Methylglyoxal Bis Research Articles

Essential role of polyamines in growth of cultured carrot cells

Increase in cell number of growing cultured carrot was inhibited in the presence of 0.5 mM methylglyoxal bis-(guanylhydrazone) (MGBG), a compound known to prevent the conversion of putrescine to spermidine and spermine. MGBG-induced inhibition of growth of carrot cells was almost restored to the level of the untreated control by the addition of spermidine or spermine to the culture, but not by putrescine. These results suggested that spermidine and/or spermine, but not putrescine, play an important role in growth of cultured carrot cells.

Spermine prevents endonuclease activation and apoptosis in thymocytes

Glucocorticoid hormones, Ca 2+ ionophores, and some toxic chemicals activate a suicide process in thymocytes, known as apoptosis or programmed cell death. A crucial event in apoptosis is the activation of a Ca 2+- and Mg 2+-dependent endonuclease that promotes extensive DNA fragmentation. In this study, we investigated the effect of various polyamines on endonuclease activation leading to thymocyte apoptosis. We found that both glucocorticoid- and Ca 2+ ionophore-induced DNA fragmentation and apoptosis were prevented by spermine. Other polyamines such as putrescine or spermidine had moderate or no effect. Moreover, spermine, and to a lesser extent spermidine, but not putrescine, prevented endonuclease activation in permeabilized liver nuclei incubated in the presence of Ca 2+ and Mg 2+, indicating that spermine efficiency in blocking DNA fragmentation was related to the interaction of this polyamine with the endonuclease or its substrate, DNA. Experiments with the fluorescent dye, ethidium bromide, and a purified preparation of liver endonuclease revealed that the protective effect of spermine on DNA fragmentation was related to its ability to modify the chromatin arrangement. Thymocytes incubated with methyl glyoxal bis(guanylhydrazone) to deplete intracellular spermine exhibited spontaneous DNA fragmentation, which suggests that modulation of the intracellular polyamine content and regulation of chromatin structure may play a critical role in the early phases of apoptosis. Finally, these results demonstrate that inhibition of DNA fragmentation also prevents the onset of apoptosis, directly linking endonuclease activation and cell death.

A Simplified Method for the Determination of Methylglyoxal Bis(Guanylhydrazone), Mgbg, in Biological Fluids by Reversed-Phase Ion-Pair HPLC

Abstract Methylglyoxal bis(guanylhydrazone, MGBG, is described as an important agent in the fight against certain types of leukemia and other proliferative processes. For this study, we developed a paired-ion HPLC method in order to measure MGBG concentrations in serum and urine samples. MGBG recovery in serum and urine being 95–98%, ultrafiltered samples were then injected onto a C18 column and eluted isocratically with a mobile phase consisting of a mixture of phosphate buffer 0.05 M, pH = 3.0, methanol (80:20) and 1.0 mM sodium heptane sulfonate, with UV (280 nm) detection. The lower limit of MGBG detection was 1.0 μg/ml in urine and 0.1 μg/ml in serum.

Mitochondrial effects of the guanidino group-containing cytostatic drugs, m-iodobenzylguanidine and methylglyoxal bis (guanylhydrazone)

