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At least 19 recordsLinked to original sources
PubMed1987

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.

Adenosylmethionine Decarboxylase↗
PubMed1993

Enhancement of the spermidine uptake system and lethal effects of spermidine overaccumulation in ornithine decarboxylase-overproducing L1210 cells under hyposmotic stress.

The D-R cell subline, an ornithine decarboxylase-overproducing variant of L1210 mouse leukemia cells, shows a growth advantage at low osmolality due to its high putrescine content. We tested the ability of spermidine to fulfill the role of putrescine under hyposmotic conditions. Although spermidine (1-30 microM) had no effect on growth under normosmotic conditions (325 mosm/kg), it was strongly inhibitory to D-R cell proliferation at 150 mosm/kg in a concentration-dependent manner. Hypotonic shock greatly increased the rate of spermidine uptake in D-R cells. The increased spermidine content enhanced total putrescine synthesis through a large induction of cytosolic spermidine/spermine N1-acetyltransferase activity but also promoted the excretion of most of the putrescine synthesized by the cells. Delaying the addition of spermidine until 24 h after hypotonic shock resulted in a much sharper decrease in D-R cell viability and strongly depressed polyamine contents. These lethal effects occurred between 8 and 24 h after spermidine addition and followed a dramatic increase in the rate and extent of spermidine accumulation which overrode the metabolic capacity of the N1-acetyltransferase/polyamine oxidase (PAO) pathway. Inhibition of PAO partly reversed the effect of spermidine on growth when the polyamine was added at the time of hypotonic shock, but not 24 h later. Similar experiments performed with alpha-methylspermidine, a metabolically resistant analog, which can completely fulfill cellular requirements for spermidine in normosmotic media, suggested that the lethal effect of a delayed spermidine addition is caused predominantly by excessive accumulation with a minor contribution resulting from stress due to polyamine oxidase activity. In contrast, in hypotonically shocked L1210 cells, spermidine stimulated cell proliferation (albeit less effectively than putrescine), there was no lethal effect of a delayed addition of alpha-methylspermidine, and there was no time-dependent increase in the rate of alpha-methylspermidine uptake. Thus, the spermidine transport system is strongly enhanced by hyposmotic shock in D-R cells, which can result in extensive cell death from overaccumulation of the polyamine and, to a lesser extent, from stress related to the PAO-catalyzed degradation of N1-acetylspermidine. The absence of these effects in parental L1210 cells indicates that the acquisition of an ornithine decarboxylase-overproducing phenotype also involves major modifications in the expression and/or regulation of polyamine transport.

Animals↗
PubMed2000

Spermidine acetyltransferase is required to prevent spermidine toxicity at low temperatures in Escherichia coli.

Polyamines are required for optimal growth in most cells; however, polyamine accumulation leads to inhibition of cellular growth. To reduce intracellular polyamine levels, spermidine is monoacetylated in both prokaryotes and eukaryotes. In Escherichia coli, the speG gene encodes the spermidine acetyltransferase, which transfers the acetyl group to either the N-1 or N-8 position. In addition to polyamine accumulation, stress conditions, such as cold shock, cause an increase in the level of spermidine acetylation, suggesting an adaptive role for reduced polyamine levels under stressful growth conditions. The effect of spermidine accumulation on the growth of E. coli at low temperature was examined using a speG mutant. At 37 degrees C, growth of the speG mutant was normal in the presence of 0. 5 or 1 mM spermidine. However, following a shift to 7 degrees C, the addition of 0.5 or 1 mM spermidine resulted in inhibition of cellular growth or cell lysis, respectively. Furthermore, at 7 degrees C, spermidine accumulation resulted in a decrease in total protein synthesis accompanied by an increase in the synthesis of the major cold shock proteins CspA, CspB, and CspG. However, the addition of 50 mM Mg(2+) restored growth and protein synthesis in the presence of 0.5 mM spermidine. The results indicate that the level of spermidine acetylation increases at low temperature to prevent spermidine toxicity. The data suggest that the excess spermidine replaces the ribosome-bound Mg(2+), resulting in ribosome inactivation at low temperatures.

