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Polyamines are organic cations shown to control gene expression at the transcriptional, posttranscriptional, and translational levels. Multiple cellular oncogenic pathways are involved in regulation of transcription and translation of polyamine-metabolizing enzymes. As a consequence of genetic alterations, expression levels and activities of polyamine-metabolizing enzymes change rapidly during tumorigenesis resulting in high levels of polyamines in many human epithelial tumors. This review summarizes the mechanisms of polyamine regulation by canonical tumor suppressor genes and oncogenes, as well as the role of eukaryotic initiation factor 5A (EIF5A) in cancer. The importance of research utilizing pharmaceutical inhibitors and cancer chemopreventive strategies targeting the polyamine pathway is also discussed.

Polyamines are ubiquitous cellular components that are involved in normal and neoplastic growth. Polyamine biosynthesis is very highly regulated in mammalian cells by the activities of two key decarboxylases acting on ornithine and S-adenosylmethionine. Recent studies, which include crystallographic analysis of the recombinant human proteins, have provided a detailed knowledge of their structure and function. Ornithine decarboxylase is a PLP-requiring decarboxylase, whereas S-adenosylmethionine decarboxylase (AdoMetDC) contains a covalently bound pyruvate prosthetic group. Both enzymes have a key cysteine residue, which is involved in protonation of the Schiff base intermediate C(alpha) to form the product. These residues, Cys360 in ornithine decarboxylase (ODC) and Cys82 in AdoMetDC, react readily with nitric oxide (NO), which is therefore a potent inactivator of polyamine synthesis. The inactivation of these enzymes may mediate some of the antiproliferative actions of NO.

The role of ornithine decarboxylase (ODC) and polyamines in kidney hypertrophy is controversial. Since part of this controversy could be related to differences in the model system used by the different authors, we studied the changes in renal ODC and polyamines in six different models of kidney hypertrophy in mice, including compensatory renal hypertrophy produced by unilateral nephrectomy, experimental diabetes, potassium depletion and treatment with hormones such as testosterone, thyroxine and fluorocortisone. Only in the case of renal hypertrophy produced by testosterone administration was there a significant increase in ODC activity and putrescine content in the kidneys. However, the concomitant treatment with difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, as a 2% solution in the drinking water completely abolished the increase of renal ODC, but the kidney weights increased and other androgenic effects, such as the induction of renal beta-glucuronidase, were not affected. Moreover, DFMO-treatment did not prevent the kidney enlargement produced in other types of hypertrophy, even in the cases associated with hyperplasia. The present results support the premise that, at least in mice, the increase in ODC activity and polyamine biosynthesis is not required for kidney growth, and also that in most cases renal enlargement is not accompanied by any increase in the polyamine content.

This study was performed to assess the potential role of polyamines in the alterations in vascular structure and function in spontaneously hypertensive rats (SHR). The effects of chronic administration of eflornithine (alpha-difluoromethylornithine; DFMO), a highly specific inhibitor of ornithine decarboxylase (the rate limiting enzyme in polyamine biosynthesis), on vascular polyamine contents, vascular structure and function, and blood pressure was studied. Male SHR (16-17 weeks of age) with an average systolic blood pressure (SBP) of 161 +/- 3 mmHg were used. The rats were divided into two groups and received either tap water or a 1% DFMO solution to drink for 6 weeks. SBP and body weight were recorded prior to and once-a-week during the experiment. Standard in vitro vascular reactivity studies on ring segments of aorta and tail artery were performed. Ring segment weight, arterial medial thickness, and vascular polyamine contents were also determined. Body weights were not significantly affected by the DFMO treatment. SBP in control SHR rose progressively to an average value of 185 +/- 5 mmHg by the sixth experimental week. Although DFMO treatment did not cause a significant decrease in SBP compared to pretreatment values, it did prevent a further increase in SBP. Aortic and tail artery responsiveness to norepinephrine and electrical stimulation, respectively, ring segment weight, arterial medial thickness, and vascular polyamine contents were all significantly less in SHR receiving the DFMO treatment. These data are the first to demonstrate the effectiveness of DFMO to lower polyamine contents in the vasculature of hypertensive SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Aim of the present study was to evaluate the relevance of various intracellular regulatory mechanisms of polyamine metabolism in normal and camostate (FOY 305)-induced pancreatic growth in rats. Furthermore it was investigated whether the simultaneous inhibition of the polyamine interconversion pathway by the potent polyamine oxidase inhibitor MDL-72527 together with the ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) is able to enhance and prolong the only initial and transient inhibitory effects of DFMO on camostate-induced pancreatic growth. Simultaneous administration of DFMO and MDL-72527 resulted in a significant inhibition of the camostate-induced increases in pancreatic putrescine, ODC and DNA over 5 days, while the initial significant inhibition of pancreatic weight, protein content, DNA-polymerase and especially spermidine was absent after 5 days and spermine as well as N1-acetylspermidine were even increased. Diamine oxidase (DAO) activity in pancreas is very low and the potent DAO inhibitor aminoguanidine failed to alter any of the measured parameters except DAO. Therefore, oxidative degradation of putrescine by DAO--which is essential in the gut--is irrelevant in the pancreas. Simultaneous inhibition of the pancreatic polyamine interconversion pathway extended the initial inhibitory effects of DFMO only slightly and failed to exert a potent long-lasting inhibitory effect on spermidine and pancreatic growth.