The involvement of mitochondrial damage in the antiproliferative effects of m-iodobenzylguanidine [MIBG] and methylglyoxal bis (guanylhydrazone) [methylGAG] was studied in human neuroblastoma SK-N-SH, mouse neuroblastoma N 1E115 and mouse lymphosarcoma S49 cells. Proliferation of SK-N-SH cells was insensitive to MIBG (100 μM gave 15% inhibition), but sensitive to methylGAG ( ic 50 = 50 μ M) . MIBG and methlyGAG were approximately equitoxic to N 1E115 cells ( ic 50 of 92 and 87 μM, respectively). S49 cells were most sensitive to both MIBG ( ic 50 = 11 μ M) and methylGAG ( ic 50 = 5 μ M) . In isolated sonicated mitochondria, MIBG inhibited respiration at complex I of the respiratory chain ( ec 50 = 0.5 mM) , whereas methylGAG was much less effective ( ec 50 > 15 mM ). In intact cells, MIBG at 31 μM impaired mitochondrial respiration and stimulated the glycolytic flux. In contrast, equimolar concentrations of methylGAG had no effect on oxygen consumption, ATP content, glucose consumption and lactate production. MethylGAG significantly increased putrescine levels in N 1E115 and S49 cells within 12 hr via inhibition of S-adenosylmethionine decarboxylase. No such effects were seen in SK-N-SH cells for up to 48 hr. Equimolar concentrations of MIBG had no effect on the putrescine levels in the various cell lines, suggesting that MIBG did not inhibit S-adenosylmethionine decarboxylase. It is concluded that the antiproliferative mechanisms of the guanidino compounds are essentially different. MIBG inhibited mitochondrial respiration at complex I with concomitant stimulation of the glycolytic flux but was essentially without effect on polyamine levels. On the other hand, cytotoxicity of methylGAG was not associated with mitochondrial dysfunction.

Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus suspension cultures

Various inhibitors of polyamine biosynthesis were used to study the role of polyamines in DNA synthesis and cell division in suspension cultures of Catharanthus roseus (L.) G. Don. Arginine decarboxylase (ADC; EC 4.1.1.19) was the major enzyme responsible for putrescine production. DL α-difluoromethylarginine inhibited ADC activity, cellular putrescine content, DNA synthesis, and cell division. The effect was reversible by exogenous putrescine. Ornithine decarboxylase (ODC; EC 4.1.1.17) activity was always less than 10% of the ADC activity. Addition of DL α-difluoromethylornithine had no effect on ODC activity, cellular polyamine levels, DNA synthesis, and cell division within the first 24 h but by 48 to 72 h it did inhibit these activities. Methylglyoxal bis(guanyl-hydrazone) inhibited S-adenosylmethionine decarboxylase (EC 4.1.1.50) activity without affecting DNA synthesis and cell division.

Genetic characterization of adenovirus transformed cell revertants resistant to methylglyoxal bis(guanylhydrazone): Evidence for the involvement of three genetic loci

Five new independent rat somatic cell mutants resistant to the antileukemic drug methylglyoxal bis(guanylhydrazone) (MGBG) were isolated after mutagen treatment. The mutants were 7- to 10-fold more resistant to MGBG than were the parental wild-type cells. When the MGBG-resistant (MGR) mutants were exposed to the drug in the presence of Tween-80, a nonionic detergent, they became as sensitive (MGS) to MGBG as the wild-type cells, indicating that they were probably permeability mutants. Genetic analysis of hybrids between MGR mutants and wild-type cells showed that MGR and the nontransformed alleles to be recessive to the MGS (wild-type) and transformed phenotype, respectively. Complementation analysis of the seven mutants revealed three functional genetic units or loci responsible not only for the MGR phenotype but also for tumorigenicity as determined in nude mice. Only the MGS hybrids produced tumors in the nude mice, whereas the MGR hybrids and mutants did not. Our results suggest the existence of cellular membrane components that are responsible both for cellular tumorigenicity and resistance to MGBG.

Evaluation of the antimicrobial activity of methylglyoxal bis(guanylhydrazone) analogues, the inhibitors for polyamine biosynthetic pathway

Metabolic and antiproliferative effects of methylglyoxal bis(butylamidinohydrazone) (MGBB) and methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP), inhibitors for polyamine biosynthetic pathway, on Escherichia coli, Shigella sonnei, Aeromonas sobria, Aeromonas hydrophila and Vibrio cholerae were investigated. MGBB at the concentration of 100 mumol/l depleted intracellular putrescine and spermidine concentrations of E. coli to 25 and 20% of the controls, respectively, while MGBCP depressed their concentrations to 38 and 24%, respectively. In these polyamine-depleted E. coli cells the syntheses of RNA, DNA and protein decreased to 13, 54 and 29% of the control, respectively, with MGBB and to 23, 71 and 55%, respectively, with MGBCP. The minimum inhibitory concentrations (MIC) of MGBB for the growth of A. sobria, E. coli, A. hydrophila, V. cholerae and Sh. sonnei were estimated to be 50, 160, 240, 285 and 320 mumol/l, respectively, whereas those of MGBCP were slightly higher for respective bacteria.

