Structure-function correlations of polyamine analog-induced increases in spermidine/spermine acetyltransferase activity

The cytosolic enzyme, spermidine/spermine acetyltransferase (SSAT), is distinguished by its role in polyamine interconversion and by its high inducibility in response to a variety of physiological and pharmacological stimuli. Among a series of fifteen polyamines and polyamine analogs, the most potent inducers of SSAT activity in cultured L1210 cells were found to be N1,N8-bis(ethyl)spermidine (BES) and N1,N12-bis(ethyl)spermine (BESm). Over a 24-hr exposure at 10 microM, enzyme activity rose 13- and 16-fold with BES and BESm, respectively, compared to 2- to 3-fold with the anticancer agent, methylglyoxal bis(guanylhydrazone). The increase in enzyme activity by BESm began rapidly and continued steadily with time so that by 48 hr it increased to about twenty times control. By inhibitor studies, the increase was found to be due to elevated protein synthesis predominantly at the level of translation and to an apparent prolongation of enzyme half-life related to enzyme stabilization. Among the analogs, the structural requirements for maximum enzyme induction were found to be critically dependent on aminopropyl moieties and on the presence, size and location of the alkyl groups. By structure-function comparisons, it was deduced that the known abilities of BES and BESm to regulate ornithine and S-adenosylmethionine decarboxylase activities or to inhibit cell growth occur independently of their effects on SSAT activity in L1210 cells.     

Polyamine levels and ornithine decarboxylase activity in blood and erythrocytes in human diseases

Serum and erythrocyte levels of the polyamines spermine, spermidine and putrescine, as well as ornithine decarboxylase in erythrocytes, were studied in patients with different neoplasms (breast, lung and colon cancer) and in those with a nonmalignant proliferative disease (familial polyposis). The blood levels of polyamines and the spermine/putrescine ratio were significantly higher in all tumors and in nonmalignant colon polyposis. In erythrocyte ornithine decarboxylase activity, spermine and spermidine levels, as well as spermidine/putrescine and spermine/putrescine ratios showed a significant decrease after surgery and chemotherapy. Our data suggest that high levels of blood polyamines and erythrocyte ornithine decarboxylase activity are related to cell proliferation and cancer treatment, but that levels of polyamines in serum and erythrocytes are still significantly high after cancer treatment and are similar to those in polyposis disease. Polyamines are related to nuclear activity during differentiation; therefore, the altered turnover of polyamines could be a sign of abnormal nuclear function. Since polyamines stimulate protooncogene expression, their high levels could be considered an important cofactor in malignant cell transformation.     

Stabilization of ornithine decarboxylase in mouse liver and lung by methylglyoxal bis(cyclohexylamidinohydrazone)

The intraperitoneal injection of methylglyoxal bis(cyclohexylamidinohydrazone) (MGBC), an inhibitor of S-adenosylmethionine decarboxylase and spermidine synthase, markedly increased (7-fold of the basal level at 4 hr) ornithine decarboxylase (ODC) activity in normal mouse liver. ODC activity was also increased 2.5-fold over the basal level in mouse lung at 6 hr after the injection. The effect of MGBC on ODC activity occurred in a dose-dependent manner. Measurement of the apparent half-life of ODC induced in the liver and lung by MGBC treatment revealed a clear decrease in the decay rate of the enzyme in both the tissues. Activities of S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine/spermine N1-acetyltransferase (SAT) were not increased by the intraperitoneal injection of MGBC. There was a large rise in putrescine and a fall in spermidine and spermine in the liver and lung except for brain within an 8 hr period in response to MGBC, suggesting that these changes resulted from the stabilization of ODC and inhibitions of AdoMetDC and spermidine synthase.     

