to enable them to enter from G₁ into S phase and traverse S phase at normal rates in the absence of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content.