Two transfected cell lines, one carrying a mammalian ornithine decarboxylase (ODC) that is suppressed by polyamines and one carrying a trypanosomal ODC that is not, were used to ask whether ODC suppression is necessary for the antiproliferative activities of two polyamine analogs, N1,N8-bis(ethyl)spermidine (BES) and N1,N14-bis(ethyl)homospermine (BE444). Both analogs accumulated within cells and suppressed S-adenosylmethionine decarboxylase, as well as polyamine-sensitive mouse ODC activity. Neither drug was able to suppress the activity of the polyamine-refractory trypanosome ODC. But, whereas BE444 was able to inhibit growth of both cell lines, BES could inhibit only growth of cells carrying the polyamine-sensitive ODC, under conditions that cause prolonged depletion of endogenous polyamines. We conclude from these studies that the antiproliferative activity of BES, a less potent drug, requires the suppression of ODC. The efficacy of BE444 is enhanced by its ability to suppress ODC. However, it can function without ODC suppression, whereas BES cannot.

The possible regulatory interactions of purified ornithine decarboxylase with DNA-directed RNA polymerases in isolated macronuclei from the ciliated protozoan Tetrahymena thermophila were studied. It has been found that highly purified ODC (specific activity 10.2 mumols CO2 x h-1 x mg-1), even at activities of 37,500 nmol CO2 x h-1 per ml failed to alter RNA polymerase activity in the in vitro transcription assay in the presence or absence of the substrate L-ornithine at 20mM. The naturally occurring di- and polyamines putrescine, spermidine, and spermine stimulated in-vitro-transcription in isolated macronuclei more at optimal Mg2+/Mn(2+)-concentrations than at suboptimal concentrations, suggesting that polyamines act via a mechanism which is distinct from that of the inorganic cations. Of the monovalent amine compounds tested, (NH4)+ at high concentrations between 40 and 50mM slightly stimulated activity whereas the onset of stimulation by the organic amine compounds, piperidine and cyclohexylamine, was inversely related to the hydrophobicity of each particular compound. In the series of divalent amines, the correct distance between the N-atoms appeared to be very important since ethylenediamine and piperazine did not stimulate significantly but did inhibit at concentrations above 5 mM. 1,3-Diaminopropane stimulated slightly but inhibited above 10 mM, whereas the 1,4-diamino compounds putrescine and 1,4-diaminocyclohexane (DAC) were equally potent stimulators with the more hydrophobic one, DAC, reaching the maximum at lower concentrations than putrescine. For the trivalent amines, the influence of correct spacing seems not to be as important: N-(2-aminoethyl)piperazine stimulated very similar to spermidine.(ABSTRACT TRUNCATED AT 250 WORDS)

The polyamines putrescine, spermidine and spermine represent a group of naturally occurring compounds exerting a bewildering number of biological effects, yet despite several decades of intensive research work, their exact physiological function remains obscure. Chemically these compounds are organic aliphatic cations with two (putrescine), three (spermidine) or four (spermine) amino or amino groups that are fully protonated at physiological pH values. Early studies showed that the polyamines are closely connected to the proliferation of animal cells. Their biosynthesis is accomplished by a concerted action of four different enzymes: ornithine decarboxylase, adenosylmethionine decarboxylase, spermidine synthase and spermine synthase. Out of these four enzyme, the two decarboxylases represent unique mammalian enzymes with an extremely short half life and dramatic inducibility in response to growth promoting stimuli. The regulation of ornithine decarboxylase, and to some extent also that of adenosylmethionine decarboxylase, is complex, showing features that do not always fit into the generally accepted rules of molecular biology. The development and introduction of specific inhibitors to the biosynthetic enzymes of the polyamines have revealed that an undisturbed synthesis of the polyamines is a prerequisite for animal cell proliferation to occur. The biosynthesis of the polyamines thus offers a meaningful target for the treatment of certain hyperproliferative diseases, most notably cancer. Although most experimental cancer models responds strikingly to treatment with polyamine antimetabolites--namely, inhibitors of various polyamine synthesizing enzymes--a real breakthrough in the treatment of human cancer has not yet occurred. It is, however, highly likely that the concept is viable. An especially interesting approach is the chemoprevention of cancer with polyamine antimetabolites, a process that appears to work in many experimental animal models. Meanwhile, the inhibition of polyamine accumulation has shown great promise in the treatment of human parasitic diseases, such as African trypanosomiasis.