Effects of polyamines on the binding of [3H]MK-801 to the N-methyl-D-aspartate receptor: pharmacological evidence for the existence of a polyamine recognition site

A heat-stable factor of low molecular weight that increases the binding of [3H]MK-801 to rat brain membranes in the presence of maximally effective concentrations of L-glutamate and glycine was purified from bovine brain by reverse phase and ion-exchange high pressure liquid chromatography. The stimulatory activity was due to the presence of spermidine in the active fractions. Polyamines including spermine and spermidine are found in high concentrations in mammalian tissue. These compounds increase the affinity of N-methyl-D-aspartate (NMDA) receptors for [3H]MK-801 when assays are carried out in the presence of 100 microM L-glutamate and 100 microM glycine. At concentrations of 1 to 300 microM, a number of di- and triamines, including NH2(CH2)3NH2, NH2(CH2)3NH(CH2)2NH2, and NH2(CH2)3NH(CH2)3NH2, have partial or full agonist-like activity similar to that of spermidine. Other polyamines, including putrescine, cadaverine, NH2(CH2)2NH(CH2)2NH2, and CH3NH(CH2)3NHCH3, at concentrations of 1 to 100 microM, inhibited the binding of [3H]MK-801 in the presence of spermine, L-glutamate, and glycine but not in the presence of only L-glutamate and glycine. It is concluded that these compounds are selective antagonists of the effects of spermine at the NMDA receptor. These results suggest that there may be a polyamine recognition site on the NMDA receptor complex.     

Inhibition by polyamines of methylthiopropylamine-induced ornithine decarboxylase in human lymphoid leukemia Molt 4B cells

Methylthiopropylamine (MTPA), an inhibitor of spermidine synthase, markedly induced ornithine decarboxylase (ODC) activity (about 30-fold of the basal level) in human lymphoid leukemia Molt 4B cells. This induction was blocked by the addition of spermidine, spermine or putrescine simultaneously with MTPA. Inhibition by spermidine or spermine of the MTPA-induced ODC activity was larger than that by putrescine. The increase of ODC activity by MTPA led to the large increase of cellular putrescine content. This increase of putrescine content was abolished drastically by the simultaneous addition of spermidine or spermine. The increase of ODC activity was almost completely blocked by the addition of cycloheximide or actinomycin D. This finding suggested that the increase of ODC activity was not due to activation of ODC preformed in Molt 4B cells. The ODC induction by MTPA was dose-dependently blocked by adding the calcium channel blockers (verapamil and nifedipine) or protein kinase C inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine and palmitoyl carnithine). These results suggested that calcium and protein kinase C (PKC) were involved in MTPA-associated induction of ODC.     

Allylglycine affects acetylation of putrescine and spermidine in mouse brain

Administration of allylglycine to mice (.8 nmole/kg, 1. p.) results in a depletion of GABA levels, and it is accompanied by a decrease in SAM-DC activity and spermidine and spermine levels (Pajunen et al., 1979). Here we describe a biphasic effect on the acetylation of putrescine and spermidine in mouse brain homogenate. There appears to be an inverse correlation between the initial decrease in spermidine levels at 2 hours and the increase in the acetylation of spermidine. This is suggestive of a conversion of spermidine, probably through N -acetylspermidine to putrescine. The peak of putrescine acetylation observed by us at 4 hours may also reflect a conversion of putrescine, via acetylputrescine to GABA. The inter conversion hypothesis is supported by the fact that putrescine levels remain essentially stable in spite of a significant depletion of spermidine and spermine. In addition, there is a decrease in putrescine and spermidine acetylation at 8 hours, which coincides with the increase in ODC activity and the increase towards control levels of GAD activity (Pajunen et al., 1979). Such inverse correlations suggest a mechanism for replenishment of polyamines once GAD activity returns to control levels.     