Purification of methylglyoxal bis(guanylhydrazone)-induced spermidine N-acetyltransferase from baby hamster kidney cells (BHK-21/C13)

Purification of methylglyoxal bis(guanylhydrazone)-induced spermidine N-acetyltransferase from baby hamster kidney cells (BHK-21/C13)

α-dl-Difluoromethylornithine, a Specific, Irreversible Inhibitor of Putrescine Biosynthesis, Induces a Phenotype in Tobacco Similar to That Ascribed to the Root-Inducing, Left-Hand Transferred DNA of Agrobacterium rhizogenes

alpha-dl-Difluoromethylarginine (DFMA) and alpha-dl-difluoromethylornithine (DFMO), specific irreversible inhibitors of putrescine biosynthesis were applied to Nicotiana tabacum var. Xanthi nc during floral induction. DFMO, but not DFMA, induced a phenotype in tobacco that resembles the transformed phenotype attributed to the root-inducing, left-hand, transferred DNA of Agrobacterium rhizogenes, including wrinkled leaves, shortened internodes, reduced apical dominance, and retarded flowering. Similar treatment of transformed plants (T phenotype) accentuated their phenotypic abnormalities. Cyclohexylammonium and methylglyoxal bis (guanylhydrazone), inhibitors of spermidine and spermine biosynthesis, produced reproductive abnormalities, but did not clearly mimic the transformed phenotype. This work strengthens the previously reported correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway.

Effects of Combinations of Polyamine Biosynthetic Inhibitors on Cellular Polyamines in Carrot Cell Cultures

Summary The effects of cyclohexylammonium phosphate (CHAP) in combination with other inhibitors of polyamine biosynthesis were studied on cellular polyamine levels in carrot ( Daucus carota L.) cell suspension cultures. A combined treatment with CHAP and difluoromethylornithine increased cellular putrescine and spermine levels but had little effect on spermidine levels. Difluoromethylarginine (DFMA)+CHAP decreased cellular putrescine and spermidine levels but increased spermine levels. A combination of CHAP and methylglyoxal bis(guanylhydrazone) caused a significant increase (up to four-fold) in putrescine and a decrease in cellular spermidine and spermine levels.

Regulation of S-adenosylmethionine decarboxylase during the germination of sporangiospores of Mucor rouxii

Summary General properties of S-adenosylmethionine decarboxylase (SAMDC) from Mucor rouxii were studied. Dormant spores of the fungus did not contain detectable levels of the enzyme, but it started to be synthesized at early stages of spore germination. Kinetics of synthesis changed before emergence of the germ tube, with a corresponding increase in a second SAMDC activity which, in contrast to the one originally synthesized, was not activated by putrescine. Development of the second enzyme activity required de novo protein synthesis. Neither enzymic activity was stimulated by Mg2+. Addition of the SAMDC inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG) stopped fungal development in growth phase Ia: cells became spherical and showed ultrastructural alterations. Although MGBG inhibited polyamine formation, it barely inhibited protein and RNA biosynthesis during the first hour of incubation. However, at later periods, biosynthesis of both macromolecules was strongly decreased. When MGBG was added to growth media 3 h after inoculation of spores, it did not affect spore germination and outgrowth. A hypothesis for two different roles of spermidine and putrescine in spore germination is discussed.

Separation of palladium(II) and platinum(II) as their dithiosemicarbazone chelates by reversed-phase HPLC.

