Properties of purified recombinant human polyamine oxidase,PAOh1/SMO

The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.     

Aminooxy analogues of spermidine as inhibitors of spermine synthase and substrates of hepatic polyamine acetylating activity

Aminooxy analogues of spermidine, 1-aminooxy-3-N-[3-aminopropyl]- aminopropane (AP-APA) and N-[2-aminooxyethyl]-1,4-diaminobutane (AOE-PU), were tested as substrates or inhibitors of the enzymes involved in methionine and polyamine metabolism. Both compounds were good competitive inhibitors and poor substrates of spermine synthase, good substrates of cytosolic polyamine acetyltransferase, inactivators of S-adenosylmethionine decarboxylase and inhibitors of ornithine decarboxylase. AP-APA and AOE-PU showed K1-values of 1.5 and 186 microM as inhibitors of purified spermine synthase, and Km-values of 1.4 and 2.1 mM as substrates of the crude hepatic polyamine acetyltransferase activity. AP-APA was more potent than AOE-PU in crude enzyme preparations. Neither drug had any significant effect at 1 mM concentration on the activities of spermidine synthase, methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, and methylthioadenosine phosphorylase. The results suggest that compounds of this type are valuable tools in unraveling the physiology of polyamines.     

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.     

THE UPTAKE OF SPERMIDINE AND SPERMINE BY SLICES OF MOUSE CEREBRAL HEMISPHERES

Abstract— Spermidine and spermine are taken up into mouse cerebral hemisphere slices by active transport and can be accumulated well above the medium concentration. The uptake process shows saturation kinetics and resembles that for amino acid uptake in that it is sensitive to temperature and inhibited by cyanide, 2,4-dinitrophenol or by the absence of glucose from the medium. However, at low initial medium concentrations spermine is taken up by a process which is insensitive to metabolic inhibitors or to temperature. It is suggested that either physical binding to a cellular constituent or exchange transport may account for this uptake. Ouabain does not inhibit polyamine uptake. Spermidine or spermine uptake is inhibited by cadaverine and putrescine. Spermine is the most potent inhibitor of spermidine uptake and vice-versa. Polyamine uptake differs from that of amino acids in that it is increased by a reduction in medium sodium or calcium content and decreased by an increase in medium potassium content. Recently taken up spermine undergoes heteroexchange with spermidine and homoexchange with recently entered spermine. Spermidine undergoes neither heteroexchange with spermine nor homoexchange.     

Hydroxylamine derivatives for regulation of spermine and spermidine metabolism

The biogenic polyamines spermine, spermidine, and their precursor putrescine are present in micro-to-millimolar concentrations in all cell types and are vitally important for their normal growth. High intracellular content of spermine and spermidine determines the multiplicity of the cellular functions of the polyamines. Many of these functions are not well characterized at the molecular level, ensuring the ongoing development of this field of biochemistry. Tumor cells have elevated polyamine level if compared with normal cells, and this greatly stimulates the search for new opportunities to deplete the intracellular pool of spermine and spermidine resulting in decrease in cell growth and even cell death. O-Substituted hydroxylamines occupy their own place among chemical regulators of the activity of the enzymes of polyamine metabolism. Varying the structure of the alkyl substituent made it possible to obtain within one class of chemical compounds highly effective inhibitors and regulators of the activity of all the enzymes of putrescine, spermine and spermidine metabolism (with the exception of FAD-dependent spermine oxidase and acetylpolyamine oxidase), effectors of the polyamine transport system, and even actively transported in cells “proinhibitor” of ornithine decarboxylase. Some principles for the design of specific inhibitors of these enzymes as well as the peculiarities of cellular effects of corresponding O-substituted hydroxylamines are discussed.     

Xylylated dimers of putrescine and polyamines: influence of the polyamine backbone on spermidine transport inhibition

Dimeric norspermidine and spermidine derivatives are strong competitive inhibitors of polyamine transport. A xylyl tether was used for the dimerization of various triamines and spermine via a secondary amino group, and of putrescine via an ether or an amino group. Dimerization of putrescine moieties potentiates their ability to compete against spermidine transport to a much greater extent than for triamine dimers.     

