Effects of Ethyl and Benzyl Analogues of Spermine on Escherichia coli Peptidyltransferase Activity, Polyamine Transport, and Cellular Growth

Various ethyl and benzyl spermine analogues, including the anticancer agent N1,N12-bis(ethyl)spermine, were studied for their ability to affect the growth of cultured Escherichia coli cells, to inhibit [3H]putrescine and [3H]spermine uptake into cells, and to modulate the peptidyltransferase activity (EC 2. 3. 2. 12). Relative to other cell lines, growth of E. coli was uniquely insensitive to these analogues. Nevertheless, these analogues conferred similar modulation of in vitro protein synthesis and inhibition of [3H]putrescine and [3H]spermine uptake, as is seen in other cell types. Thus, both ethyl and benzyl analogues of spermine not only promote the formation and stabilization of the initiator ribosomal ternary complex, but they also have a sparing effect on the Mg2+ requirements. Also, in a complete cell-free protein-synthesizing system, these analogues at low concentrations stimulated peptide bond formation, whereas at higher concentrations, they inhibited the reaction. The ranking order for stimulation of peptide-bond formation by the analogues was N4,N9-dibenzylspermine > N4, N9-bis(ethyl)spermine congruent with N1-ethylspermine > N1, N12-bis(ethyl)spermine, whereas the order of analogue potency regarding the inhibitory effect was inverted, with inhibition constant values of 10, 3.1, 1.5, and 0.98 microM, respectively. Although the above analogues failed to interact with the putrescine-specific uptake system, they exhibited high affinity for the polyamine uptake system encoded by the potABCD operon. Despite this fact, none of the analogues could be internalized by the polyamine transport system, and therefore they could not influence the intracellular polyamine pools and growth of E. coli cells.     

Regulation of polyamine transport in Chinese hamster ovary cells

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with alpha-difluoromethylornithine in CHO cells or via polyamine starvation of CHODC- cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.     

Facilitation of the cellular uptake of a triplex-forming oligonucleotide by novel polyamine analogues: structure-activity relationships.

The inefficient uptake of oligodeoxynucleotides, including that of TFO, through the cell membrane is a limiting factor in developing gene therapy approaches for cancer and other diseases. To develop a new strategy for oligonucleotide delivery into the nucleus, we synthesized a series of novel polyamine analogues and examined their effects on the uptake of a 37-mer [32P]-labeled TFO, targeted to the promoter region of c-myc oncogene. We used MCF-7 breast cancer cells to investigate the efficacy of polyamines on the internalization of the TFO. The uptake of TFO was enhanced by complexing it with several unsubstituted polyamine analogues at 0. 1-5 microM concentrations, with up to 6-fold increase in TFO uptake in the presence of a hexamine, 1,21-diamino-4,9,13, 18-tetraazahenicosane (H2N(CH2)(3)NH(CH2)(4)NH(CH2)(3)NH(CH2)(4)NH(CH2)(3)NH(2) or 3-4-3-4-3). TFO uptake increased with the cationicity of the polyamines; however, bis(ethyl) substitution and structural features of the methylene bridging region had significant effects on TFO uptake. The majority of labeled TFO was recovered from the nuclear fraction containing genomic DNA. Electrophoretic mobility shift assay revealed enhanced binding of TFO to a target duplex containing promoter region sequence of c-myc oncogene. Treatment of MCF-7 cells with the TFO complexed with 0.5 microM 3-4-3-4-3 suppressed c-myc mRNA level by 65%, as determined by Northern blot analysis. These data indicate a novel approach to deliver oligodeoxynucleotides to the cell nucleus, and suppress the expression of target genes, and provide new insights into the mechanism of oligonucleotide transport in living cells.     

TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.     

Selectivity of polyamines on the stability of RNA-DNA hybrids containing phosphodiester and phosphorothioate oligodeoxyribonucleotides.