The complex formation of palladium(II) and platinum(II) with glyoxal bis(4-phenyl-3-thiosemicarbazone) (GBS), methylglyoxal bis(4-phenyl-3-thiosemicarbazone) (MBS), dimethylglyoxal bis(4-phenyl-3-thiosemicarbazone) (DMBS) and methylethylglyoxal bis(4-phenyl-3-thiosemicarbazone) (MEBS) were studied in 80 v/v% N, N-dimethyl-formamide-water mixture. And reversed-phase HPLC with a spectrophotomertric de-tection was applied to the separation of their Pd-and Pt-chelates by using an ODS col-umn (6 mm i.d.×250 mm in length). Palladium(II) and platinum(II) formed stable chelates with each reagent in weak acidic medium, though it needed to be heated at 80°C for 15 min on the formation of Pt-chelate. The elution peaks of Pd and Pt-chelates on their chromatogram were not successfully separated by using methanol-water and acetonitrile-water as mobile phases. The resolved separation of Pd- and Pt-chelates were achieved by using an acetone-water mobile phase. The Pt-chelates were eluted more rapidly than the Pd-chelates in all reagent systems. In both metal systems, the retention time of each metal chelate was longer in order of GBS, MBS, DMBS and MEBS. The linear relationship was found between the logarithms of capacity factor (log k') of Pdand Pt-chelates and all alkyl carbon number of glyoxal residue in dithiosemicarbazone compounds.

Effects of DL-α-difluoromethylornithine, 4-deoxypyridoxine and methylglyoxal bis(guanylhydrazone) on allograft prolongation

DL-α-difluoromethylornithine (DFMO), 4-deoxypyridoxine (4-DOP), and methylglyoxal bis(guanylhydrazone) (MGBG) were tested as inhibitors of acute skin graft rejection. Proximal full thickness tail skins were exchanged between C57BL/6 and Balb/C mice. Distal autografts were placed to monitor healing. Inhibitors were given singly or in combination, either orally or by injection, in various schedules to 10 groups of mice. Compared to controls, singly treated mice had significant mean prolongation of allografts ranging from 126% to 141%. Likewise, DFMO plus MGBG extended mean time of complete rejection ranging from 172% to 206%. Autografts remained intact. Some grafts persisted after discontinued immunosuppression. Complete rejection was preceded by a decline in vascularity of the graft bed and/or gradual replacement by host tissue. Graft protection in such stringent circumstances i.e., the use of skin in strains with complete histoincompatibility at the H-2 MHC loci, clearly indicate the anti-rejection effects of polyamine synthesis inhibitors. Moreover, primary and secondary effects of DFMO establish the critical role of polyamine pathway activation in acute rejection. In doses and schedules used, toxicity was encountered when DFMO and 4-DOP were used in combination and when increased amounts of MGBG were administered.

Transglutaminase is involved in the fusion of mouse alveolar macrophages induced by 1α,25-dihydroxyvitamin D 3

We have reported that 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3] induces fusion of mouse alveolar macrophages directly by a mechanism involving spermidinedependent protein synthesis (Tanaka, H. et al., 1989, Exp. Cell Res. 180, 72–83). The macrophage fusion induced by 1α,25(OH) 2D 3 occurred in a calcium-dependent manner (Jin, C. H. et al., 1988, J. Cell. Physiol. 137, 110–116). In the present study, we examined the possibility that transglutaminase, a calcium-dependent enzyme, is involved in the fusion of macrophages induced by 1α,25(OH) 2D 3. The activity of transglutaminase increased greatly 12 h after 1α,25(OH) 2D 3 was added and reached a maximum at 48 h. Western blot analysis of the cell lysate using an anti-transglutaminase antibody showed that 1α,25(OH) 2D 3 induced a 77-kDa protein corresponding to transglutaminase. When spermidine synthesis was inhibited by adding methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase, the increase in the transglutaminase synthesis by 1α,25(OH) 2D 3 was markedly inhibited with concomitant inhibition of fusion. Adding more spermidine restored both the synthesis of transglutaminase and the fusion. The treatment of macrophages with cystamine, an inhibitor of transglutaminase, inhibited the fusion in parallel with the suppression of transglutaminase activity, both induced by 1α,25(OH) 2D 3. These results clearly indicate that 1α,25(OH) 2D 3 induces transglutaminase by a spermidine-dependent mechanism and that this enzyme is involved in a biological reaction(s) essential for inducing macrophage fusion.