Norepinephrine has been hypothesized as a trophic factor influencing postnatal development of the cerebellum. In the current study, neonatal rats were given 6-hydroxydopamine (6-OHDA) to destroy noradrenergic projections and the effects on the ornithine decarboxylase (ODC)/polyamine system were evaluated; ODC initiates the synthesis of polyamines, which are known to control cellular development in the cerebellum, and neonatal ODC activity is regulated in part by beta 2-adrenergic receptors. Intracisternal administration of 6-OHDA resulted in complete and permanent depletion of cerebellar norepinephrine and a deficit in ODC, polyamine levels and cerebellar growth. Subcutaneous administration of 6-OHDA, which caused only a small initial reduction in cerebellar norepinephrine, did not affect ODC and had only minor effects on tissue growth. Indeed, levels of the polyamines tended to be elevated after subcutaneous 6-OHDA, associated with postweaning elevations in norepinephrine, results which are probably indicative of axonal regeneration. In contrast to the effects of 6-OHDA on cerebellar development, neither the intracisternal nor subcutaneous drug treatment had any effect on cerebral cortical polyamines or growth, although the intracisternal treatment did impair ODC activity early in development. These data suggest that postnatal noradrenergic input, acting through the ODC/polyamine pathway, plays a selective role in cerebellar development.

This chapter reviews available data concerning the role of polyamines in pancreatic growth. Like in many other mammalian cells stimulation of pancreatic proliferation by trophic factors is associated with an early increase in ornithine decarboxylase (ODC) activity with consequent putrescine formation. ODC and putrescine play probably an important role in the initiation but not maintenance of pancreatic growth. The application of the specific ODC inhibitor alpha-difluoromethylornithine (DFMO) alone is not sufficient to judge the role of polyamines in pancreatic growth. For that purpose a combination of substances which inhibit ODC (DFMO) and polyamine oxidase (N1,N4-bisallenylputrescine) and/or S-adenosylmethionine-decarboxylase (SAM-DC) simultaneously has to be investigated. Ideally polyamines from dietary sources and intestinal bacterial flora should be abolished. The uptake of polyamines from the circulation has still to be considered. Studies will be presented proving that though ODC is completely inhibited by DFMO putrescine is still formed via spermidine/spermine N1-acetyltransferase (SAT). The fast increase of ODC in response to growth stimulating factors like cholecystokinin (CCK) is regulated at a pretranslational level by increasing ODC mRNA.

The intracellular concentrations of the polyamines are highly regulated and high polyamine concentrations are associated with rapidly proliferating cells. Hormones, nutrients and growth factors that stimulate the proliferation of the intestinal epithelium, increase the intracellular polyamine concentration mainly by activating ODC expression. Other cell types stimulated to proliferate satisfy their requirement for polyamines by increasing polyamine uptake. In the present study, we investigated polyamine uptake by a human colon carcinoma cell line, CaCo-2. Uptake of putrescine, spermidine and spermine by CaCo-2 cells was saturable and temperature dependent and all polyamines appear to share a common carrier. The carrier of differentiated cells had an apparently higher affinity and lower activity than the carrier of replicating cells. Culture of CaCo-2 cells on porous filters showed that polyamine accumulation occurred mainly through the basolateral membrane in replicating cells, while an increase in the rate of apical uptake was observed after differentiation. A significant increase in polyamine uptake and in ODC expression resulted from fresh medium replacement, a well-known stimulus to proliferation; no change in uptake occurred after ODC inhibition by DFMO. We conclude that CaCo-2 cells are able to increase their polyamine concentration by both enhanced synthesis and increased polyamine uptake.

To study the role of the polyamines putrescine, spermidine and spermine and of the enzymes controlling their synthesis (ornithine decarboxylase; ODC) and degradation (diamine oxidase; DAO) along the villus:crypt axis at the crucial early stage of the ileal adaptive response to jejunectomy, we measured polyamine concentrations and the activities of ODC, DAO and alkaline phosphatase (a marker of enterocyte maturity) in epithelial cells isolated by the Weiser technique from villus tips, mid villi, lower villi and crypts 4 days after surgery in transected control (TRC) and jejunectomised rats untreated or given the specific ODC blocker, alpha-difluoromethyl ornithine (DFMO, 2% in drinking water beginning 3 days before surgery). In the TRCs, there was a diminishing villus tip-to-crypt gradient not only in alkaline phosphatase but also in ODC and DAO activities. After jejunectomy, there were up to 93% increases in mean enterocyte ODC activity when compared with the corresponding cell fractions from the TRCs, but in both the control and jejunectomised rats, DFMO treatment markedly inhibited ODC activity (p less than 0.05-0.01) and reduced spermidine and particularly putrescine concentrations (p less than 0.005-0.001) in all four cell fractions. Only 4 days post-operation, jejunectomy stimulated a significant increase in ileal wet weight but DFMO treatment completely prevented this adaptive response and significantly reduced segmental intestinal weight (mg/cm) in the TRCs. These results (i) extend our knowledge of polyamines and related enzymes along the villus:crypt gradient in the normal intestine, (ii) provides the first data on these variables after resection, and (iii) lend further support to the hypothesis that changes in enterocyte ODC activity and in putrescine and spermidine concentrations play an important role in initiating the ileal adaptive response to proximal small bowel resection in the rat.