Spermidine/spermine N1-acetyltransferase (Spd/Spm acetyltransferase) is the rate-limiting enzyme in the catabolism of polyamines. This enzyme is highly inducible by several stimuli, including the natural polyamines and their structural analogues. To investigate the underlying mechanism responsible for the control of this enzyme a cDNA which codes for an active human Spd/Spm acetyltransferase has been isolated from a random primed cDNA library constructed from mRNA of a polyamine analogue treated large cell lung carcinoma line, NCI H157. The 972-base pair cDNA was identified using a 32-fold degenerate, 20-base oligomer probe to a 7-amino acid polypeptide sequence derived from the purified protein. The cDNA has a 513-base open reading frame that codes for a protein of 171 amino acids with a predicted molecular weight of 20,023. In vitro translation studies demonstrated the protein product of this cDNA to be a biologically active enzyme. The cDNA recognizes a 1.5-kilobase transcript in human cells which is highly induced in the human large cell lung carcinoma NCI H157 line following treatment with the polyamine analogue. The unusually high expression of Spd/Spm acetyltransferase mRNA by the NCI H157 cells in response to treatment does not appear to be a result of an amplification of the Spd/Spm acetyltransferase gene.

Changes in the metabolism of polyamines, which seem to be involved in transcription and translation in animal systems, have been studied in cultured cells of Daucus carota (carrot) undergoing embryogenesis. Putrescine levels were elevated by as much as 2-fold over the control within 24 hours after transfer of the cells to embryogenic medium. Spermidine levels were elevated also but spermine levels appeared to be lower in embryogenic cells. Embryogenic cells incorporated [(14)C]arginine into putrescine at two times the rate of control cells. These changes suggest that polyamines may be involved in cellular differentiation during embryogenesis.

Pseudomonas aeruginosa is an opportunistic human pathogen. Treatment is complicated by frequent acquired resistance to antipseudomonal therapies. Polyamines (cadaverine, putrescine, spermidine, and spermine) are ubiquitous polycationic compounds essential for all living organisms. In a dose-dependent manner, polyamines increased the susceptibility of P. aeruginosa to 14 beta-lactam antibiotics, chloramphenicol, nalidixic acid, and trimethoprim as demonstrated by a reduction in MIC of up to 64-fold. This effect was partially antagonized (25 to 50%) by the presence of 10 mM of Mg(2+) or Ca(2+). In contrast, the effects of the outer membrane permeabilizers, polymyxin B nonapeptide and EDTA, were completely abolished by 3 mM Mg(2+) or Ca(2+). Changes on the outer membrane barrier by these compounds were assessed by activity measurements of periplasmic beta-lactamase. The results showed that while EDTA and polymyxin B serve as outer membrane disorganizing agents as expected, exogenous spermidine and spermine did not exhibit any apparent effect on outer membrane permeability or rupture. In summary, these results strongly suggest that the increased antibiotic susceptibility by polyamines is exerted by a mechanism that differs from that of EDTA and polymyxin B. Polyamines might be potentially useful in antipseudomonal therapies by increasing the effectiveness of certain beta-lactam antibiotics.

Spermidine is essential for viability in eukaryotes but the importance of the longer polyamine spermine has not been established. Spermine is formed from spermidine by the action of spermine synthase, an aminopropyltransferase, whose gene (SpmS) is located on the X chromosome. Deletion of part of the X chromosome that include SpmS in Gy mice leads to a striking phenotype in affected males that includes altered phosphate metabolism and symptoms of hypophosphatemic rickets, circling behavior, hyperactivity, head shaking, inner ear abnormalities, deafness, sterility, a profound postnatal growth retardation, and a propensity to sudden death. It was not clear to what extent these alterations were due to the loss of spermine synthase activity, since this chromosomal deletion extends well beyond the SpmS gene and includes at least one other gene termed Phex. We have bred the Gy carrier female mice with transgenic mice (CAG/SpmS mice) that express spermine synthase from the ubiquitous CAG promoter. The resulting Gy-CAG/SpmS mice had extremely high levels of spermine synthase and contained spermine in all tissues examined. These mice had a normal life span and fertility and a normal growth rate except for a reduction in body weight due to a loss of bone mass that was consistent with the observation that the derangement in phosphate metabolism is due to the loss of the Phex gene and was not restored. These results show that spermine synthesis is needed for normal growth, viability, and fertility in male mice and that regulation of spermine synthase content is not required.