Polyamine interconversion in rat retina: Effect of intravitreous administration of methylglyoxal bis[guanylhydrazone

The uneven distribution of polyamines in the different cell types of mammalian retina documents the functional accumulation of spermine in photoreceptor cells. Polyamine interconversion in non-growing organs is responsible for the maintenance of putrescine levels and polyamine turnover. Administration of methylglioxal bis[guanylhydrazone] was chosen because this compound is known to inhibit S-adenosylmethionine decarboxylase and to induce spermidine/spermine N 1-acetyltransferase. The combined treatment with both, an inhibitor of S-adenosylmethionine decarboxylase and of polyamine oxidase caused a significant accumulation of N 1-acetylspermine, greater than that of N 1-acetylspermidine. These results are in agreement with the observations that polyamine reutilization in the adult nervous tissues is a major source of spermidine and putrescine.

Ion-Paired Liquid Chromatographic Determination of Phenylglyoxal Bis(Guanylhydrazone) in Serum and Urine

Abstract Phenylglyoxal bis(guanylhydrazone) (PGBG),shows a strong inhibitory activity of the same order and pattern as Methylglyoxal bis (guanylhydrazone) (MGBG), against Diamine oxidase from the small intestine of the rats and against S-adenosylmethionine decarboxylase (one of the key enzymes of polyamine biosinthesis) from Eimeria stiedai cysts, and from partially purified rabbit liver enzyme. The therapeutic interest of polyamine biosynthesis inhibitors in tumoral, infectious and parasitic processes and the lesser toxicity of PGBG than of MGBG favour the use of this compound as an alternative treatment for this kind of disease. For this study a paired-ion HPLC method was developed to measure concentrations of PGBG in serum and urine samples. The method involved ultrafiltration of diluted serum and urine through a 30,000-Dalton molecular weight cut-off filter. Recoveries for PGBG in serum and urine were 97–99%. Ultrafiltered samples were then injected into a C18 column and eluted isocratically with a mo...

Inhibition of human breast cancer cell growth by methylglyoxal bis(guanylhydrazone)

Inhibition of human breast cancer cell growth by methylglyoxal bis(guanylhydrazone)

The regulation of G 0-S transition in mouse T lymphocytes by polyamines

While the role of polyamines in DNA synthesis during the S phase of the cell cycle has been repeatedly postulated, recent studies point also to polyamine involvement in the early phase of the G 0-S transition. In order to determine polyamine-dependent steps in the cell cycle we have studied the effects of inhibitors of polyamine biosynthesis and exogenous polyamines on the proliferation of T lymphocytes as well as on the expression of some growth-regulated genes. The ability of Con A-stimulated mouse T lymphocytes to enter DNA synthesis was markedly inhibited by methylglyoxal bis(guanylhydrazone) in a dose-dependent manner. This inhibitory effect was stronger in the presence of fetal calf serum containing a high level of activities of polyamine oxidases than in the presence of horse serum. Putrescine and spermine added to T splenocyte culture instead of mitogen-Con A stimulated [ 3H]thymidine incorporation with kinetics similar to that observed with Con A. The growth-stimulating effects of polyamines were concentration-dependent. Polyamines at optimal growth-stimulating concentrations (10 μ M spermine and 80 μ M putrescine) induced the expression of genes encoding the cytoskeletal proteins β-actin, vimentin, and α-tubulin to an extent and with kinetics similar to those of Con A. The results presented herein suggest that polyamines are capable of stimulating the transition of G 0 cells to the S phase and that this effect may be mediated by their influence on the gene expression.