Acetyltransferases↗
PubMed1980

Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase.

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.

Animals↗
PubMed1995

Decrease in cell viability due to the accumulation of spermidine in spermidine acetyltransferase-deficient mutant of Escherichia coli.

Physiological functions of spermidine acetyltransferase in Escherichia coli have been studied using the spermidine acetyltransferase (speG) gene-deficient mutant CAG2242 and the cloned speG gene. The growth of E. coli CAG2242 in the defined M9 medium was normal in the presence and absence of 0.5mM spermidine. However, cell viability of E. coli CAG2242 at 48 h after the onset of growth decreased greatly by the addition of 0.5 mM spermidine. The amount of spermidine accumulated in the cells was approximately 3-fold that in the cells grown in the absence of spermidine. Transformation of the cloned speG gene to E. coli CAG2242 recovered the cell viability. Decreased in cell viability of E. coli CAG2242 was observed even when 0.5mM spermidine was added at 24 h after the onset of growth. The results indicate that accumulated spermidine functions at the late stationary phase of growth. The accumulation of spermidine caused a decrease in protein synthesis but not in DNA and RNA synthesis at 28 h after the onset of growth. The synthesis of several kinds of proteins was particularly inhibited. They included ribosome modulation factor and OmpC protein. Since the ribosome modulation factor is essential for cell viability at the stationary phase of growth (Yamagishi, M., Matsushima, H., Wada, A., Sakagami, M., Fujita, N., and Ishihama, A. (1993) EMBO J. 12, 625-630), the decrease in the protein was thought to be one of the reasons for the decrease in cell viability. The decrease in the ribosome modulation factor mainly occurred at the translational level.

Acetyltransferases↗
PubMed2003

Spermidine but not spermine is essential for hypusine biosynthesis and growth in Saccharomyces cerevisiae: spermine is converted to spermidine in vivo by the FMS1-amine oxidase.

In our earlier work we showed that either spermidine or spermine could support the growth of spe2Delta or spe3Delta polyamine-requiring mutants, but it was unclear whether the cells had a specific requirement for either of these amines. In the current work, we demonstrate that spermidine is specifically required for the growth of Saccharomyces cerevisiae. We were able to show this specificity by using a spe3Delta fms1Delta mutant that lacked both spermidine synthase and the FMS1-encoded amine oxidase that oxidizes spermine to spermidine. The polyamine requirement for the growth of this double mutant could only be satisfied by spermidine; i.e., spermine was not effective because it cannot be oxidized to spermidine in the absence of the FMS1 gene. We also showed that at least one of the reasons for the absolute requirement for spermidine for growth is the specificity of its function as a necessary substrate for the hypusine modification of eIF5A. Spermine itself cannot be used for the hypusine modification, unless it is oxidized to spermidine by the Fms1 amine oxidase. We have quantified the conversion of spermine in vivo and have shown that this conversion is markedly increased in a strain overexpressing the Fms1 protein. We have also shown this conversion in enzymatic studies by using the purified amine oxidase from yeast.

Base Sequence↗
PubMed2005

Control of spermidine and spermine levels in rat tissues by trans-4-methylcyclohexylamine, a spermidine-synthase inhibitor.

In rat tissues, a decrease in spermidine, accompanied by an increase in spermine was induced by the oral administration (once daily for either 1 week or 1 month) of trans-4-methylcyclohexylamine (4MCHA), a spermidine synthase inhibitor. This is similar to the changes observed in polyamine content when cell growth is arrested. The body-weight gain of the rats tended to decrease with increasing doses of 4MCHA. A decrease in spermidine, combined with a moderate increase in spermine, was observed dose-dependently in all of the tissues tested, with a relatively fast clearance of 4MCHA. Manipulating the polyamine content of tissues, by daily administration of 100 mumol 4MCHA for 1 week, made it possible to estimate the effects of simultaneously added spermidine or spermine on endogenous polyamine contents. The altered polyamine levels, obtained after daily administration for 1 week, were maintained during the extended 1-month period, with growth-dependent alteration. The results show it is possible to produce experimental rats with a higher spermine:spermidine ratio than control rats to investigate the physiological significance of spermidine downregulation and spermine upregulation in vivo.