Purified preparations of the "exonuclease" specified by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) possess an endonuclease activity. The exonuclease and endonuclease activities copurify and cosediment in a sucrose density gradient. Endonuclease activity is only observed in the presence of a divalent cation, and Mg(2+) or Mn(2+) is equally effective as a cofactor with an optimal concentration of 2 mM. A slight amount of endonuclease activity is observed in the presence of Ca(2+), whereas no activity occurs in the presence of Zn(2+). In the presence of Mg(2+), Ca(2+) and Zn(2+) are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn(2+) greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg(2+). The endonuclease was affected by putrescine and spermidine to the same extent as the exonuclease activity, but in marked contrast the endonuclease was inhibited by a 10-fold-lower concentration of spermine compared to the exonuclease. The activity specified by HSV-1 and HSV-2 has very similar properties. HSV-1 and HSV-2 endonuclease cleave covalently closed circular DNA to yield, firstly, nicked circles and then linear DNA which is subsequently hydrolyzed to small oligonucleotides. Cleavage does not appear to be base sequence specific. Conversion of nicked circles to linear DNA and subsequent degradation of linear DNA occurs more rapidly in the presence of Mg(2+) than Mn(2+) presumably by virtue of the presence of the exonuclease activity. Nonsuperhelical covalently closed circular duplex DNA is cleaved by the endonucleases at a rate 60 times slower than the rate observed on the supercoiled form. These data indicate that the HSV-1 and HSV-2 endonuclease preferentially recognize single-stranded DNA regions.

Conditions of formation of DNA aggregates by the addition of spermidine were determined with 146 base pair DNA fragments as a function of spermidine and NaCl concentration. Two different phases of spermidine-DNA complexes are obtained: a cholesteric liquid crystalline phase with a large helical pitch, with interhelix distances ranging from 31.6 to 32.6 A, and a columnar hexagonal phase with a restricted fluidity in which DNA molecules are more closely packed (29.85 +/- 0.05 A). In both phases, the DNA molecule retains its B form. These phases are always observed in equilibrium with the dilute isotropic solution, and their phase diagram is defined for a DNA concentration of 1 mg/ml. DNA liquid crystalline phases induced by spermidine are compared with the DNA mesophases already described in concentrated solutions in the absence of spermidine. We propose that the liquid crystalline character of the spermidine DNA complexes is involved in the stimulation of the functional properties of the DNA reported in numerous experimental articles, and we discuss how the nature of the phase could regulate the degree of activity of the molecule.

An endonuclease has been purified more than 300-fold from Escherichia coli infected with bacteriophage T4. The enzyme degrades rapidly sedimenting (greater than 1000 S) DNA in vitro by introducing a limited number of breaks. The substrate is the replicative DNA isolated from cells infected with gene-49-defective phage [Kemper, B, and Janz, E. (1976) J. Virol. 18, 992-999]. Molecules of approximately a third the size of unit-length T4 DNA are exclusively found in a limit digest. The enzyme also reacts with single-stranded DNA from various sources. Heat-denatured T4 DNA is converted into acid-soluble oligonucleotides. Circular single-stranded M13 DNA is linearized by endonucleolytic cleavage causing a reduction of infectivity during transfection. The enzyme behaves like a typical late-gene product. Its activity is 100-fold reduced in cells infected with gene-55-defective phage (defect in expression of late functions). A 30-fold reduction in its specific activity was found in cells infected with gene-49-defective phage suggesting that gene 49 codes for the enzyme or controls its expression. The purified enzyme binds to native or denatured DNA from various sources. The protein has a molecular weight of 42000 as determined by gel filtration and sedimentation analysis. Optimal activity on rapidly sedimenting DNA is obtained at pH 8.6 in Tris/HCl buffer in the presence of 10 mM MgCl2. Some 75% of the activity can be obtained with 7 mM MnCl2. 5 mM CaCl2 has a stimulatory effect on the reaction with MgCl2 or MnCl2 each present at its individual optimal concentration. The enzyme does not require the addition of sulfhydryl reagent for full activity. The reaction can be inhibited by compounds like KCl, spermidine, p-hydroxymercuribenzoate or tRNA.