RNA-DNA hybrid stabilization is an important factor in the efficacy of oligonucleotide-based antisense gene therapy. We studied the ability of natural polyamines, putrescine, spermidine, and spermine, and a series of their structural analogues to stabilize RNA-DNA hybrids using melting temperature (Tm) measurements, circular dichroism (CD) spectroscopy, and the ethidium bromide (EB) displacement assay. Phosphodiester (PO) and phosphorothioate (PS) oligodeoxyribonucleotides (ODNs) (21-mer) targeted to the initiation codon region of c-myc mRNA and the corresponding complementary RNA oligomer were used for this study. In the absence of polyamines, the Tm values of RNA-PODNA and RNA-PSDNA helices were 41 +/- 1 and 35 +/- 1 degrees C, respectively, in 10 mM sodium cacodylate buffer. In the presence of a hexamine analogue of spermine at a concentration of 25 microM, the hybrids were stabilized with Tm values of 80 and 78 degrees C, for RNA-PODNA and RNA-PSDNA, respectively. The d(Tm)/d(log[polyamine]) values, representing the concentration-dependent stabilization of hybrid helices by polyamines, increased from 10 to 24 for both the RNA-PODNA and RNA-PSDNA helices. Bisethyl substitution of the primary amino groups of the polyamines reduced the hybrid stabilizing potential of the polyamines. Among the homologues of spermidine [H2N(CH2)3NH(CH2)nNH2, where n = 2-8; n = 4 for spermidine] and spermine [H)N(CH2)3NH(CH2)nNH(CH2)3NH2, where n = 2-8; n = 4 for spermine], spermidine and spermine were the most effective agents for stabilizing the hybrid helices. At a physiologically compatible concentration of 150 mM NaCl, the hybrid helix formed from PODNA was more stable than that formed from PSDNA in the presence of polyamines. CD spectroscopic studies showed that the hybrids were stabilized in a conformation close to A-DNA in the presence of polyamines. The relative binding affinity of the polyamine homologues for the hybrid helices, as measured by the EB displacement assay, followed the same order in which they stabilized the hybrids. These results are important in the antisense context and in the general context of polyamine-nucleic acid interactions, and suggest that pentamine and hexamine analogues of spermine might be useful in improving the efficacy of therapeutic ODNs.     

Inhibition of mammalian spermine synthase by N-alkylated-1,3-diaminopropane derivatives in vitro and in cultured rat hepatoma cells

A number of N-alkylated-1,3-diaminopropane derivatives [H2N-(CH2)3-NH-(CH2)nH, where n = 1-9] have been tested as potential inhibitors of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase. Among the compounds described in this paper, the most potent competitive inhibitor of spermine synthase, with respect to spermidine, is N-butyl-1,3-diaminopropane with Ki values of 11.9 nM and 10.4 nM for the HTC cell and bovine spleen enzymes respectively. Inhibition of spermine synthase by this alkylated amine is selective since spermidine synthase activity is not affected up to 100 microM N-butyl-1,3-diaminopropane at a range of 5-200 microM putrescine. Added to the culture medium of growing HTC cells, N-butyl-1,3-diaminopropane causes the expected changes in the polyamine levels with a marked decrease of spermine and an increase of spermidine. Under these conditions cell growth continues unabated. Such N-alkylated-1,3-diaminopropane derivatives may have considerable potential as tools for studying the role of polyamines and in particular the functions of spermine in cell multiplication and differentiation.     

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

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

Regulation of spermidine/spermine N1-acetyltransferase in L6 cells by polyamines and related compounds.

Exposure of rat L6 cells in culture to exogenous polyamines led to a very large increase in the activity of spermidine/spermine N1-acetyltransferase. Spermine was more potent than spermidine in bringing about this increase, but in both cases the elevated acetyltransferase activity increased the cellular conversion of spermidine into putrescine. The N1-acetyltransferase turned over very rapidly in the L6 cells, with a half-life of 9 min after spermidine and 18 min after spermine. A wide variety of synthetic polyamine analogues also brought about a substantial induction of spermidine/spermine N1-acetyltransferase activity. These included sym-norspermidine, sym-norspermine, sym-homospermidine, N4-substituted spermidine derivatives, 1,3,6-triaminohexane, 1,4,7-triaminoheptane and deoxyspergualin, which were comparable with spermidine in their potency, and N1N8-bis(ethyl)spermidine, N1N9-bis(ethyl)homospermidine, methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone) and 1,1'-[(methylethanediylidene)dinitrilo]bis(3-amino-guanidine ), which were even more active than spermidine. It is suggested that these polyamine analogues may bring about a decrease in cellular polyamines not only by inhibiting biosynthesis but by stimulating the degradation of spermidine into putrescine.     

