Premalignant alterations in rat colonic N1-acetylspermidine levels induced by 1,2-dimethylhydrazine: effects of a high corn oil dietary regimen

Recently, our laboratory has demonstrated that elevations in the levels of N1-acetylspermidine could be detected in the colonic mucosa of rats after administration of 1,2-dimethylhydrazine for 15 weeks, i.e., before the development of colon tumors. Since prior studies have indicated that diets high in fat, particularly unsaturated fat, promote the development of dimethylhydrazine-induced tumors, it was of interest to examine the effect of a corn oil dietary regimen (20% by weight) on colonic N1-acetylspermidine levels in this model of colonic adenocarcinoma. Four groups of rats were used in these studies: chow, chow + carcinogen, corn oil and corn oil + carcinogen. The carcinogen groups received weekly s.c. injections of 1,2-dimethylhydrazine (20 mg/kg body wt) for 15 weeks, while the control groups received diluent. 1 week after the last injection, animals from each group were killed, and their proximal and distal colons were resected, examined and compared with respect to polyamine levels, including N1-acetylspermidine, as well as the activities of ornithine decarboxylase, spermidine N1-acetyltransferase, and polyamine oxidase. In view of previous studies which suggested that N1-acetylspermidine levels may be elevated in the urine of patients with various malignancies, it was also of interest to examine and compare the urinary levels of this acetylated polyamine in animals from each group. The results of these experiments demonstrated that: (1) the levels of N1-acetylspermidine in the distal colonic segment were found to be increased approx. 25 and 80% in the chow + carcinogen and corn oil + carcinogen groups, respectively, compared to their control counterparts; (2) the activities of spermidine N1-acetyltransferase in the distal colonic segments of chow + carcinogen and corn oil + carcinogen animals were increased 1.5- and 2-fold, respectively, compared to control values; (3) dimethylhydrazine administration did not affect the levels of this acetylated polyamine or spermidine N1-acetyltransferase activities in the proximal colon, but, in general, did increase the levels of putrescine and spermidine as well as ornithine decarboxylase activities in both colonic segments of animals fed chow or corn oil diets; and (4) elevated urinary levels of N1-acetylspermidine did not appear to be a reliable 'premalignant' marker in this experimental model of colonic adenocarcinoma.     

Induction of spermidine/spermine N1-acetyltransferase in needle-punctured rat lens as a model of traumatic cataract

Isolated rat lens was punctured with a needle at a single point in the equatorial region and was incubated at 37 degrees C. Spermidine/spermine N1-acetyltransferase activity was increased about 5-fold at 8 h after the puncture. Concomitantly, putrescine content in the lens increased markedly at 8-16 h after the puncture, while spermidine levels were slightly depressed. Pretreatment of the lens with actinomycin D or cycloheximide blocked the increases of spermidine/spermine N1-acetyltransferase activity and putrescine content. Ornithine decarboxylase, on the other hand, was not induced to a detectable degree by this stimulus and 5 mM difluoromethylornithine could not block the increase of putrescine content. Polyamine oxidase showed a relatively constant activity that was sufficient for the metabolism of newly formed N1-acetylspermidine. These results suggested that, in the punctured lens, the polyamine levels were regulated predominantly by the activity of spermidine/spermine N1-acetyltransferase, but not by the induction of ornithine decarboxylase.     

