Inhibitors of tri- and tetra- polyamine oxidation, but not diamine oxidation, impair the initial stages of wound-induced suberization.

The mechanisms regulating, and modulating potato wound-healing processes are of great importance in reducing tuber infections, reducing shrinkage and maintaining quality and nutritional value for growers and consumers. Wound-induced changes in tuber polyamine metabolism have been linked to the modulation of wound healing (WH) and in possibly providing the crucial amount of H2O2 required for suberization processes. In this investigation we determined the effect of inhibition of specific steps within the pathway of polyamine metabolism on polyamine content and the initial accumulation of suberin polyphenolics (SPP) during WH. The accumulation of SPP represents a critical part of the beginning or inchoate phase of tuber WH during closing-layer formation because it serves as a barrier to bacterial infection and is a requisite for the accumulation of suberin polyaliphatics which provide the barrier to fungal infection. Results showed that the inhibitor treatments that caused changes in polyamine content generally did not influence wound-induced accumulation of SPP. Such lack of correlation was found for inhibitors involved in metabolism and oxidation of putrescine (arginine decarboxylase, ornithine decarboxylase, and diamine oxidase). However, accumulation of SPP was dramatically reduced by treatment with guazatine, a potent inhibitor of polyamine oxidase (PAO), and methylglyoxal-bis(guanylhydrazone), a putative inhibitor of S-adenosylmethione decarboxylase which may also cross-react to inhibit PAO. The mode of action of these inhibitors is presumed to be blockage of essential H2O2 production within the WH cell wall. These results are of great importance in understanding the mechanisms modulating WH and ultimately controlling related infections and associated postharvest losses.

Biological activity of antitumoural MGBG: the structural variable.

The present study aims at determining the structure-activity relationships (SAR's) ruling the biological function of MGBG (methylglyoxal bis(guanylhydrazone)), a competitive inhibitor of S-adenosyl-L-methionine decarboxylase displaying anticancer activity, involved in the biosynthesis of the naturally occurring polyamines spermidine and spermine. In order to properly understand its biochemical activity, MGBG's structural preferences at physiological conditions were ascertained, by quantum mechanical (DFT) calculations.

Inhibitors of polyamine metabolism: review article.

The identification of increased polyamine concentrations in a variety of diseases from cancer and psoriasis to parasitic infections has led to the hypothesis that manipulation of polyamine metabolism is a realistic target for therapeutic or preventative intervention in the treatment of certain diseases. The early development of polyamine biosynthetic single enzyme inhibitors such as alpha-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) showed some interesting early promise as anticancer drugs, but ultimately failed in vivo. Despite this, DFMO is currently in use as an effective anti-parasitic agent and has recently also been shown to have further potential as a chemopreventative agent in colorectal cancer. The initial promise in vitro led to the development and testing of other potential inhibitors of the pathway namely the polyamine analogues. The analogues have met with greater success than the single enzyme inhibitors possibly due to their multiple targets. These include down regulation of polyamine biosynthesis through inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase and decreased polyamine uptake. This coupled with increased activity of the catabolic enzymes, polyamine oxidase and spermidine/spermine N1-acetyltransferase, and increased polyamine export has made the analogues more effective in depleting polyamine pools. Recently, the identification of a new oxidase (PAO-h1/SMO) in polyamine catabolism and evidence of induction of both PAO and PAO-h1/SMO in response to polyamine analogue treatment, suggests the analogues may become an important part of future chemotherapeutic and/or chemopreventative regimens.

Biochemical and chemical characterization of phenylglyoxal bis(guanylhydrazone), an aromatic analogue of mitoguazone.