Evaluation method for polyamine uptake by N 1-dansylspermine

Polyamine uptake by the polyamine transport system (PTS) in HTC cells was studied without the use of radioisotope-labeled polyamines. N¹-Dansylspermine (DNS343) was selected as a candidate probe to examine the PTS. DNS343 was incorporated into HTC cells, and its distribution in the cells was confirmed by fluorescence microscopy. The incorporation of DNS343 via PTS was confirmed by a competition study with bis(3-aminopropyl)amine, which is incorporated into cells via the PTS. In addition, the temperature dependency of DNS343 uptake and studies with inhibitors of ornithine decarboxylase and proteoglycan synthesis supported the use of DNS343 as a fluorescent probe for the PTS. The kinetics studies for HTC cells treated with or without an ornithine decarboxylase inhibitor indicated that DNS343 uptake was saturable and that the apparent Km values for the PTS were approximately 1.5 μM in both types of cells at 37°C. Thus, we developed an assay method for the PTS by high-performance liquid-chromatography with DNS343. The inhibitory effect of polyamine analogs and related compounds on DNS343 uptake was then examined and discussed.     

Altered spermidine/spermine N1-acetyltransferase activity as a mechanism of cellular resistance to bis(ethyl)polyamine analogues

To develop a model system to investigate mechanisms of antiproliferative action of bis(ethyl)polyamine analogues, intermittent analogue treatments followed by recovery periods in drug-free medium were used to select an N(1), N(12)-bis(ethyl)spermine-resistant derivative of the Chinese hamster ovary cell line C55.7. The resulting C55.7Res line was at least 10-fold resistant to N(1),N(12)-bis(ethyl)spermine and N(1), N(11)-bis(ethyl)norspermine. The stability of the resistance in the absence of selection pressure was >/=9 months, indicating that a heritable genotypic change was responsible for the resistance phenotype. Polyamine transport alterations and multi-drug resistance were eliminated as causes of the resistance. Spermidine/spermine N(1)-acetyltransferase (SSAT) activity and regulation were altered in C55.7Res cells as basal activity was decreased, and no activity induction resulted from exposure to analogue concentrations, which caused 300-fold enzyme induction in parental cells. SSAT mRNA levels in the absence and presence of analogue were unchanged, but no SSAT protein was detected in C55.7Res cells. A point mutation, which results in the change leucine156 (a fully conserved residue) to phenylalanine, was identified in the C55.7Res SSAT cDNA. Expression of wtSSAT activity in C55.7Res cells restored sensitivity to bis(ethyl)polyamines. These results provided definitive evidence that SSAT activity is a critical target of the cytotoxic action of these analogues.     

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

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

Studies on the interaction of homologues of spermine with deoxyribonucleic acid and with bacterial protoplasts

Four homologues of the naturally occurring polyamine spermine, of the type H(2)N.[CH(2)](3).NH.[CH(2)] (n).NH.[CH(2)](3).NH(2) where n=2, 3, 5 and 6, have been synthesized. Their ability to stabilize Escherichia coli protoplasts against osmotic lysis was compared with that of spermine. All homologues were approximately as effective as spermine. The effect of low concentrations of the homologues on the T(m) of calf thymus DNA and of Aerobacter aerogenes DNA in 0.03m-sodium chloride-1mm-potassium dimethylglutarate buffer, pH6.2, was tested. The increase in T(m) for a given concentration of amine was found to be n=5>n=4 and n=6> n=3>n=2. When calf thymus DNA in 0.15m-sodium chloride-15mm-sodium citrate was used spermine gave the highest increase in T(m). It is concluded that the stabilization of E. coli protoplasts by tetra-amines is a non-specific effect independent of chain length, whereas the elevation of T(m) of DNA is a more specific effect which depends on chain length.     