1,2-Dimethylhydrazine-induced alterations in lipid peroxidation in preneoplastic and neoplastic colonic tissues

To determine whether alterations in lipid peroxidation existed in the preneoplastic and neoplastic colonic tissues of animals treated with the procarcinogen 1,2-dimethylhydrazine, rats were injected subcutaneously with this agent (20 mg/kg body weight per week) or diluent for 5, 10, 15 and 26 weeks. At each of these time periods, animals from both groups were sacrificed, their distal colonic mucosa and/or tumors harvested, and examined and compared with respect to malondialdehyde and lipofuscin-like pigments levels. Additionally, at 26 weeks, the fatty acid composition of microsomes prepared from control, 'uninvolved' and tumor colonic tissues were analyzed and compared. The results of these experiments demonstrated that: (1) the levels of these products of lipid peroxidation were similar in the distal colons of all animals at 5 and 10 weeks; (2) at 15 weeks, however, lipid peroxidation was decreased in the distal colons of animals treated with dimethylhydrazine; (3) at 26 weeks, the levels of these products of lipid peroxidation remained lower in dimethylhydrazine-treated distal 'uninvolved' colonic mucosa and was, moreover, markedly decreased in colonic tumors; and (4) at this latter time period, differences in the fatty acid composition between tumor, 'uninvolved' and control tissues were found. These differences, however, did not appear to underlie the changes noted in the lipid peroxidation products seen in these tissues. Taken together, these findings suggest that alterations in lipid peroxidation may be involved in the colonic malignant transformation process in this experimental model.     

Secretin-induced accumulation of N1-acetylspermidine and putrescine in the rat pancreas

The effects of secretin on polyamine metabolism in rat pancréas were investigated. Single injections of secretin increased ornithine decarboxylase activity only very slightly. However a substantial time- and dose-dependent increase of acetyl CoA: polyamine N¹-acetyltransferase activity was observed. The concentrations of N¹-acetylspermidine, putrescine and β-alanine increased concomitantly, but spermidine and spermine remained unchanged. These results suggest that, in this model, the accumulated putrescine was formed from spermidine, via its acetylation, rather than from ornithine.     

Excretion of acetylated and free polyamines by polyamine depleted Chinese hamster ovary cells

1. Cultured Chinese hamster ovary cells (CHO) and their ornithine decarboxylase deficient mutant cells (C55.7) were found to excrete small amounts of N8-acetylspermidine and free polyamines, putrescine and spermidine into the culture medium. 2. The concentration of N8-acetylspermidine in the control cells was 2-3% of that of spermidine. In the medium, however, the amount of N8-acetylspermidine was about 2-fold that of spermidine and 2- to 3-fold higher than the intracellular amount. N1-acetylspermidine or acetylated spermine were never detected in the cells or in the media. 3. Confluent CHO cells treated with 2 mM difluoromethylornithine stopped the excretion when the intracellular spermidine concentration had decreased to 20% of control while there was no decrease in spermine concentration. At low cell density, neither polyamine depleted CHO cells nor the C55.7 cells excreted any polyamines into the culture media.     

Spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in 1 alpha,25-dihydroxyvitamin D3-induced putrescine synthesis

We have reported that a single injection of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the active form of vitamin D3, into vitamin D-deficient chicks produces a marked increase in the formation of duodenal putrescine by two pathways, one from ornithine and one from spermidine (Shinki, T., Takahashi, N., Kadofuku, T., Sato, T., and Suda, T. (1985) J. Biol. Chem. 260, 2185-2190). In this work, the conversion of [3H]ornithine into [3H]putrescine catalyzed by ornithine decarboxylase was compared with the conversion of [14C]spermidine into [14C]putrescine catalyzed by spermidine N1-acetyltransferase and polyamine oxidase. Using the in situ duodenal loop method in the presence or absence of alpha-difluoromethylornithine, we evaluated the relative contributions of these two pathways in the 1 alpha,25(OH)2D3-induced duodenal synthesis of putrescine. Prior administration of alpha-difluoromethylornithine inhibited neither the 1 alpha,25(OH)2D3-induced increase in duodenal spermidine N1-acetyltransferase activity nor the vitamin-induced enhancement of the duodenal putrescine content, although it completely suppressed the duodenal ornithine decarboxylase activity induced by 1 alpha,25(OH)2D3. The duodenal content of spermidine decreased time-dependently after injection of 1 alpha,25(OH)2D3. The increase of duodenal putrescine by 1 alpha,25(OH)2D3 coincided quantitatively with the amount of putrescine synthesized from spermidine but not from ornithine after injection of the vitamin. These unexpected results clearly indicate that spermidine N1-acetyltransferase has a larger role than ornithine decarboxylase in the increase of duodenal putrescine synthesis induced by 1 alpha,25(OH)2D3. The polyamine metabolism reported here may be related to the characteristics of intestinal epithelial cells such as the short lifetime (90-108 h) and typical gradient of differentiation from the crypt to villus regions.     