Putrescine or spermidine binding site of aminopropyltransferases and competitive inhibitors

A model of the active site of aminopropyltransferases was proposed based on the study of a number of monoamino and diamino compounds as potential inhibitors and substrates, respectively, of spermidine synthase purified from pig liver. The active site seems to have a relatively large hydrophobic cavity adjacent to a negatively charged site, to which a protonated amino group of putrescine binds, with another amino group of putrescine being situated in the hydrophobic cavity as a free form to be aminopropylated by decarboxylated S-adenosylmethionine. On the basis of the above-mentioned model, another modified one was proposed for spermine synthase, and several compounds mentioned model, another modified one was proposed for spermine synthase, and several compounds designed according to the modified model were found to potently inhibit spermine synthase, purified from rat brain, in competition with spermidine. The newly developed inhibitors were about two orders of magnitude more potent in vitro than a known inhibitor of spermine synthase, dimethyl(5'-adenosyl)sulfonium perchlorate.     

Allyglycine affects acetylation of putrescine and spermidine in mouse brain

Administration of allylglycine to mice (.8 mmole/kg, i.p.) results in a depletion of GABA levels, and it is accompanied by a decrease in SAM-DC activity and spermidine and spermine levels (Pajunen et al., 1979). Here we describe a biphasic effect on the acetylation of putrescine and spermidine in mouse brain homogenate. There appears to be an inverse correlation between the initial decrease in spermidine levels at 2 hours and the increase in the acetylation of spermidine. This is suggestive of a conversion of spermidine, probably through N¹-acetylspermidine to putrescine. The peak of putrescine acetylation observed by us at 4 hours may also reflect a conversion of putrescine, via acetylputrescine to GABA. The interconversion hypothesis is supported by the fact that putrescine levels remain essentially stable in spite of a significant depletion of spermidine and spermine. In addition, there is a decrease in putrescine and spermidine acetylation at 8 hours, which coincides with the increase in ODC activity and the increase towards control levels of GAD activity (Pajunen et al., 1979). Such inverse correlations suggest a mechanism for replenishment of polyamines once GAD activity returns to control levels.     

Observations of the fluorometric assay of histamine, spermidine and spermine in brain

The separation of histamine, spermidine and spermine in brain extracts by cation-exchange chromatography on Bio-Rex 63 is described. The tissue extraction has been investigated and trichloroacetic acid has been shown to be superior to perchloric acid. Incubation at 25°C has been shown to be an essential requirement if reproducible results are to be produced when forming histamine or spermidine o-phthaldialdehyde fluorophores and a modified procedure for forming spermine phenylacetaldehyde fluorophores is described.     

Influence of polyamine architecture on the transport and topoisomerase II inhibitory properties of polyamine DNA-intercalator conjugates

An efficient five-step synthetic method was developed to access a series of spermine derivatives containing appended acridine, anthracene, and 7-chloroquinoline motifs. The derivatives were composed of a spermine fragment covalently tethered at its N4 and N9 positions to an aromatic nucleus via an aliphatic chain (e.g., 8: acridine -[C4 aliphatic tether]-spermine-[C4 aliphatic tether]-acridine). The distance separating the spermine and aromatic nuclei was altered via different tethers composed of four or five methylene units. These bis ligands (8, 9, 12, and 13) were shown to inhibit human DNA topoisomerase II (topo II) activity at 5 microM. Enzymatic activity was assessed as the ability to unknot (decatenate) and cleave kinetoplast DNA (kDNA). Polyamine conjugation did not disrupt the ability of the acridine-spermine conjugates 8 and 9 to inhibit topo II activity as compared with the 9-aminoacridine and 9-(N-butyl)aminoacridine controls (at 5 microM). The parent polyamines, spermine (5 microM) and spermidine (10 microM), had little effect on topo II activity. In general, the bis-substituted spermine derivatives (8, 9, 12, and 13) were more efficient topo II inhibitors at 5 microM than their monosubstituted spermidine counterparts (22-25) at 10 microM. Within the bisintercalator spermine series, insertion of an additional methylene unit (i.e., C5 tethers) increased potency 2-fold (8, bis-C4-acridine, 47 h IC(50) = 40 microM; 9, bis-C5-acridine, IC(50) = 17 microM). Comparison of the bis- and monoacridine spermine motifs (8 and 17) revealed a 4-fold increase in potency for the latter architecture (94 h IC(50) for 8, 74 microM; for 17, 17 microM). In general the bisintercalators (8, 9, 12, and 13) behaved as cytostatic agents, while the monosubstituted acridine and anthracene derivatives (22-25) were cytotoxic. Anthracene-containing conjugates were generally more toxic than their acridine counterparts in an L1210 (murine leukemia) cell assay. Of the conjugates tested the (monointercalator)-spermine motif (e.g., 17) had the highest affinity for the L1210 polyamine transporter as revealed by spermidine protection experiments.     