Elevated polyamine and nitric oxide levels (both derived from arginine) promote tumorigenesis, whereas non-steroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal cancer (CRC) incidence in experimental and epidemiologic studies. We investigated dietary arginine-induced intestinal tumorigenesis and NSAID-inhibitory effects in Apc(Min/+) mice differentially expressing nitric oxide synthase-2 (Nos2). We also studied effects of estimated arginine exposures through meat consumption on tumor characteristics and survival in human CRC cases. Dietary arginine increased high-grade colon adenoma incidence in Apc(Min/+)Nos2(+/+) mice, but not in Nos2 knockout mice. Additionally, celecoxib suppressed intestinal steady state ornithine decarboxylase RNA levels (p < 0.001), induced steady state spermidine/spermine N(1)-acetyltransferase RNA levels (p = 0.002), decreased putrescine levels (p = 0.04) and decreased tumor number in the small intestines of Apc(Min/+)Nos2(+/+) mice (p = 0.0003). Five hundred and eleven cases from our NCI-supported CRC gene-environment study were analyzed based on self-reported meat (as a surrogate for arginine) consumption. Familial CRC cases (n = 144) in the highest meat consumption quartile (Q4) had no statistically significant differences in tumor grade compared to cases in Q1-Q3 (p = 0.32); however, they were observed to have decreased overall survival (OS) (10-year OS = 42% vs. 65%; p = 0.017), and increased risk of death in an adjusted analysis (hazards ratio [HR] = 2.24; p = 0.007). No differences in tumor grade, OS or adjusted HR were observed for sporadic CRC cases (n = 367) based on meat consumption. Our results suggest important roles for arginine and meat consumption in CRC pathogenesis, and have implications for CRC prevention.

Our previous results indicate that during protoplast isolation an oxidative burst occurs [A.K. Papadakis and KA Roubelakis-Angelakis (1999) Plant Physiol 127:197-205] and that suppression of totipotency is correlated with reduced antioxidant activity and low redox state [A.K. Papadakis et al. (2001b) Plant Physiol 126:434-444]. Polyamines are known to affect cell development and to act as antioxidants. Polyamines applied during isolation of tobacco (Nicotiana tabacum L.) protoplasts reduced the accumulation of O2*- but not that of H2O2. This antioxidant effect is probably due to the inhibition of microsomal membrane NADPH oxidase, which occurred in a concentration-dependent manner, with spermine exerting the highest inhibitory effect. However, during protoplast culture, polyamine oxidase activity increased severalfold in spermidine- and spermine-treated protoplasts, concomitant with H2O2 titers. A cell death program was executed in untreated protoplasts, as documented by membrane malfunction, induced DNase activity, DNA fragmentation and a positive TUNEL reaction. Protoplast cell death was prevented in protoplasts treated with putrescine, but not by treatment with spermidine or spermine, which rather had the opposite effect. The data presented suggest that PAs may be implicated in the expression of plant protoplast totipotency.