Alkenes↗
PubMed1997

Spermidine biosynthesis in Saccharomyces cerevisae: polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase).

The Saccharomyces cerevisiae SPE3 gene, coding for spermidine synthase, was cloned, sequenced, and localized on the right arm of chromosome XVI. The deduced amino acid sequence has a high similarity to mammalian spermidine synthases, and has putative S-adenosylmethionine binding motifs. To investigate the effect of total loss of the SPE3 gene, we constructed a null mutant of this gene, spe3delta, which has no spermidine synthase activity and has an absolute requirement for spermidine or spermine for the growth. This requirement is satisfied by a very low concentration of spermidine (10(-8) M) or a higher concentration of spermine (10(-6) M).

Amino Acid Sequence↗
PubMed1986

Increased activity of spermidine N1-acetyltransferase in the adrenal cortex of rats following administration of exogenous spermidine, carbamylcholine, 2-deoxyglucose and dopamine agonists.

The activity of N1-acetyltransferase was increased in the dissected adrenal cortex of the rat following a single administration of spermidine. The activity was maximal 6-8 h after the onset of treatment. The increase in enzyme activity was abolished when the rats were given cycloheximide 2 h after spermidine; this suggests that increased activity results from an augmentation in the synthesis of the enzyme. Adrenocortical spermidine N1-acetyltransferase was also induced by carbamylcholine, 2-deoxyglucose, apomorphine and piribedil, drugs that are known to cause induction of ornithine decarboxylase in that organ. Hypophysectomized rats showed reduced activity of spermidine N1-acetyltransferase when compared to sham-operated controls, and carbamylcholine no longer elicited an increase in enzyme activity in such animals. Adrenocortical spermidine N1-acetyltransferase activity of hypophysectomized rats is induced by corticotropin (ACTH). These results suggest a hormonal control over the activity of the enzyme in the adrenal cortex with ACTH acting as a mediator.

Acetyltransferases↗
PubMed1995

Aminooxy analogues of spermidine evidence the divergent roles of the charged amino nitrogens in the cellular physiology of spermidine.

Two recently devised spermidine analogues, N-[2-aminooxyethyl]-1,4-diaminobutane (AOEPU) and 1-aminooxy-3-N-[3-aminopropyl]-aminopropane (APAPA), were used to elucidate the role of charge distribution in the functions of spermidine in cultured baby hamster kidney cells. The drugs did not affect cell proliferation nor did they relieve the growth-arrest but potentiated the metabolic disturbances caused by DL-alpha-difluoromethyl-ornithine (DFMO). Neither drug affected spermidine uptake but both competed with putrescine uptake. Neither drug could replace spermidine in the control of S-adenosylmethionine decarboxylase and accumulation of the reaction product. APAPA prevented spermine synthesis and showed that modest putrescine synthesis take place in the presence of DFMO. AOEPU, but not APAPA, interfered with cellular constituents resulting in enzymatic formation, accumulation and excretion to culture medium of UV-absorbing catabolites.

Animals↗
PubMed1984

Studies of DNA organization in hydrated spermidine-condensed DNA toruses and spermidine-DNA fibres.

We have investigated in vitro spermidine-condensed DNA preparations by both biochemical and freeze-etch electron microscopic approaches. These studies lead us to the conclusion that the reversibly condensed preparations, qualitatively described by Manning's counterion condensation theory, contain disk-like torus structures largely comprised of unidirectional, circumferentially wrapped DNA. Stereoscopic measurements on stereomicrographs of DNA torus and fibre objects have demonstrated the feasibility of directly measuring DNA writhe or, for that matter, mapping any secondary, tertiary or quaternary structure features of a hydrated macromolecular array in which the features can be differentially highlighted by low replica metal shadow levels.