Ionic and structural specificity effects of natural and synthetic polyamines on the aggregation and resolubilization of single-, double-, and triple-stranded DNA

DNA condensation, precipitation, and aggregation are related phenomena involving DNA-DNA interactions in the presence of multivalent cations, and studied for their potential implications in DNA packaging in the cell. Recent studies have shown that the condensation/aggregation is a prerequisite for the cellular uptake of DNA for gene therapy applications. To elucidate the ionic and structural factors involved in DNA aggregation, we studied the precipitation and resolubilization of high molecular weight and sonicated calf thymus DNA, two therapeutic oligonucleotides, and poly(dA).2Poly(dT) triplex DNA in the presence of the tetravalent polyamine spermine using a centrifugation assay, Tm measurements, and CD spectroscopy. The ability of spermine to provoke DNA precipitation was in the following order: triplex DNA > duplex DNA > single-stranded DNA. In contrast, their resolubilization at high polyamine concentrations followed a reverse order. The effective concentration of spermine to precipitate DNA increased with Na+ in the medium. Tm data indicated the DNA stabilizing effect of spermine even in the resolubilized state. CD spectroscopy revealed a series of sequential conformational alterations of duplex and triplex DNA, with the duplex form regaining the B-DNA conformation at high concentrations (approximately 200 mM) of spermine. The triplex DNA, however, remained in a Psi-DNA conformation in the resolubilized state. Chemical structural specificity effects were exerted by spermidine and spermine analogues in precipitating and resolubilizing sonicated calf thymus DNA, with N4-methyl substitution of spermidine and a heptamethylene separation of the imino groups of spermine having the maximal difference in the precipitating ability of the analogues compared to spermidine and spermine, respectively. Therapeutically important bis(ethyl) substitution reduced the precipitating ability of the analogues compared to spermine. The effect of the cationicity of polyamines was evident with the pentamines being much more efficacious than the tetramines and triamines. These results provide new insights into the mechanism of DNA precipitation by polyamines, and suggest the importance of polyamine structure in developing gene delivery vehicles for therapeutic applications.     

Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.     

Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter.

We present evidence that polyamine uptake into rat liver mitochondria is mediated by a specific polyamine uniporter. Polyamine transport is not mediated by the ornithine, lysine, or Ca2+ transporters of mitochondria. Polyamine transport is a saturable process, with apparent Km values of 0.13 mM for spermine, 0.26 mM for spermidine, and 1 mM for putrescine. These substrates are mutually competitive inhibitors, indicating a common transport system. Polyamine transport is strictly dependent on membrane potential and insensitive to medium pH, showing that these polycations are transported electrophoretically. Spermine, spermidine, and putrescine are taken up by rat liver mitochondria at rates that increase with increasing valence of the transported species. The activation enthalpies for transport were 24, 32, and 59 kJ/mol for putrescine, spermidine, and spermine, respectively. These values, which amount to about 12 kJ/mol per charge transferred, may be compared to a value of 76 kJ/mol observed for monovalent tetraethylammonium cation. Flux-voltage analysis is consistent with the hypothesis that the mitochondrial polyamine transporter catalyzes transport via a channel mechanism.     

Polyamine analogues: potent inducers of nucleosomal array oligomerization and inhibitors of yeast cell growth

Polyamines are naturally occurring intracellular polycations that are essential for viability and growth of eukaryotes. Dysregulation of polyamine metabolism is a hallmark of cancer and the carcinogenic process, and consequently development of polyamine analogues has emerged as a viable strategy for therapeutic intervention. Previously, we showed that the naturally occurring polyamines spermidine and spermine were quite effective at inducing the oligomerization of nucleosomal arrays in vitro, suggesting that polyamines may play a key role in regulating higher order chromatin structures in vivo. Here, we analyse the ability of a number of synthetic polyamine analogues to potentiate formation of higher order chromatin structures in vitro. We find that a class of long-chain polyamines called oligoamines are potent inducers of nucleosomal array oligomerization in vitro and that these same polyamine analogues rapidly block yeast cell growth.     