Metabolic and antiproliferative consequences of activated polyamine catabolism in LNCaP prostate carcinoma cells

Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid approximately 10-fold increase in SSAT mRNA and an increase of approximately 20-fold in enzyme activity. SSAT products N(1)-acetylspermidine, N(1)-acetylspermine, and N(1),N(12)-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor alpha-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition.     

The role of polyamine reutilization in depletion of cellular stores of polyamines in non-proliferating tissues

It was known from previous work that specific inhibition of neither ornithine decarboxylase activity nor polyamine oxidase activity produces spermidine depletion by more than 20% in non-growing organs, which are in a steady state with regard to polyamine metabolism. Combined treatment with inactivators of both ornithine decarboxylase and polyamine oxidase for a prolonged time caused, however, a gradual decrease of spermidine levels in liver, kidney and brain of mice by 50% and more. The method is in accordance with the previously suggested role of polyamine interconversion. Inhibition of polyamine oxidase prevents the reutilization for de novo polyamine biosynthesis of putrescine and spermidine, which are formed by oxidative splitting of N1-acetylspermine and N1-acetylspermidine, respectively, and the ornithine decarboxylase inhibitor prevents the compensatory increase of putrescine from ornithine. The findings are further evidence for the physiological significance of polyamine reutilization.     

Comparison of 3-isobutyl-1-methylxanthine-induced accumulation of putrescine in rat pancreas and liver

3-Isobutylmethylxanthine (IBMX), a potent phosphodiesterase inhibitor, causes accumulation of putrescine of same magnitude in rat pancreas and liver. IBMX produces increases of acetyl CoA: polyamine N'-acetyltransferase (PAT) and of ornithine decarboxylase (ODC) activities in both organs. However ODC activity is 300 times higher in liver than in pancreas. In the latter organ, there is a transient increase of N1-acetylspermidine, followed by a decrease of spermidine, alpha-Difluoromethylornithine (DFMO), a potent ODC inhibitor, impairs the accumulation of putrescine in liver but not in pancreas. These results suggest that in pancreas the accumulated putrescine is essentially formed from spermidine, via N1-acetylation and oxidation, while in liver it is formed from decarboxylation of ornithine. A possible involvement of cAMP in the stimulation of the polyamine interconversion pathway is discussed.     

Glucocorticoid Regulation of Spermidine Acetylation in the Rat Brain

The effect of glucocorticoids on polyamine metabolism has been elucidated further by measuring putrescine, spermidine, and spermine levels as well as ornithine decarboxylase, S-adenosylmethionine decarboxylase, and N1-acetylspermidine transferase activities in the hippocampus, cerebellar cortex, vermis, and deep nuclei of adrenalectomized rats. At 6 h after corticosterone or dexamethasone administration, the specific activities of ornithine decarboxylase and N1-acetylspermidine transferase showed the greatest increases in all brain tissues examined, and at 12 h, S-adenosylmethionine decarboxylase activity was not increased significantly. The hippocampus and cerebellar regions displayed different responses to corticosterone and dexamethasone, corresponding to the distribution of glucocorticoid and mineralocorticoid receptors. Corticosterone and dexamethasone increased ornithine decarboxylase and N1-acetylspermidine transferase activities in a dose-dependent manner, with dexamethasone being more active than corticosterone in all tissues. However, estradiol, progesterone, testosterone, and aldosterone were only active at doses greater than 5 mg/kg. The great increases in ornithine decarboxylase and N1-acetylspermidine transferase activities were accompanied by a marked increase in putrescine level and a small decrease in spermidine level. Our data confirm that the hippocampus and cerebellum are glucocorticoid target tissues and suggest that the increase in the content of putrescine, following acute treatment with glucocorticoids, is dependent on ornithine decarboxylase as well as N1-acetylspermidine transferase induction.     

Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury

The polyamine system is very sensitive to different pathological states of the brain and is perturbed after CNS injury. The main modifications are significant increases in ornithine decarboxylase activity and an increase in tissue putrescine levels. Previously we have shown that the specific polyamine oxidase (PAO) inhibitor N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) reduced the tissue putrescine levels, edema, and infarct volume after transient focal cerebral ischemia in spontaneously hypertensive rats and traumatic brain injury of Sprague-Dawley rats. In the present study, N1-acetyl-spermidine accumulation was greater in injured brain regions compared with sham or contralateral regions following inhibition of PAO by MDL 72527. This indicates spermidine/spermine-N1-acetyltransferase (SSAT) activation after CNS injury. The observed increase in N1-acetylspermidine levels at 1 day after CNS trauma paralleled the decrease in putrescine levels after treatment with MDL 72527. This suggests that the increased putrescine formation at 1 day after CNS injury is mediated by the SSAT/PAO pathway, consistent with increased SSAT mRNA after transient ischemia.     

Polyamine homeostasis in arginase knockout mice

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine-N¹-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues. ornithine; putrescine; spermidine; spermine; decarboxylase     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Polyamines in mycoplasmas and in mycoplasma-infected tumour cells.

Three out of four different mycoplasma strains analysed for the polyamine contents contained relatively high concentrations of putrescine, cadaverine, spermidine and spermine. In addition to ornithine decarboxylase (EC 4.1.1.17) activity, the mycoplasmas also exhibited comparable or higher lysine decarboxylase (EC 4.1.1.18) activity fully resistant to the action of 2-difluoromethylornithine, an irreversible inhibitor of eukaryotic ornithine decarboxylase. 2-Difluoromethylornithine did not modify the polyamine pattern of actively growing mycoplasmas. Ehrlich ascites carcinoma cells and L1210 mouse leukemia cells infected with any of the four mycoplasma strains contained, in addition to putrescine, spermidine and spermine, and also easily measurable concentrations of cadaverine; the latter diamine was absent in uninfected cultures. When the infected cells were exposed to difluoromethylornithine, the accumulation of cadaverine was markedly enhanced. The modification of cellular polyamine pattern by mycoplasmas, especially in the presence of inhibitors of eukaryotic ornithine decarboxylase, could conceivably be used as an indicator of mycoplasma infection in cultured animal cells.     

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine activities in rat spleen

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N1-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N1-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N1-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N1-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.     

Polyamine Synthesis and Interconversion by the Microsporidian Encephalitozoon cuniculi

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.     

Genetic manipulation of polyamine catabolism in rodents

Activation of polyamine catabolism through the overexpression of spermidine/spermine N1-acetyltransferase (SSAT) in transgenic rodents does not only lead to distorted tissue polyamine homeostasis, manifested as striking accumulation of putrescine, appearance N1-acetylspermidine and reduction of tissue spermidine and/or spermine pools, but likewise creates striking phenotypic changes. The latter include loss of hair, lipoatrophy and female infertility. Forced expression of SSAT modulates skin, prostate and intestinal carcinogenesis, induces acute pancreatitis and blocks early liver regeneration. Although many of these features are directly attributable to altered tissue polyamine pools, some of them are more likely related to the greatly accelerated flux of the polyamines caused by activated catabolism and compensatorily enhanced biosynthesis.     

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.     

Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase.

Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1-acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S-adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.