Ornithine decarboxylase activity in extracts of phytohaemagglutinin-stimulated human lymphocytes is rapidly and extensively inhibited by additions of micromolar concentrations of putrescine or spermidine to the culture medium. This inhibition is not due to feedback inhibition of the enzyme by putrescine, spermidine or their metabolites. Inhibition is dependent on the continuation of protein synthesis, but does not require RNA synthesis. The effect of putrescine is abolished when its conversion into spermidine by the cells is prevented.

The activity of T4 polynucleotide kinase (EC 2.7.1.78) was found to be greatly stimulated by salts, such as NaCl and KCl, and polyamines such as spermine and spermidine. Up to a sixfold increase in initial rates was observed with a variety of different single-stranded DNAs and mono- and oligonucleotides. The optimal concentrations of salts were 0.125 M, corresponding to a total ionic strength of mu equals 0.19. For polyamines the optimal concentrations were found to be at approximately 2 mM. With low enzyme concentration and in the absence of activators complete phosphorylation was not achieved for a number of substrates. In the presence of salts or polyamines or high concentration of enzyme the phosphorylation proceeded to completion. Addition of salt led to an increase in both the apparent V-max and the Michaelis constant for the DNA substrate whereas the Michaelis constant of ATP remained unchanged. Polyamines had a similar influence on the kinetic constants for the DNA substrate whereas a decrease was found for the apparent Michaelis constant for ATP. The overall mechanism in the presence of activators was found to be sequential but probably of a rapid equilibrium random type. Of the inorganic anions tested both P-i and PP-i inhibited the enzyme in a competitive manner with both substrates.

Polyamines are essential for cell migration during early mucosal restitution after wounding in the gastrointestinal tract. Activity of voltage-gated K(+) channels (Kv) controls membrane potential (E(m)) that regulates cytoplasmic free Ca(2+) concentration ([Ca(2+)](cyt)) by governing the driving force for Ca(2+) influx. This study determined whether polyamines are required for the stimulation of cell migration by altering K(+) channel gene expression, E(m), and [Ca(2+)](cyt) in intestinal epithelial cells (IEC-6). The specific inhibitor of polyamine synthesis, alpha-difluoromethylornithine (DFMO, 5 mM), depleted cellular polyamines (putrescine, spermidine, and spermine), selectively inhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression, and resulted in membrane depolarization. Because IEC-6 cells did not express voltage-gated Ca(2+) channels, the depolarized E(m) in DFMO-treated cells decreased [Ca(2+)](cyt) as a result of reduced driving force for Ca(2+) influx through capacitative Ca(2+) entry. Migration was reduced by 80% in the polyamine-deficient cells. Exogenous spermidine not only reversed the effects of DFMO on Kv1.1 channel expression, E(m), and [Ca(2+)](cyt) but also restored cell migration to normal. Removal of extracellular Ca(2+) or blockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These results suggest that polyamine-dependent intestinal epithelial cell migration may be due partially to an increase of Kv1.1 channel expression. The subsequent membrane hyperpolarization raises [Ca(2+)](cyt) by increasing the driving force (the electrochemical gradient) for Ca(2+) influx and thus stimulates cell migration.

The mechanism of the antiproliferation effect of spermidine and spermine was studied using a cell culture system of mouse FM3A cells. The addition of either 10 mM spermidine or 2 mM spermine to the growth medium containing 0.9 mM Mg2+ greatly inhibited cell growth (more than 90%). A decrease in the Mg2+ concentration to 50 microM in the growth medium, but without the polyamine addition, did not influence cell growth. However, the concentrations of spermidine and spermine necessary for the inhibition of cell growth when cells were cultured in the presence of 50 microM Mg2+ were much smaller (2 mM spermidine and 0.15 mM spermine). Nevertheless, the amount of polyamines accumulating in cells which could cause the inhibition of cell growth was almost the same, regardless of the large difference in the added polyamine concentrations. At the early stage of polyamine accumulation, the inhibition of cell growth correlated with the decrease of Mg2+ content, but not with a decrease of the ATP content. The decrease in Mg2+ content correlated well with the inhibition of macromolecular synthesis, especially protein synthesis. Thus, the inhibition of cell growth at the early stage of polyamine accumulation was thought to be due to the inactivation of ribosomes through the replacement of Mg2+ on magnesium-binding sites by polyamines. The decrease in Mg2+ content was mainly caused by the inhibition of Mg2+ transport by polyamines. At the later stage of polyamine accumulation, a decrease in ATP content was also observed. This was followed by swelling of the mitochondria, which may be a symptom of the subsequent cell death.