Control of plant disease by perturbation of fungal polyamine metabolism

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous in nature and are essential for cell proliferation. Since polyamine biosynthesis in plants can start from either ornithine or arginine, while fungal polyamine biosynthesis appears to utilise only the ornithine route, it was suggested that specific inhibition of fungal polyamine biosynthesis should be lethal. Indeed, inhibitors of polyamine biosynthesis, e.g. the ornithine decarboxylase inhibitor α-difluoromethylornithine, have been shown to inhibit fungal growth in vitro and to control fungal infections on a variety of plants under glasshouse and field conditions. It is now known that polyamine analogues can perturb polyamine metabolism leading to powerful antiproliferative effects in cancer cells. This paper reviews the results of a research programme focused on the synthesis and evaluation of putrescine analogues as novel fungicides. A number of aliphatic, alicyclic and cyclic diamines have been shown to possess considerable fungicidal activity, but although many of these compounds perturb polyamine metabolism in fungal cells, such changes are not considered sufficient to account for the observed antifungal effects. More recent work on spermidine analogues is also described.     

Polyamine transport,accumulation, and release in brain

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.     

Putrescine derivatives as substrates of spermidine synthase

1. Derivatives of 1,4-butanediamine (putrescine) were studied in vitro and in vivo as potential substrates of spermidine synthase. 2. Substituents in the 1-position decreased the reaction rate by steric hindrance, and in the case of electron withdrawing groups there was an additional decrease due to the lowered basicity of the vicinal amino group. 3. Substituents in the 2-position are tolerated; under saturating conditions reaction rates are comparable to those of putrescine. 4. Compounds which were identified as substrates of spermidine synthase in vitro formed derivatives of spermidine and spermine in vivo. Exception: compounds, such as 1-methylputrescine formed in vivo only a spermidine derivative, because the second aminopropylation was sterically hindered by the substituent on the carbon atom next to the amino group. 5. Administration of 2-hydroxyputrescine to alpha-difluoromethylornithine-pretreated chick embryos produced spermidine and spermine analogues in amounts exceeding spermidine and spermine formation from putrescine under comparable conditions. 6. Since the concentration of 2-hydroxyputrescine in the embryo was higher than that of putrescine and all other putrescine analogues, it appears that uptake of the polyamine precursor from the yolk may be rate limiting. 7. Three days after administration of 5 mM alpha-difluoromethylornithine there is a near-to-complete arrest of embryonal growth. 8. A series of diamines supported growth under these conditions, even if they were not substrates of spermidine synthase. 9. Survival of chick embryos was, however, only supported if the diamines were capable of forming significant amounts of spermidine and spermine analogues.     

Effect of N1,N12-bis(ethyl)spermine and related compounds on growth and polyamine acetylation, content, and excretion in human colon tumor cells

Exposure of human colon tumor (HT 29 cells) to N1,N12-bis(ethyl)spermine and analogs produced a rapid loss of intracellular polyamines. This loss was brought about predominantly by an increased excretion of spermidine. N1,N11-Bis(ethyl)norspermine and N1,N12-Bis(ethyl)spermine were potent inducers of spermidine/spermine N1-acetyltransferase, and this induction facilitated the efflux of polyamines by enhancing the conversion of spermine into spermidine. N1,N14-Bis(ethyl)homospermine, which did not induce spermidine/spermine N1-acetyltransferase, also caused the loss of spermidine from the cell but was less effective in bringing about the decline in intracellular spermine. These results indicate that cellular polyamine levels can be regulated by excretion of spermidine and that the bis(ethyl)spermine derivatives deplete intracellular polyamine content by interference with this process.     

Polyamine uptake in cultured astrocytes: characterization and modulation by protein kinases

The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high-affinity transport system for putrescine, spermine, and spermidine (K:(m) = 3.2, 1.2, and 1.8 microM:, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na(+) cotransport as in the presence of Na(+) and cholinium, polyamine uptake was much lower than when measured in a sucrose-based medium. On the other hand, ouabain, gramicidin (a Na(+) ionophore), and ionomycin (a Ca(2+) ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP-dependent protein kinase or when Ca(2+)/calmodulin-dependent protein kinase II was inhibited with KN-62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.     