Animals↗
PubMed1994

Cellular pharmacology of N1- and N8-aziridinyl analogues of spermidine.

We have previously described the synthesis and cytotoxic properties of 2 polyamine analogues in which either the N1- or N8-amino group of spermidine was replaced by the alkylating moiety, aziridine. However, the mechanisms by which these aziridinyl analogues of spermidine inhibit cell growth remain unknown. As a result, we have studied: (a) the effect of pretreatment with difluoromethyl ornithine (DFMO) and coincubation with exogenous spermidine on cytotoxicity induced by the aziridinyl spermidines; (b) the reversibility of the cytotoxicity induced by the aziridinyl spermidines; (c) the accumulation of N1- and N8-aziridinyl spermidine by cells and the effects of DFMO on this process; and (d) the impact of N1- and N8-aziridinyl spermidine on cellular polyamine pools and on cellular accumulation of spermidine. The cytotoxicity induced by these 2 aziridinyl derivatives of spermidine [concentration required to inhibit cell growth or incorporation of radiolabeled precursor into trichloroacetic acid-precipitable material by 50% (IC50) N1 = 0.2 microM, IC50 N8 = 0.4 microM)] was potentiated by pretreatment of L1210 cells for 24 h with 100 microM DFMO (IC50 N1 = 0.05 microM, IC50 N8 = 0.15 microM) and was prevented by coincubation with 3.7 microM spermidine (IC50 N1 = 1.1 microM, IC50 N8 = 2.4 microM). In contrast, similar pretreatment with DFMO or coincubation with spermidine had no effect on the cytotoxicity induced by the aziridine-containing alkylating agent, N,N',N"-triethylenethiophosphoramide (thiotepa) (IC50 = 2.4 microM). The cytotoxicity induced by 24-h incubation with either N1- or N8-aziridinyl spermidine was not altered by removal of those compounds and incubating treated cells in medium augmented with 3.7 microM spermidine. However, and as expected, similar maneuvers did not reverse the cell growth-inhibitory effect induced by 24-h incubation with 100 microM DFMO. Cellular accumulation of both N1- and N8-aziridinyl spermidine increased with increasing extracellular concentrations. N1-Aziridinyl spermidine was accumulated to a greater degree than was the N8-analogue, achieving up to 6-fold higher intracellular concentrations at the same extracellular concentration. Cellular accumulation of both aziridinyl compounds was greatly enhanced by 24-h pretreatment with DFMO. Both N1- and N8-aziridinyl spermidine inhibited the uptake of spermidine in a dose-dependent manner. The perturbation of polyamine biochemistry by the test compounds was characterized by their ability to deplete cellular putrescine, as well as spermidine and spermine. These results imply that the cytotoxic mechanism of the aziridinyl spermidine analogues is, to a great extent, dependent on their polyamine nature and may imply selectivity for rapidly growing and neoplastic cells.

Alkylating Agents↗
PubMed2006

Spermidine synthase is prominently expressed in the striatal patch compartment and in putative interneurones of the matrix compartment.

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.

Animals↗
PubMed1991

Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells.

The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28-fold) compared to that for spermidine (3.5-fold). Putrescine uptake was partially Na+ dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69-75 microM, whereas the estimated Km for putrescine uptake was only 5.36 microM. By contrast, spermidine inhibition of putrescine transport produced a non-linear Eadie-Scatchard plot suggesting that putrescine was taken up by a spermidine-sensitive and a spermidine-insensitive process. The estimated spermidine Ki for inhibition of the spermidine-sensitive process was 0.125 microM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1,12-di(N5-azido-2-nitrobenzoyl)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell-line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.

Affinity Labels↗