The cyclic nucleotide-gated (CNG) channel in retinal rods converts the light-regulated intracellular cGMP concentration to various levels of membrane potential. Blockade of the channel by cations such as Ca2+ and Mg2+ lowers its effective conductance. Consequently, the membrane potential has very low noise, which enables rods to detect light with extremely high sensitivity. Here, we report that three polyamines (putrescine, spermidine, and spermine), which exist in both the intracellular and extracellular media, also effectively block the CNG channel from both sides of the membrane. Among them, spermine has the greatest potency. Extracellular spermine blocks the channel as a permeant blocker, whereas intracellular spermine appears to block the channel in two conformations-one permeant, and the other non- (or much less) permeant. The membrane potential in rods is typically depolarized to approximately -40 mV in the dark. At this voltage, K1/2 of the CNG channel for extracellular spermine is 3 microM, which is 100-1,000-fold higher affinity than that of the NMDA receptor-channel for extracellular spermine. Blockade of the CNG channel by polyamines may play an important role in suppressing noise in the signal transduction system in rods.

OBJECTIVE

Polyamines are essential for the normal postnatal development, maintenance, and function of gastrointestinal epithelia. The extracellular Ca(2+) (Ca(2+)(o)/nutrient)-sensing receptor is expressed on both luminal and basolateral membranes of colonocytes, and, in other cell systems, this receptor has been shown to respond to polyamines. Thus, the Ca(2+)-sensing receptor could provide a mechanism for modulation of colonocyte function by dietary and systemic extracellular polyamines. In the present study, we investigated the interaction of polyamines, particularly spermine, and extracellular Ca(2+) on second messenger generation by, and on function of, rat distal colonic crypts.

METHODS

Calcium-sensing receptor activation was assessed in colonic epithelial cells and intact crypts freshly isolated from distal colon by monitoring intracellular IP(3) and Ca(2+) accumulation using radioimmunoassay and Fluo-3 fluorometry, respectively. Interactions of extracellular Ca(2+) and spermine on regulation of both basal and forskolin-stimulated fluid transport were measured in crypts microperfused in vitro.

RESULTS

Polyamine (spermine>> spermidine>> putrescine)-mediated enhancement of intracellular D-myo-inositol 1,4,5-trisphosphate (IP(3)) and Ca(2+) accumulation required extracellular Ca(2+), and the EC(50) for extracellular Ca(2+)-mediated activation of the calcium-sensing receptor was reduced by polyamines. Extracellular spermine modulated both basal and forskolin-stimulated fluid secretion in perfused colonic crypts, and the EC(50) for spermine-induced reduction in forskolin-stimulated fluid secretion was inversely dependent on extracellular Ca(2+) (Ca(2+)(o)).

CONCLUSIONS

The interactions of extracellular Ca(2+) and polyamines on second messenger accumulation and fluid secretion support a role for the luminal and basolateral calcium-sensing receptors in mediating some of the effects of polyamines on distal colonic epithelial cells.