Effect of S-adenosyl-1,12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase, on polyamine metabolism in mammalian cells

The effects of the potent spermine synthase inhibitor S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDatad) on polyamine biosynthesis have been studied in transformed mouse fibroblasts (SV 3T3 cells) and in mouse leukemia cells (L1210). A dose-dependent decrease in intracellular spermine concentration was observed in both cell lines when grown in the presence of the inhibitor. A major difference in the effects seen in these two cell lines was the cytotoxicity observed in L1210 cells exposed to the inhibitor, which contrasted with little or no effects on growth of SV 3T3 cells treated similarly. Oxidative metabolism of the drug in L1210 cells was suggested by the fact that addition of aminoguanidine, an amine oxidase inhibitor, to the cell cultures ablated the cytotoxic effects of the inhibitor. Complete analysis of intracellular polyamines was carried out, together with analysis of S-adenosylmethionine, decarboxylated S-adenosylmethionine, and the inhibitor. These analyses revealed that, although the inhibitor had a dramatic effect on spermine biosynthesis in the cells studied, a compensatory increase in spermidine biosynthesis was observed. This resulted in no change in total polyamine concentrations in cells treated with inhibitors of either spermine synthase or spermidine synthase (Pegg et al., 1982) alone or in combination. In all cases, the concentration of the aminopropyl donor decarboxylated S-adenosylmethionine increased dramatically, thus allowing for the observed maintenance of total polyamine levels even in the presence of either one or both potent inhibitors of the aminopropyltransferases. Oxidative metabolism of the inhibitor complicates the interpretation of experiments carried out in the absence of amine oxidase inhibitors such as aminoguanidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Polyamine transport and metabolism in mouse mammary gland. General properties and hormonal regulation.

Mouse mammary gland has been shown to possess a transport system for spermidine, spermine, and putrescine. The uptake system for sperimidine, as studied in detail on mammary explants in culture is a time-dependent, energy-requiring process which can be stimulated by insulin and prolactin. The stimulatory effect of insulin involves both enhancement of Vmax for spermidine influx and prevention of efflux of the polyamine, whereas prolactin, in the presence of insulin, elicits a greater increase in Vmax for spermidine. Studies are also reported on the effects of temperature, concentration, and various inhibitors on this system. The accumulated spermidine exists virtually in an unchanged form with little metabolic conversion to either spermine or putrescine or to its conjugated form. In contrast, spermine and putrescine, both of which are also taken up by mammary explants, undergo metabolic conversion to spermidine.     

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.     

Epidermal keratinocytes actively maintain their intracellular polyamine levels

Increased cellular polyamine levels are thought to be essential for epidermal keratinocyte proliferation. However, a number of studies report that the induction of keratinocyte proliferation and of ornithine decarboxylase, the rate-limiting enzyme of putrescine, spermidine and spermine biosynthesis, is not concordantly expressed. The relationship between epidermal keratinocyte polyamine synthesis and proliferation was studied in neonatal mouse keratinocyte cultures using specific inhibitors of ODC activity to decrease the intracellular polyamine levels. The ODC inhibitors alpha-methyl ornithine (alpha-Me-Orn), alpha-hydrazino ornithine (alpha-HO) and difluoro-alpha-methylornithine (alpha-DFMO) did not significantly inhibit epidermal keratinocyte proliferation at 5 X 10(-3) to 10(-4) M concentrations. At these doses, only alpha-DFMO was seen to decrease (by 70%) the cellular levels of putrescine, but not of spermidine or spermine. Epidermal keratinocyte growth in the higher dose of 20 mM alpha-DFMO, however, did not decrease the cellular levels of putrescine. Polyamine analyses of the spent medium showed that growth in 10 mM alpha-DFMO decreased the normal epidermal cell transport of putrescine and spermidine into the medium. At 20 mM alpha-DFMO concentration, the keratinocytes actually transported, intracellularly, the putrescine and spermidine that are naturally found in the foetal bovine component of the growth medium. We conclude from these studies that epidermal keratinocyte polyamine levels are determined by both the rate of synthesis, and of the transport of these amines into the extracellular medium. Since epidermal keratinocytes actively maintain specific polyamine levels, it appears that these molecules are essential for epidermal keratinocyte function.