Regulation of N-Methyl-d-aspartate receptor-mediated calcium transport and norepinephrine release in rat hippocampus synaptosomes by polyamines

The role of polyamines (PA) synthesis in NMDA receptor-mediated⁴⁵Ca²⁺ fluxes and norepinephrine release was studied in rat hippocampal synaptosomes. NMDA (50M) caused a sharp (>2-fold) transient increase in PA synthesis regulating enzyme, ornithine decarboxylase (ODC) activity with concomitant elevation in PA levels in the order putrescine>spermidine>spermine. ODC inhibitor, -difluoromethylornithine (DFMO), and NMDA antagonist, 2-amino-5-phosphonovaleric acid (D-AP5), both blocked increases in ODC activity and PA levels. Activation of NMDA receptors induced a sharp (3 to 4-fold) and quick (15 seconds) increase in⁴⁵Ca²⁺ uptake by synaptosomes within 15 seconds of exposure at 37°C. The efflux of⁴⁵Ca²⁺ and³H-norepinephrine (NE) release at 22°C from pre-loaded synaptosomes was also significantly (2 to 4-fold) enhanced by NMDA within 15 seconds. These NMDA receptor-mediated effects on calcium fluxes and NE release were blocked by NMDA receptor-antagonists (DAP-5 and MK-801) and PA synthesis inhibitor, DFMO and the DFMO inhibition nullified by exogenous putrescine. These observations establish that ODC/PA cascade play an important role in transduction of excitatory amino acid mediated signals at NMDA receptors.Special issue dedicated to Dr. Sidney Ochs.     

Excitotoxin induction of ornithine decarboxylase in cerebral cortex is reduced by phospholipase A2 inhibition.

The enzyme ornithine decarboxylase (ODC) has been shown to be induced by a number of conditions such as cold-injury, kindling, ischaemia and excitotoxin injection. In previous studies we have characterised the cortical response to kainate injection into the nucleus basalis and shown a substantial increase in both ODC mRNA and enzyme activity which reaches a maximum at 8h. This response is completely prevented by treatment with MK-801, indicating the involvement of NMDA receptors in mediating this response. Whilst NMDA receptors are known to gate a cation channel leading to increased calcium entry, an additional effect on the release of arachidonic acid has been reported. The possibility that NMDA receptor mediated activation of phospholipase A2 and release of arachidonic acid might mediate this ODC response was investigated in this study by treatment with the phospholipase inhibitors quinacrine and dexamethasone. Treatment of animals with quinacrine (100 mg/kg) at the time of injection of kainate into the nucleus basalis caused a significant attenuation of the induction of ODC in cerebral cortex of 43%. No further attenuation was seen at higher doses. A similar reduction in ODC induction was seen after treatment with dexamethasone (1 mg/kg) but a greater effect could be obtained (65% attenuation) at higher doses. The possible involvement of arachidonic acid derivatives in mediating ODC induction was further investigated by treatment with the cyclo-oxygenase inhibitor indomethacin and the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Indomethacin was able to significantly attenuate the induction of ODC (greater than 60%) whilst NDGA (30 mg/kg) was ineffective. These results indicate the possible role of arachidonic acid derivatives in the regulation of the expression of ODC in cerebral cortex after excitotoxin injection.     

Chronic neonatal blockade of NMDA receptor does not affect developmental polyamine metabolism but results in altered response to the excitotoxic induction of ornithine decarboxylase

Neonatal rats were subjected to chronic blockade of the (NMDA) receptor through daily systemic administration of increasing doses of the competitive antagonist CGP 39551 from postnatal days 1–22. Treatment did not result in any significant alteration of the levels of putrescine, spermidine and spermine or in the constitutively expressed activity of the key enzyme for polyamine biosynthesis, ornithine decarboxylase (ODC), as evaluated at 10 and 20 days of age. However, in 30-day-old rats significant differences were observed in the process of excitotoxic ODC induction in the olfactory cortex and the hippocampus of chronically-treated rats: the increase of ODC activity caused by systemic administration of kainic acid took place more rapidly but it was shorter and apparently reached a smaller peak in treated animals as compared to controls. This result, in conjunction with previous data on neurochemistry and locomotor activity of similarly treated rats, strengthens the suggestion that functional alterations of some brain circuits may be the consequence of the blockade of NMDA receptor during the critical neonatal period of brain maturation.     