The intracellular parasite Leishmania causes a wide spectrum of human disease, ranging from self-resolving cutaneous lesions to fatal visceral disease, depending on the species of Leishmania involved. The mechanisms by which different Leishmania species cause different pathologies are largely unknown. We have addressed this question by comparing the gene expression profiles of bone marrow-derived macrophages infected with either Leishmania donovani or L. major promastigotes. We found that the two species had very similar effects on macrophage gene expression. Both species caused a small (<2.5-fold) but statistically significant repression of several hundred genes. In addition, both species strongly induced and repressed about 60 genes. Comparing the effects of the two species showed that only 26 genes were regulated differently by L. major as opposed to L. donovani, including those for metallothioneins 1 and 2, HSP70 and -72, CCL4, Gadd45beta, Dsp1, matrix metalloprotease 13, T-cell death-associated gene 51 (Tdag51), RhoB, spermine/spermidine N1-acyl transferase 1 (SSAT), and Cox2. L. donovani-infected macrophages were also found to express higher levels of Cox2 protein and prostaglandin E synthase mRNA than L. major-infected macrophages. While both species have previously been shown to trigger prostaglandin E synthesis by bystander cells, this study suggests that infected macrophages themselves express prostaglandin E-synthesizing genes only in response to L. donovani.

Significant amounts of H2O2 were produced when the polyamines, spermine, or spermidine were incubated with a Krebs-Ringer phosphate buffer that contained bovine serum albumin (Fraction V). This effect was specific for certain amines and could be prevented by treatment of the albumin fraction with isoniazid or aminoguanidine. These features suggest that H2O2 is formed during oxidative deamination of the polyamines catalized by spermine oxidase, a known contaminant of Fraction V bovine serum albumin. The insulin-like effects elicited by polyamines in fat cells (e.g. enhancement of glucose transport and inhibition of cAMP-mediated lipolysis) were dependent on H2O2 production. Incubation of cells with catalase or treatment of the albumin fraction with isoniazid abolished the stimulation of glucose uptake by polyamines but did not alter the stimulatory effects of insulin or vitamin K5. The H2O2 generating activity was partially separated from the albumin by gel filtration; only those fractions which formed H2O2 provided support for the activation of glucose transport by polyamines. Also, the time needed to activate glucose uptake was markedly shortened by incubation of the albumin buffer with the polyamines before addition of the cells. These findings indicate that the polyamines do not themselves mimic the actions of insulin but that the insulin-like effects result from the formation of H2O2 which has been shown to stimulate glucose transport.

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.

The threonyl-tRNA synthetase gene (thrS) is a member of the T-box family of approximately 250 genes, found essentially in Gram-positive bacteria, regulated by a tRNA-dependent antitermination mechanism in response to starvation for the cognate amino acid. While interaction between uncharged tRNA and the untranslated leader region of these genes has been firmly established by genetic means, attempts to show this interaction or to reconstitute the antitermination mechanism in vitro using purified tRNAs have so far failed. In addition, a number of conserved sequences have been identified in the T-box leaders, for which no function has yet been assigned. This suggests that factors other than the tRNA are important for this type of control. Here we demonstrate tRNA-mediated antitermination for the first time in vitro, using the regulatory tRNA(Thr) isoacceptor isolated from Bacillus subtilis and a partially purified protein fraction. As predicted by the model, aminoacylation of tRNA(Thr(GGU)) with threonine completely abolishes its ability to act as an effector. The role of the partially purified protein fraction can be functionally substituted by high concentrations of spermidine. However, this polyamine does not play a significant role in the induction of thrS expression in vivo, suggesting that it is specific protein co-factors that promote T-box gene regulation in conjunction with uncharged tRNA.

Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.