Amino acid/spermine conjugates: polyamine amides as potent spermidine uptake inhibitors

In this paper we describe the synthesis and characterization of a series of simple spermine/amino acid conjugates, some of which potently inhibit the uptake of spermidine into MDA-MB-231 breast cancer cells. The presence of an amide in the functionalized polyamine appeared to add to the affinity for the polyamine transporter. The extensive biological characterization of an especially potent analogue from this series, the Lys-Spm conjugate (31), showed this molecule will be an extremely useful tool for use in polyamine research. It was shown that the use of 31 in combination with DFMO led to a cytostatic growth inhibition of a variety of cancer cells, even when used in the presence of an extracellular source of transportable spermidine. It was furthermore shown that this combination effectively reduced the cellular levels of putrescine and spermidine while not affecting the levels of spermine. These facts together with the nontoxic nature of 31 make it a novel lead for further anticancer development.     

Synthesis and biological evaluation of S-adenosyl-1,12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase

As part of a continuing search for specific inhibitors of the enzymes involved in polyamine biosynthesis, we have designed and synthesized a multisubstrate adduct inhibitor, S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDATAD), in which critical portions of the nucleophilic aminopropyl acceptor are covalently linked to critical portions of the electrophilic aminopropyl donor to form a potent and specific inhibitor of spermine synthase. In addition, the corresponding desamino analogue which was designed to lack activity against spermine synthase on the basis of substrate structure-activity data has been synthesized as a control. Preliminary biological results demonstrate that AdoDATAD is a potent and specific inhibitor of mammalian spermine synthase in vitro, while being almost completely devoid of inhibitory activity toward the closely related aminopropyltransferase spermidine synthase. The desamino analogue, as predicted, showed no inhibitory activity against either enzyme. AdoDATAD represents an important addition to the arsenal of specific enzyme inhibitors available for blockade of the polyamine biosynthetic pathway at specific sites.     

Transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N1-acetyltransferase show enhanced sensitivity to the polyamine analog, N1, N11-diethylnorspermine

We have recently generated transgenic mice in which polyamine catabolism has been activated by overexpressing the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT). These animals have now been tested for their sensitivity to the polyamine analog N1,N11-diethylnorspermine (DENSPM), which is currently undergoing Phase I clinical trial. The analog is known for its ability to potently induce SSAT. Treatment for 4 days with a daily dose (125 mg/kg) of analog caused profound changes in polyamine metabolism in the transgenic animals. Liver SSAT activity was increased by approximately 800-fold while hepatic mRNA increased only 4-fold. Putrescine pools increased while spermidine and spermine pools nearly disappeared, resulting in a compensatory increase in ornithine decarboxylase activity. Similar but less profound changes were also seen in other tissues (spleen, intestine, and skin). This treatment also resulted in a 50% mortality in the transgenic animals, with no apparent histopathological changes in major organs. Nontransgenic animals exhibited no toxicity, and tissue SSAT activity was unchanged or only moderately increased. Polyamine pools were only slightly altered. Greater analog toxicity in transgenic animals may be attributable to higher tissue levels of DENSPM facilitated by SSAT-mediated decreases in spermidine and spermine. To further confirm the enhanced sensitivity of the transgenic animals to the analog, groups of nontransgenic and transgenic animals were subjected to daily injections with DENSPM. On average, transgenic mice died approximately 3 days earlier than their nontransgenic litter-mates. The findings indicate a contributing role for SSAT in whole animal toxicity by SSAT-inducing polyamine analogs.     