Since little has been known about the properties of aromatic analogues of the antineoplastic agent methylglyoxal bis(guanylhydrazone) (MGBG), an investigation was performed on phenylglyoxal bis(guanylhydrazone) (PhGBG). PhGBG competitively inhibited yeast adenosylmethionine decarboxylase (AdoMetDC) with a Ki of 65 microM. As compared to MGBG (Ki 0.23 microM), PhGBG is a much weaker inhibitor, being even weaker than the unsubstituted congener glyoxal bis(guanylhydrazone) (GBG, Ki 18 microM). PhGBG inhibited porcine kidney diamine oxidase (DAO) non-competitively, being a more potent inhibitor (Ki 0.12 microM) than GBG (Ki 0.17 microM) or MGBG (Ki 0.33 microM). Thus, PhGBG has an unfavourably high ratio of Ki(AdoMetDC)/Ki(DAO) for potential use for selectively inhibiting polyamine biosynthesis. This does not exclude the possibility that PhGBG or other aromatic congeners might have therapeutic value since the corresponding ratio of the antileukaemic congeners GBG and MGBG is also high as compared to many aliphatic non-antileukaemic analogues. The pKa1 and pKa2 values of PhGBG dication were found to be 6.39 +/- 0.02 and 8.64 +/- 0.02 respectively, their difference being distinctly larger than in the case of GBG or its C-alkylated analogues. This may result from decreased stability of the dication form, caused by the resonance effect or possibly by the inductive effect of the phenyl group. The species distribution of PhGBG (proportion of free base 5.5%, predominant species the monocation) at 37 degrees C resembles that of GBG and MGBG but is clearly different from that of non-antileukaemic C-alkylated analogues. These similarities suggest that PhGBG and its derivatives may be worth antitumour screening. Depending on the conditions used in the crystallization, three different types of crystals of PhGBG sulphate were obtained. Crystallography indicated that, in two of the types, the crystal consisted exclusively of the anti-anti isomer, i.e. the same isomer as has been observed in the case of GBG and its C-alkylated congeners. One crystal type, however, consisted of a different geometrical isomer (anti-syn), suggesting that PhGBG may isomerize more easily than its aliphatic analogues. Previous concepts on the isomerism of GBG and C-alkylated bis(guanylhydrazones) thus cannot be generalized to aromatic congeners. A theory based on resonance, inductive and hyperconjugative effects and electron transfers is presented that is capable of explaining the formation of the two geometrical isomers of PhGBG that were experimentally observed. A similar theory, based on hyperconjugation of C-F bonds, is presented that is capable of explaining the previous finding of the formation of the anti-syn isomer of trifluoromethylglyoxal bis(guanylhydrazone) (CF3GBG). Like that of CF3GBG, the anti-syn isomer of the PhGBG dication is stabilized by an internal hydrogen bond. The lack of structural rigidity may affect the biological properties of PhGBG, e.g. its ability to inhibit AdoMetDC.

Peritubular paraquat transport in isolated renal proximal tubules.

To better understand the characteristics of peritubular transport of organic cations (OCs), the uptake of the polyvalent OC dimethylbipyridinium (paraquat) and the structurally similar monovalent OC 1-methyl-4-phenylpyridinium (MPP+) was measured in suspensions of rabbit renal proximal tubules. Compared to the uptake of MPP+, the uptake of paraquat across the peritubular membrane was a low affinity, low capacity carrier-mediated process with a Jmax of 0.52 +/- 0.19 nmol.mg of protein.-1 min-1 and a Km of 162 +/- 25 microM. The uptake of MPP+ was a carrier-mediated process with a measured Jmax and Km of 1.8 +/- 0.09 nmol.mg of protein.-1min-1 and 28 +/- 8 microM, respectively. To determine whether paraquat is a substrate for the monovalent OC pathway, the effect of unlabeled MPP+ and tetraethylammonium (TEA) on paraquat uptake was examined. A 1 mM concentration of the monovalent OC MPP+ and TEA reduced the uptake of [14C]paraquat and [3H]MPP+ by approximately 30 and 90%, respectively, whereas 1 mM paraquat had no effect on [3H]MPP+ or [14C]TEA uptake. Thus, MPP+, but not paraquat, appears to interact with the monovalent OC transporter. On the other hand, the polyvalent OC substrates, including the polyamines putrescine and spermine, the herbicide diquat and the divalent hexamethonium bromidehydrate had no effect on either paraquat or MPP+ uptake. However, the synthetic polyamine methylglyoxal bis(guanyl-hydrazone)dihydrochloride (MGBG; 1 mM) reduced both paraquat and MPP+ uptake (by 60 and 90%, respectively). The ability of MGBG, unlike the other polyvalent substrates, to interact with paraquat transport may be related to structural similarities in the relative location of the two charged nitronium moieties in paraquat and MGBG.(ABSTRACT TRUNCATED AT 250 WORDS)

Overproduction of S-adenosylmethionine decarboxylase in ethylglyoxal-bis(guanylhydrazone)-resistant mouse FM3A cells.