Induction of Ornithine Decarboxylase by N-Methyl-D-Aspartate Receptor Activation Is Unrelated to Potentiation of Glutamate Excitotoxicity by Polyamines in Cerebellar Granule Neurons

Abstract: Polyamines positively modulate the activity of the N -methyl- d -aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)—the key regulatory enzyme in polyamine synthesis—and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.     

Induction of Ornithine Decarboxylase in Cerebral Cortex by Excitotoxin Lesion of Nucleus Basalis: Association with Postsynaptic Responsiveness and N-Methyl-d-Aspartate Receptor Activation

The major cholinergic innervation of the rat cerebral cortex arises from the nucleus basalis in the basal forebrain. Introduction of the excitotoxins kainate or ibotenate into the nucleus basalis by stereotaxic injection results in degeneration of the cholinergic cells. We have investigated the effect of this excitotoxic action on ornithine decarboxylase (ODC) activity and cholinergic responsiveness in the cerebral cortex. A massive and rapid induction of ODC activity was seen in ipsilateral cortex after injection of excitotoxin. A maximal increase in ODC activity of 268 times the control value was seen in ipsilateral cerebral cortex 8 h after lesioning. Thereafter, ODC activity declined but remained significantly greater than control levels for 32 h. Pretreatment of animals with the irreversible ODC inhibitor difluoromethylornithine prevented the induction of ODC by kainate. Tissue content of the ODC product putrescine showed a marked increase in cerebral cortex ipsilateral to the lesion, increasing sevenfold at 24 h, the maximal concentration reached. After 24 h, the level of putrescine decreased but remained significantly elevated above control values for 5 days. Levels of the polyamines spermidine and spermine were unaffected by lesioning. Increases on ODC activity of much smaller magnitude were also seen in brain regions not directly innervated from the ipsilateral nucleus basalis. However, the response in ipsilateral cortex was found to be dependent on an intact projection from nucleus basalis to cortex. The induction of ODC was shown to be prevented by treatment of rats with MK-801, a result indicating the involvement of N-methyl-D-aspartate (NMDA) receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ornithine Decarboxylase Activity in In Vivo and In Vitro Models of Cerebral Ischemia

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis, and an increase in putrescine after central nervous system (CNS) injury appears to be involved in neuronal death. Cerebral ischemia and reperfusion trigger an active series of metabolic events, which eventually lead to neuronal death. In the present study, ODC activity was evaluated following transient focal cerebral ischemia and reperfusion in rat. The middle cerebral artery (MCA) was occluded for 2 h in male rats with an intraluminal suture technique. Animals were sacrificed between 3 and 48 h of reperfusion following MCA occlusion, and ODC activity was assayed in cortex and striatum. ODC activity was also estimated in an in vitro ischemia model using primary rat cortical neuron cultures, at 6–24 h reoxygenation following 1 h oxygen-glucose deprivation (OGD). In cortex, following ischemia, ODC activity was increased at 3 h (P < .05), reached peak levels by 6–9 h (P < .001) and returned to sham levels by 48 h reperfusion. In striatum the ODC activity followed a similar time course, but returned to basal levels by 24 h. This suggests that ODC activity is upregulated in rat CNS following transient focal ischemia and its time course of activation is region specific. In vitro, ODC activity showed a significant rise only at 24 h reoxygenation following ischemic insult. The release of lactate dehydrogenase (LDH), an indicator for cell damage, was also significantly elevated after OGD. 0.25 mM -difluoromethylornithine (DFMO) inhibited ischemia-induced ODC activity, whereas a 10-mM dose of DFMO appears to provide some neuroprotection by suppressing both ODC activity and LDH release in neuronal cultures, suggesting the involvement of polyamines in the development of neuronal cell death.     