The actions of spermidine and spermine on the central nervous system.

The effects of intraventricular injections of spermidine and spermine in mice and rabbits are described. These polyamines initially produced sedation and hypothermia. In rabbits but not in mice tachypnoea was noted. In addition, in both species there was anorexia and adipsia lasting more than 24 hr. After several hours, animals which had been given spermine in particular, became extremely hyperexcitable. Ultimately convulsions, which were sometimes lethal, were produced. The administration of spermidine or spermine resulted within a few days in the development of a quadriplegic paralysis. Subsequent histological examination revealed that paralysed animals exhibited a characteristic pattern of focal encephalomalacic lesions. Invariably, severe lesions involving the pyramidal tracts were found in the ventral medulla just under the leptomeninges. Lesions also often occurred in the cervical cord being situated superficially just under the pia mater.A large dose of putrescine given to mice by intraventricular injection produced convulsions or paralysis and a concomitant increase in brain spermidine and spermine content.     

Role of polyamines in experience-dependent brain plasticity

Enriched experience increases brain growth, neuronal differentiation and learning abilities. Polyamines are modulators of growth and differentiation. We studied the effect of difluoromethylornithine (DFMO, an inhibitor of putrescine synthesis) on brain growth of rats exposed either to a complex or an impoverished environment. In both environmental conditions, DFMO decreased cortical putrescine by 50% and increased spermine by 13%; spermidine remained constant. Cortical RNA was not affected significantly by DFMO but DNA was decreased exclusively in rats exposed to the impoverished environment. Environmental complexity increased cortical weight, RNA and spermidine content. These differences were larger in DFMO-injected rats than in saline controls. Since stimulants such as amphetamines also enhance the environmental effects it was conceivable that DFMO might act as a stimulant. We have measured the effect of DFMO on rats' exploratory activity and found it decreased by the drug. Therefore the enhancing effect of DFMO cannot be explained by its behavioral activity. We propose that DFMO enhances the experience-dependent brain plasticity by facilitating differentiation of neurons.     

Shape change of blood platelets brought about by myelin basic protein and other basic polypeptides

Summary Basic proteins and polypeptides (BPP) such as myelin basic protein (MBP), polyornithine (M.W. 40,000), polylysine and protamine, which are known to cause neuronal depolarization in the central nervous system, induced a shape change reaction in blood platelets of various species, including man. This reaction was not accompanied by platelet aggregation or marked alterations of 5-hydroxytryptamine release. Cyclic nucleotide levels were also unchanged. The shape change induced by polyornithine was inhibited by heparin but not by antagonists of 5HT, catecholamines or -aminobutyric acid, substances which are known to have no effect on the MBP-induced neuronal depolarization. Other basic substances, e. g. low molecular weight polyornithine (M.W. 4,000), cytochrome c, spermine and spermidine, did not induce either platelet shape change or (as shown before) neuronal depolarization.It is concluded, that 1) the shape change reaction of platelets seems to be a sensitive and simple means of detecting those BPP which induce functional changes in mammalian cells and 2) the use of platelets as models for neurons can be extended to include the action of BPP on the plasma membranes.     

Chemotherapeutic choice of ranimustine or nimustine on the basis of regional polyamine levels in rat brain

The aim of the present study was to improve the chemotherapeutic efficacy of anticancer drugs by choosing on the basis of the polyamine level induced by the drug in each host cancer-bearing tissue. We propose an "organ-specific therapy" in the article. The polyamines, putrescine, spermidine and spermine are strongly associated with tumor cell growth. The effects of ranimustine (MCNU) and nimustine (ACNU) on body weight, regional brain weights and concentrations of putrescine, spermidine and spermine in the cerebellum, hippocampus, corpus striatum, cortex, combined thalamus and hypothalamus and diencephalon of the brain were examined in rats. MCNU and ACNU reduced spermidine and spermine in the corpus striatum, and spermine in the diencephalon, but increased putrescine in the corpus striatum and combined thalamus and hypothalamus. These results indicate that both MCNU and ACNU are suitable for the treatment of cancers of the corpus striatum, but ACNU is not suitable for cancers of the corpus striatum, thalamus and hypothalamus.     