Inhibitors of polyamine metabolism: Review article

Summary. 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 -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.     

Tolerance to Putrescine Toxicity in Chinese Hamster Ovary Cells Is Associated with Altered Uptake and Export

When Chinese hamster ovary (CHO) cells were cultured with low concentrations of putrescine (< 5 mM) their cell cycle time increased significantly and a fraction of the cells died. A cell line tolerant to the cytotoxic and growth inhibitory effects of millimolar concentrations of putrescine was developed by growing CHO cells over many months in increasing concentrations of the polyamine. A putrescine-tolerant cell line was obtained which was capable of growing in concentrations up to 25 mMputrescine and displayed growth and cell division rates similar to the original untreated/parental CHO cells. The tolerant cells grown in putrescine displayed relatively high intracellular putrescine yet the cell-associated putrescine concentration was estimated to be 10-fold less than the culture medium level. This high concentration of cellular putrescine diminished within 60 min when the cells were changed to non-putrescine-containing media. The putrescine-tolerant phenotype was further characterized in regards to the mechanisms involved in putrescine uptake, efflux, and biosynthesis. The parental and tolerant cell lines had similar or identical levels of cellular spermidine and spermine and no differences in the acetylated polyamine pools or diamine oxidase activity. The activity of ornithine decarboxylase was also similar in the two cell lines in both the presence and the absence of ornithine. The tolerant cells, however, had a decreased uptake rate for putrescine. The tolerant cell line also showed a greatly enhanced ability to export putrescine, especially when treated with ornithine, suggesting that an upregulated polyamine export system may be present in the tolerant cells which could be responsible for the increased cell survival in high putrescine concentrations. The data are discussed in regard to the potential for identifying the transport protein(s) responsible for the maintenance of nontoxic intracellular concentrations of putrescine in a tolerant cell line grown in putrescine.     

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.     

Structure-activity investigations of polyamine-anthracene conjugates and their uptake via the polyamine transporter

Summary. A series of polyamine conjugates were synthesized and evaluated for their ability to target the polyamine transporter (PAT) in two Chinese hamster ovary (CHO) cell lines (PAT-active CHO and PAT-inactive CHOMG). This systematic study identified salient features of the polyamine architecture required to target and enter cells via the PAT. Indeed, the separation of charges, the degree of N-alkylation, and the spacer unit connecting the N¹-terminus to the appended cytotoxic component (anthracene) were found to be key contributors to optimal delivery via the PAT. Using the CHO screen, the homospermidine motif (e.g., 4,4-triamine) was identified as a polyamine vector, which could enable the selective import of large N¹-substituents (i.e., naphthylmethyl, anthracenylmethyl and pyrenylmethyl), which were cytotoxic to cells. The cell selectivity of this approach was demonstrated in B-16 murine melanoma cells and normal melanocytes (Mel-A). Three polyamine areas (recognition and transport, vesicle sequestration and polyamine-target interactions) were identified for future research.     

Characteristics of the polyamine transporter TPO1 and regulation of its activity and cellular localization by phosphorylation

The subcellular localization of the polyamine transporter TPO1 of Saccharomyces cerevisiae was determined by sucrose gradient centrifugation and indirect immunofluorescence microscopy. When expressed from a multi-copy vector, TPO1 was located mainly on the plasma membrane, but with some localization on the vacuolar membrane. Polyamine transport by TPO1 was dependent on pH. Uptake of spermidine and spermine occurred at alkaline pH (pH 8.0), whereas inhibition of spermidine uptake, but not spermine uptake, was observed at acidic pH (pH 5.0). This suggests that TPO1 catalyzes polyamine excretion at acidic pH, similar to the PotE transporter in Escherichia coli. Paraquat, a polyamine analogue, was excreted by TPO1 at a rate comparable with the excretion of spermidine (deduced from the inhibition of spermidine uptake) at pH 5.0. However, excretion of preloaded radiolabeled spermidine and spermine was not observed in intact cells, suggesting that preloaded spermidine (or spermine) exists mainly as spermidine (or spermine)-ribosome complex in cells. The transport activity of TPO1 was enhanced through phosphorylation at Ser19 by protein kinase C and at Thr52 by casein kinase 1. Sorting of TPO1 from the endoplasmic reticulum to the plasma membrane was enhanced through phosphorylation at Ser342 by cAMP-dependent protein kinases 1 and 2.     