EFFECT OF POLYAMINE DEPLETION ON DNA DAMAGE and REPAIR FOLLOWING UV IRRADIATION OF HeLa CELLS

Treatment of HeLa cells with the polyamine biosynthesis inhibitors, methylglyoxal bis(guanylhydrazone) (MGBG), difluoromethylornithine (DFMO) or a combination of the two, resulted in reduction in cellular polyamine levels. Analysis of UV light-induced DNA damage and repair in these polyamine depleted cells revealed distinct differences in the repair process relative to that seen in cells possessing a normal polyamine complement. Initial yield of thymine dimers and rate of removal of these lesions from cellular DNA appeared normal in polyamine-depleted cells. However, depleted cells exhibited retarded sealing of DNA strand breaks resulting from cellular repair processes, reduced repair synthesis and an increased sensitivity to UV killing. Incision at damaged sites was not affected since ara-C repair-dependent breaks accumulated in a normal fashion. Molecular analysis of inhibited repair sites by exonuclease III and T4 DNA ligase probes suggest that the strand interruptions consist of gaps rather than ligatable nicks, consistent with an interpretation of the repair defect being at the gap-filling stage rather than the ligation step. Observed patterns of differential polyamine depletion by DFMO and MGBG, and partial reversal of repair inhibition by polyamine supplementation, suggests that polyamine depletion per se, rather than some secondary effect of inhibitor treatment, is responsible for the inhibition of repair.

Regulation of the Na+‐dependent and the Na+‐independent polyamine transporters in renal epithelial cells (LLC‐PK1)

We have studied the regulation of the Na(+)-dependent and Na(+)-independent polyamine transport pathways in the renal LLC-PK1 cell line. Most of the experiments were performed in the presence of 5 mM DL-2-difluoromethylornithine (DFMO) in order to inhibit the cellular synthesis of polyamines. The activity of both transporters as measured by putrescine uptake was increased by growth-promoting stimuli and decreased by exogenous polyamines. The time course of the increase in uptake activity induced by fetal calf serum could be fitted by a single exponential, and the process was three times faster for the Na(+)-dependent than for the Na(+)-independent transporter. Maximum activity was reached after more than 24 h. This increase could be inhibited by actinomycin D and by cycloheximide. Other growth-promoting stimuli, such as subconfluent cell density, as well as growth factors also induced an increase in the transport activity. Particularly, there was a marked stimulation of the Na(+)-dependent pathway by epidermal growth factor in combination with insulin. On the other hand, the transport activity decayed very rapidly upon addition of exogenous polyamines (t1/2 less than 60 min). The diamine putrescine was much less effective in this respect than the polyamines spermidine and spermine. The non-metabolizable substrate methylglyoxal bis(guanylhydrazone) did not induce a decay of the transport activity, but it protected the Na(+)-dependent pathway against the polyamine-induced decay. Inhibition of the protein synthesis by cycloheximide did not induce a rapid decrease of the transport activity; neither did it affect the polyamine-induced decay. These observations suggest that this polyamine-induced decay is not owing to an inhibitory effect on the rate of synthesis of the transporters, but rather to a degradation or an inactivation of the transporters. The polyamine-induced decay slowed down at lower cell density. This effect was particularly pronounced for the Na(+)-dependent transporter. Since the uptake of polyamines was increased at low cell density, the decreased rate of decay in this condition pleads against a simple mechanism of transinhibition by the substrate. In conclusion, both transport pathways were similarly affected by the regulatory parameters, but the Na(+)-dependent transporter was more rapidly and more effectively regulated. The numerous interacting regulatory steps furthermore suggest a physiological role for these transporters, such as an involvement in urinary polyamine disposal.