A variant cell line, termed SAM-1, which overproduced S-adenosylmethionine decarboxylase (AdoMetDC), was isolated by treatment of mouse FM3A cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequent incubation with ethylglyoxal bis(guanylhydrazone), an inhibitor of the enzyme. The cells were resistant to ethylglyoxal bis(guanylhydrazone), and showed AdoMetDC activity approximately five-times higher than control cells. The rate of AdoMetDC synthesis and the amount of AdoMetDC existing in SAM-1 cells were about five-times those in control cells. The amount of AdoMetDC mRNA existing in SAM-1 cells was five-times more than that in control cells. The amount of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine, an irreversible inhibitor of AdoMetDC, necessary to inhibit cell growth was also five-times more in SAM-1 cells than in control cells. However, the following were the same in both SAM-1 and control cells; the amount of genomic DNA for AdoMetDC, the size and nucleotide sequence of 5' untranslated region of AdoMetDC mRNA, the deduced amino acid sequence (334 residues) from the nucleotide sequence of AdoMetDC cDNA and the degradation rate (t1/2 = about 4 h) of AdoMetDC. In addition, AdoMetDC mRNA in control cells was slightly more stable than that in SAM-1 cells. The results indicate that the overproduction of AdoMetDC in SAM-1 cells was caused by the increase of AdoMetDC mRNA. The variant cell line is convenient for studying the regulation of AdoMetDC and the physiological function of polyamines.

Cationic anticancer drugs and their modes of action.

This report focuses on two groups of cationic cancerostatics, anthracycline antibiotics and 1,4-benzoquinone-guanylhydrazone-thiosemicarbazone (ambazone), lining up biophysical and biochemical effects on the level of membranes and membrane constituents. The interaction of both drugs with multilamellar liposomes consisting of phosphatidylcholine used as a simple model membrane system could be ensured by means of steady state and nanosecond time-resolved fluorometric investigations. The biochemical effect on membranes is underlined by the inhibition of the neuraminidase activity of the Sendai virus, modification of the CAMP phosphodiesterase activity of leukemia L 1210 cells of mice and reduction of the lymphocyte blast transformation.

Properties and physiological function of the polyamine transport system.

Polyamine transport was examined in Chinese hamster ovary (CHO) cells because of the unique potential these cells hold for utilizing genetic approaches to study the mechanisms of polyamine transport, its regulation, and its function. Parental (control) CHO cells were shown to contain a polyamine transport system with characteristics consistent with polyamine-uptake properties described in other cell types. Polyamines appear to cross the plasma membrane via an energy-requiring transport system specific for putrescine, spermidine, spermine, and their analogues. A mutant line, CHOMG, selected for resistance to the toxicity of methylglyoxal bis(guanylhydrazone), was shown to lack a functional polyamine transport system. CHOMG cells provided the negative controls necessary to examine the role of polyamine transport in maintenance of intracellular polyamine levels and in the regulation of the polyamine metabolic enzymes. It was found that the repression of ornithine decarboxylase activity by polyamines and the induction of spermidine/spermine-N1 acetyltransferase by polyamine analogues including bis(ethyl)spermine derivatives required the presence of a functional polyamine transport system. The CHO-CHOMG model was also shown to provide a means for establishing the importance of the polyamine transport system in the toxicity of polyamine analogues. The inability of alpha-difluoromethylornithine-treated CHOMG cells to utilize extracellular polyamines to replenish depleted intracellular polyamine levels suggested a means by which polyamine transport-positive cells may be identified. Such a selection procedure will permit the use of CHOMG cells in the isolation of genes encoding proteins involved in polyamine transport.

Acetylation of spermidine and methylglyoxal bis(guanylhydrazone) in baby-hamster kidney cells (BHK-21/C13).

Treatment of BHK-21/C13 cells with methylglyoxal bis(guanylhydrazone) (MGBG) induced the cytosolic form of spermidine N1-acetyltransferase. It stabilized the enzyme against proteolytic degradation, but the drug did not affect the enzyme activity in vitro. MGBG was itself acetylated by BHK-21/C13 cells, but at only one-tenth the rate at which spermidine was acetylated. Acetylation occurred almost exclusively in the nuclear fraction. The product was identified as N-acetyl-MGBG by h.p.l.c., by using [3H]acetyl-CoA and [14C]MGBG as co-substrates. The results suggest that the acetylation of MGBG by BHK-21/C13 cells occurs by a different acetyltransferase enzyme from that which acetylates spermidine.

Endogenous ADP-ribosylation in skeletal muscle membranes.