Previous results indicate that the polyphenol resveratrol inhibits cell growth of colon carcinoma cells via modulation of polyamine metabolic key enzymes. The aim of this work was to specify the underlying molecular mechanisms and to identify a possible role of transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). Cell growth was determined by bromodeoxyuridine incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Spermine/spermidine acetyltransferase (SSAT) activity was determined by a radiochemical assay. PPARgamma ligand-dependent transcriptional activity was measured by a luciferase assay. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Resveratrol inhibits cell growth of both Caco-2 and HCT-116 cells in a dose- and time-dependent manner (P < 0.001). In contrast to Caco-2-wild type cells (P < 0.05), resveratrol failed to increase SSAT activity in dominant-negative PPARgamma cells. PPARgamma involvement was further confirmed via ligand-dependent activation (P < 0.01) as well as by induction of cytokeratin 20 (P < 0.001) after resveratrol treatment. Coincubation with SB203580 abolished SSAT activation significantly in Caco-2 (P < 0.05) and HCT-116 (P < 0.01) cells. The involvement of p38 mitogen-activated protein kinase (MAPK) was further confirmed by a resveratrol-mediated phosphorylation of p38 protein in both cell lines. Resveratrol further increased the expression of PPARgamma coactivator PGC-1alpha (P < 0.05) as well as SIRT1 (P < 0.01) in a dose-dependent manner after 24 hours of incubation. Based on our findings, p38 MAPK and transcription factor PPARgamma can be considered as molecular targets of resveratrol in the regulation of cell proliferation and SSAT activity, respectively, in a cell culture model of colon cancer.

It has been shown previously (Porter et al., Cancer Res., 45: 2050-2057, 1985) that the N1,N8-bis(ethyl) derivative of spermidine has significant antiproliferative activity which appears to derive from its regulatory effects on the polyamine biosynthetic pathway, particularly on ornithine decarboxylase activity. In the present study, N1,N4-bis(ethyl)putrescine (BEP) and N1,N12-bis(ethyl)spermine (BESm) were compared with N1,N8-bis(ethyl)spermidine (BES) in their ability to inhibit cell growth and regulate polyamine biosynthesis. With cultured L1210 murine leukemia cells, the IC50 values at 48 h were approximately 2 mM for BEP, 30 microM for BES, and 1 microM for BESm making the latter the most effective polyamine inhibitor or analogue thus far identified. At concentrations which approximated IC50 values and yielded similar intracellular concentrations at 48 h (1500-2000 pmol/10(6) cells), the effects of the analogues on polyamine biosynthesis generally correlated with their antiproliferative activity. BEP, at 1 mM, exerted relatively minor effects on polyamine biosynthesis. By contrast, 100 microM BES totally eliminated ornithine decarboxylase activity, depleted putrescine and spermidine pools, and decreased spermine pools by 40%. AdoMet decarboxylase activity was lowered slightly. The most impressive effects were obtained with 10 microM BESm which decreased ornithine and AdoMet decarboxylase activities by 99 and 84%, respectively; depleted putrescine and spermidine pools; and decreased spermine pools by 73%. None of the analogues, at 1 or 3 mM, had significant direct inhibitory effects on the decarboxylase activities from untreated cells with the exception of BESm which inhibited ornithine but not AdoMet decarboxylase activity. Thus, the effects of the analogues on these enzymes in treated cells are presumed to be mainly mediated by regulatory mechanisms. In this regard, BESm was superior to BES since both ornithine and AdoMet decarboxylase activities were suppressed. Given its unique activities, BESm would seem to have potential as both an antiproliferative agent and also as an experimental probe for studying regulation of the polyamine pathway, particularly AdoMet decarboxylase.

The natural polyamines putrescine, spermidine and spermine are intimately involved in growth-related processes. More and more evidence indicates that the excessive accumulation of putrescine and spermidine favors malignant transformation of cells. Selective depletion of putrescine has been shown to restore in some transformed cells the normal phenotype. Inhibition of polyamine formation appears, therefore, a rational target in chemoprevention. Clinical trials with 2-(difluoromethyl)ornithine, a selective inactivator of ornithine decarboxylase, a key enzyme of polyamine biosynthesis, are promising. Structural analogs of the polyamines with polyamine-mimetic or antagonist properties, and calmodulin antagonists are other types of drugs which affect several key reactions of polyamine metabolism, and appear to be candidates for the prevention of carcinogenesis especially of the gastrointestinal tract.