Uptake and disposition of putrescine, spermidine, and spermine by rabbit lungs

The pulmonary uptake and accumulation of the three polyamines in intact, ventilated, and perfused rabbit lungs was investigated. Lungs were perfused using Krebs-Ringer bicarbonate buffer with albumin in which putrescine, spermidine, or spermine were included at an initial concentration of 10(-3), 10(-2), 10(-1), 5, 10, or 20 mM. At a 5 mM concentration of spermidine and spermine, the uptake by isolated lungs reached a steady state equilibrium in 20-30 min of perfusion. This did not occur for putrescine, which showed linear uptake throughout the entire period of the 60-min perfusion. The lung uptake of putrescine for all perfusate concentrations was greater than that of spermidine or spermine, but all three showed concentration-dependent linear uptake. In the presence of harmaline (1 mM) and ouabain (1 mM), isolated perfused rabbit lungs showed a decrease in uptake of putrescine although no effect was seen for spermidine and spermine. Perfusate containing decreased sodium showed no effect on putrescine uptake by isolated rabbit lungs, but there was a significant increase in the uptake of spermidine and spermine. Significant uptake of all three polyamines was also observed when incubated separately with rabbit lung slices for 60 min. HPLC analysis of lung, the perfusate samples, lung slices, and the incubation medium after a 60-min incubation did not indicate the presence of metabolites of these polyamines. Likewise, the analysis of the lung homogenate incubated with polyamines did not show any metabolites confirming the absence of detectable pulmonary metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of some polyamines on putative behavioural indices of mesolimbic versus striatal dopaminergic function

The presence of polyamines in the brain, together with the previous reports of a structural similarity with neuroleptics, has led to the hypothesis that polyamines may have a modulatory role in the control of cerebral dopamine function. In this study, the effects of two polyamines, spermine and spermidine, were therefore tested on indices of dopamine-mediated behaviour in rats and mice. Spermine and spermidine caused a dose-dependent inhibition of mouse spontaneous climbing behaviour and wheel running at doses between 5 and 40 mg/kg IP but failed to cause catalepsy in the rat or to antagonise the stereotyped behaviour induced by apomorphine. When polyamines were given by intracerebral injection a similar regional selectivity was seen. Both spermine and spermidine (5–20 μg) when given bilaterally into the nucleus accumbens inhibited the hyperactivity caused by amphetamine injected into the same nucleus. However, when injected into the rat corpus striatum, neither polyamine was able to initiate any asymmetry or circling either spontaneously or after apomorphine injection IP. These results indicated a selective action of polyamines on mesolimbic dopamine behaviour. Possible implications for the understanding of psychosis and future work are suggested.     

Effects of spermine on the relaxation response of rat detrusor smooth muscles

Endogenous polyamines are known to influence excitation-contraction coupling in smooth muscle. This study was designed to determine the effects of the polyamines spermine, spermidine, and putrescine on the contractile responses of rat detrusor smooth muscles. Under physiological conditions, isometric tension recordings were made of isolated bladder strips from excised rat bladder. The effects of spermine, spermidine, and putrescine (1 mM each) on the bladder contractions induced by various agents, i.e., acetylcholine, bethanechol, high-K, and tetraethylammonium (TEA) were measured. A conventional patch clamp technique was used in whole cell mode with single smooth muscle cells of rat bladder. Calcium channel currents were recorded to determine the effects of spermine on channel activities. Polyamines elicited a concentration-dependent relaxations on the contractile agents induced contractures. Spermine showed the most potent relaxation effect of the polyamines examined, and relaxed the contractions induced by the agents. Calcium channel activities were significantly reduced by adding 1 mM spermine to the bath. We concluded that spermine exerts a potent relaxant effect on rat bladder smooth muscle, and this effect appears to be mediated by calcium channel antagonism.