The characteristics of ADP-ribosyltransferase activity in skeletal muscle membranes have been studied. The membrane enzymes can ADP-ribosylate exogenous substrates such as guanylhydrazones, polyarginine, lysozyme, and histones. The properties of the enzyme are investigated by using diethylaminobenzylidineaminoguanidine as a model substrate. Incubation of the membranes with [32P]adenylate-labeled NAD results in the labeling of a number of cellular proteins. Magnesium ions, detergents, and diethylaminobenzylidineaminoguanidine stimulated the ADP-ribosylation of membrane proteins, whereas L-arginine methyl ester and arginine inhibited ADP-ribosylation. The labeling of specific proteins in the sarcoplasmic reticulum and glycogen pellet is influenced significantly by detergents, nucleotides, and thiols. The hydroxylamine sensitivity of the ADP-ribose linkage in the membrane proteins is similar to that reported for (ADP-ribose)-arginine linkage. Snake venom phosphodiesterase digestion of the ADP-ribosylated membranes produces 5'-AMP as the major acid-soluble digestion product. The results suggest that the primary mode of modification is mono(ADP-ribosyl)ation. The ADP-ribosyltransferase activity in the membrane preparations is not extracted under conditions used for solubilization of extrinsic proteins, suggesting that the activity is associated with some integral membrane protein.

Isolation and uptake characteristics of adenovirus transformed cell revertants resistant to the antiproliferative effects of methylglyoxal bis(guanylhydrazone).

Four independent variant cell lines resistant to the toxic action of methylglyoxal bis(guanylhydrazone), an anticancer drug and inhibitor of polyamine synthesis, have been isolated by single step selection from an adenovirus type 2-transformed rat brain cell line. Drug-resistance was accompanied by loss of tumorigenic potential in athymic nude mice. MGBG resistance was attributable to decreased drug uptake.

Multiple pathways for uptake of paraquat, methylglyoxal bis(guanylhydrazone), and polyamines.

The uptake of polyamines, methylglyoxal bis(guanylhydrazone) (MGBG), and paraquat [N,N-dimethyl-4,4'-bipyridylium] into control Chinese hamster ovary (CHO) cells and a mutant CHO cell line selected for resistance to the toxicity of MGBG was examined. In contrast to control CHO cells, the mutant cells had no detectable uptake of MGBG or any of the polyamines. There was no difference between the two cell lines in the uptake of alpha-aminoisobutyric acid (AIB), which indicates that there was no general change in membrane transport processes. The mutant cells were also found to be resistant to the toxicity of paraquat and to have a reduced capability to take up the herbicide. This finding confirms that the uptake of paraquat is necessary for the toxicity of this compound and that the paraquat is taken up by a transport system that also transports MGBG. Competition experiments showed that an excess of unlabeled paraquat inhibited uptake of MGBG and, to a lesser extent, uptake of putrescine and spermidine, but no inhibitory action on spermine uptake could be detected. Studies with type II cells isolated from rat lung also demonstrated uptake of paraquat and spermidine, but paraquat was only a weak inhibitor of spermidine uptake in this system. These results suggest that there may be multiple systems for the uptake of MGBG and polyamines and that paraquat is taken up by at least one but not by all of these systems.

Difluoromethylornithine enhances the uptake of methylglyoxal-bis(guanylhydrazone) prior to inhibiting leukemic cell proliferation.

Difluoromethylornithine (DFMO) is a nonreversible inhibitor of ornithine decarboxylase (ODC), the initial rate-limiting enzyme in the polyamine biosynthetic pathway. When HL60 leukemic cells were incubated in the presence of concentrations of DFMO from 0.05 mM to 5 mM, there was a concentration-dependent inhibition of ODC activity apparent within 24 h. Likewise, cellular polyamine levels were reduced by the presence of DFMO in a concentration-dependent manner after 4 days. The growth of cells incubated with 0.5 mM or greater was inhibited after 3-4 cell doublings. When the concentration of DFMO was less than 0.5 mM, growth was not inhibited. Methylglyoxal-bis(guanylhydrazone) (MGBG) uptake was enhanced in cells treated with concentrations of 0.05-0.5 mM DFMO, but not enhanced in cells treated with DFMO concentrations of 1 mM or greater. DFMO-induced cellular polyamine depletion does enhance MGBG uptake into HL60 cells, but treatment with high concentrations of DFMO, which deplete polyamines to the extent that growth is inhibited, negate this effect.

DNA-binding abilities of bisguanylhydrazones of anthracene-9,10-dicarboxaldehyde.

DNA-binding strengths of three anthracene-9,10-dicarboxaldehyde hydrazones were examined by spectral shifts of the drug-DNA combinations, spectral titration by Scatchard plots, elevation of DNA melting temperature during complexation and comparison of spectral shifts in the presence of DNA's having variable base contents. Dissociation of the DNA-complexes was also observed. The results showed a strong degree of binding by the three compounds. They did not exhibit noticeable base-pair specificity, but both associated with and dissociated rapidly from DNA. Scatchard plots for DNA association indicated two types of binding; the stronger was most likely due to intercalation of the planar anthracene ring into the DNA double helix. No direct correlation can be drawn between the observed anti-cancer activities and DNA binding affinities of these compounds in vitro.