Spermine inhibition of monocyte activation and inflammation.

The innate immune system functions as a defensive front line against pathogenic invasion, but the proinflammatory products of activated monocytes and macrophages (e.g., TNF and NO) can also injure normal cells. Anti-inflammatory mediators restrain the innate immune response and prevent excessive collateral tissue damage. Spermine, a ubiquitous biogenic polyamine, specifically and reversibly suppresses the synthesis of monocyte proinflammatory cytokines. This may provide a counterregulatory mechanism to restrain monocyte activation in injured or infected tissues and in tumors where spermine levels are significantly increased. Here we show that monocyte spermine uptake was significantly increased following lipopolysaccharide stimulation. The polyamine analogue 1, 4-bis(3-aminopropyl)-piperazine (BAP) inhibited LPS-stimulated monocyte spermine uptake via the "nonselective" polyamine transporter. BAP fully restored macrophage TNF synthesis despite the presence of spermine, indicating that the mechanism of monocyte deactivation by spermine is dependent on spermine uptake. Administration of BAP in vivo significantly augmented the development of carrageenan-induced paw edema and nitric oxide release. Thus, endogenous spermine normally inhibits the innate inflammatory response by restraining macrophages.     

Synthesis of bis-spermine dimers that are potent polyamine transport inhibitors.

A series of novel spermine dimer analogues was synthesized and assessed for their ability to inhibit spermidine transport into MDA-MB-231 breast carcinoma cells. Two spermine molecules were tethered via their N(1) primary amines with naphthalenedisulfonic acid, adamantanedicarboxylic acid and a series of aliphatic dicarboxylic acids. The linked spermine analogues were potent polyamine transport inhibitors and inhibited cell growth cytostatically in combination with a polyamine synthesis inhibitor. Variation in the linker length did not alter polyamine transport inhibition. The amount of charge on the molecule may influence the molecular interaction with the transporter since the most potent spermidine transport inhibitors contained 5-6 positive charges.     

Spermine oxidase SMO(PAOh1), Not N1-acetylpolyamine oxidase PAO, is the primary source of cytotoxic H2O2 in polyamine analogue-treated human breast cancer cell lines

The induction of polyamine catabolism and its production of H2O2 have been implicated in the response to specific antitumor polyamine analogues. The original hypothesis was that analogue induction of the rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) provided substrate for the peroxisomal acetylpolyamine oxidase (PAO), resulting in a decrease in polyamine pools through catabolism, oxidation, and excretion of acetylated polyamines and the production of toxic aldehydes and H2O2. However, the recent discovery of the inducible spermine oxidase SMO(PAOh1) suggested the possibility that the original hypothesis may be incomplete. To examine the role of the catabolic enzymes in the response of breast cancer cells to the polyamine analogue N1,N1-bis(ethyl)norspermine (BENSpm), a stable knockdown small interfering RNA strategy was used. BENSpm differentially induced SSAT and SMO(PAOh1) mRNA and activity in several breast cancer cell lines, whereas no N1-acetylpolyamine oxidase PAO mRNA or activity was detected. BENSpm treatment inhibited cell growth, decreased intracellular polyamine levels, and decreased ornithine decarboxylase activity in all cell lines examined. The stable knockdown of either SSAT or SMO(PAOh1) reduced the sensitivity of MDA-MB-231 cells to BENSpm, whereas double knockdown MDA-MB-231 cells were almost entirely resistant to the growth inhibitory effects of the analogue. Furthermore, the H2O2 produced through BENSpm-induced polyamine catabolism was found to be derived exclusively from SMO(PAOh1) activity and not through PAO activity on acetylated polyamines. These data suggested that SSAT and SMO(PAOh1) activities are the major mediators of the cellular response of breast tumor cells to BENSpm and that PAO plays little or no role in this response.