Increased pulmonary uptake of exogenous polyamines after unilateral pneumonectomy.

Alterations in pulmonary uptake of the naturally occurring polyamine spermidine and of an exogenous polyamine substrate analogue, methylglyoxal bis(guanylhydrazone) (MGBG), were investigated during the early phase of compensatory lung growth after partial pneumonectomy (PNX) in rats. In lungs perfused in situ 3 days after left PNX, when a small (14%) but significant (P less than 0.01) increase in right lung mass could be detected, uptake of [14C]spermidine and of [14C]MGBG from the pulmonary circulation was increased. MGBG uptake exhibited saturation kinetics (1-50 microM MGBG), both in lungs of control animals apparent Km, 11.3 microM; Vmax, 479 pmol X g-1 X min-1) and on the 1st and 3rd post-PNX days. In both PNX groups, the apparent Km of the uptake pathway was decreased somewhat (8.5 microM), while Vmax increased progressively to 584 and 678 pmol X g-1 X min-1 at days 1 and 3, respectively. The effects of PNX on MGBG uptake were detected as early as 3 h after lung resection and were no longer evident when compensatory lung growth was completed 14 days after surgery. In rats adrenalectomized 5 days before left PNX, an accelerated onset and increased rate of lung restoration were associated with a doubling of the effect of PNX alone on MGBG uptake.

Some aspects of clearance of mitoguazone in cancer patients and experimental cancer models.

Mitoguazone (methylglyoxal-bis(guanyl-hydrazone), MGBG) was studied by its first-pass mechanism in both cancer patients and experimental cancer models. It appears from the study that 90% of MGBG is cleared from the plasma within minutes. 24-h recovery in the urine, however, did not exceed 16% so that 84% of the drug seems to be bound to subcellular compartments. Tissue levels of MGBG in the normal prostate ranged higher than in experimental prostate cancer type 3327 M/G, i.e. enhanced clearance from cancer tissues: polyamine biosynthetic enzymes ornithine decarboxylase as well as S-adenosylmethionine decarboxylase are contrarily affected by MGBG.

Biochemical characterization of propylglyoxal bis(guanylhydrazone). Facile synthesis of monoalkylglyoxal bis(guanylhydrazones).

Propylglyoxal bis(guanylhydrazone) sulfate, a novel analog of the well-known antileukemic drug methylglyoxal bis(guanylhydrazone), has been prepared from 2,2-dibromopentanal, and the compound has been characterized biochemically. Although it is a powerful inhibitor of S-adenosylmethionine decarboxylase, its Ki value (0.2 microM) is considerably higher than that of ethylglyoxal bis(guanylhydrazone) (0.06 microM). The compound is only poorly taken up by tumor cells, and its accumulation is not stimulated by a prior exposure of the tumor cells to difluoromethylornithine, a compound that causes polyamine depletion. Thus, the uptake characteristics of the compound are similar to those of ethylglyoxal bis(guanylhydrazone), but in striking contrast to those of methylglyoxal and glyoxal bis(guanylhydrazones). Since the configuration of the double bonds in glyoxal, methylglyoxal and propylglyoxal bis(guanylhydrazones) has been shown to be identical, the different uptake characteristics are probably only due to differences in side chain size and/or hydrophobicity.

Interaction of paraquat and amine uptake by rat lungs perfused in situ.

The kinetics of [14C]paraquat (N,N-di[14C]methyl-4,4'-bipyridylium) uptake from the pulmonary circulation were investigated in rat lungs perfused in situ. During the 1st h of exposure to the herbicide paraquat entered the lungs primarily by diffusion; no evidence was obtained from concentrative uptake, saturation kinetics (1-3,500 microM paraquat), or inhibition by the amines methyl-glyoxal bis(guanylhydrazone) or spermidine, both of which were expected to compete for paraquat transport sites. In contrast, after 60 min of exposure, uptake rates increased two- to three-fold, and paraquat was accumulated to an apparent intracellular concentration greater than that in the perfusate. The latter phase of paraquat uptake was saturable and was inhibited by methylglyoxal bis(guanylhydrazone); it did not appear to reflect a progressive paraquat-induced alteration in cellular permeability but rather predominance of a rapid, carrier-mediated uptake pathway.