In vivo inhibition of polyamine biosynthesis and growth in tobacco ovary tissues

Post fertilization growth of tobacco ovary tissues treated with inhibitors of polyamine (PA) biosynthesis was examined in relation to endogenous PA titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17). DL-alpha-Difluoromethylornithine (DFMO) and DL-alpha-difluoromethylarginine (DFMA), specific, irreversible ("suicide") inhibitors of ODC and ADC in vitro, were used to modulate PA biosynthesis in excised flowers. ODC represented >99% of the total decarboxylase activity in tobacco ovaries. In vivo inhibition of ODC with DFMO resulted in a significant decrease in PA titers, ovary fresh weight and protein content. Simultaneous inhibition of both decarboxylases by DFMO and DFMA produced only a marginally greater depression in growth and PA titers, indicating that ODC activity is rate-limiting for PA biosynthesis in these tissues. Paradoxically, DFMA alone inhibited PA biosynthesis, not as a result of a specific inhibition of ADC, but primarily through the inactivation of ODC. In vivo inhibition of ODC by DFMA appears to result from arginase-mediated hydrolysis of this inhibitor to urea and DFMO, the suicide substrate for ODC. Putrescine conjugates in tobacco appear to function as a storage form of this amine which, upon hydrolysis, may contribute to Put homeostasis during growth.     

Changes in Polyamine Biosynthesis Associated with Postfertilization Growth and Development in Tobacco Ovary Tissues

Polyamine (PA) titers and the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17), enzymes which catalyze rate-limiting steps in PA biosynthesis, were monitored during tobacco ovary maturation. In the period between anthesis and fertilization, the protein content of ovary tissues rapidly increased by about 40% and was accompanied by approximately a 3-fold increase in ODC activity, while ADC activity remained nearly constant. PA titers also remained relatively unchanged until fertilization, at which time they increased dramatically and the DNA content of ovary tissues doubled. This increase in PA biosynthesis was correlated with a further 3-fold increase in ODC activity, reaching a maximum 3 to 4 days after fertilization. During this time, ADC activity increased only slightly and accounted for approximately 1% of the total decarboxylase activity when ODC activity peaked. The postfertilization burst of biosynthetic activities slightly preceded a period of rapid ovary enlargement, presumably due to new cell division. During later stages of ovary development, DNA levels fell precipitously, while PA titers and decarboxylase activities decreased to preanthesis levels more slowly. In this period, growth producing a 300% increase in ovary fresh weight appears to be the result of cell enlargement.

Synchronous changes in PA titers and in the rates of PA biosynthesis, macromolecular synthesis, and growth in the tobacco ovary suggest that PAs may play a role in the regulation of postfertilization growth and development of this reproductive organ.

     

Ornithine decarboxylase activity as a marker of androgen and antiandrogen action in the rat epididymis

After castration, there was a marked decrease in serum androgen concentration at 6 h, and a dramatic inhibition of ornithine decarboxylase (ODC) at 12 h. Administration of testosterone propionate to castrated rats at a dose of 0.05 mg/animal restored ODC activity to the normal value. However, no change was observed when intact rats were treated with testosterone even at a 40-fold higher dose, indicating that endogenous androgens present in intact rats are far in excess for maintenance of maximal levels of activity. Administration of the antiandrogen flutamide to intact rats caused a moderate decrease in epididymal weight, whereas this effect was more pronounced in castrated, androgen-treated rats. In the latter, the effect of flutamide was significant at the lowest dose used (0.5 mg/day). ODC activity was significantly decreased by flutamide treatment of intact rats, but even at the highest dose used (10 mg/day) only a 39% inhibition was observed. In flutamide-treated rats, LH concentrations were markedly increased, as were serum and epididymal androgens. In androgen-treated castrated rats, flutamide caused epididymal ODC to fall to undetectable values. These results show that: (1) androgens are essential for the maintenance of ODC activity in the epididymis; (2) epididymal ODC activity is maximally stimulated by endogenous androgens, at least in the pubertal rat; (3) the apparent potency of flutamide is substantially lowered by an increase in epididymal androgens. We suggest that ODC is a sensitive marker of the action of androgens and antiandrogens in the epididymis.     

Hyperbaric oxygen treatment: the influence on the hippocampal superoxide dismutase and Na+,K+-ATPase activities in global cerebral ischemia-exposed rats

The influence of hyperbaric oxygen (HBO) treatment on the activities of superoxide dismutase (SOD) and Na⁺,K⁺-ATPase was determined during different time periods of reperfusion in rats exposed to global cerebral ischemia. Ischemic animals were either sacrificed or exposed to the first HBO treatment 2, 24, 48 or 168 h after ischemic insult (for SOD activities measurement) or immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na⁺,K⁺-ATPase activities measurement). Hyperbaric oxygenation procedure was repeated for seven consecutive days. The results of presented experiments demonstrated the statistically significant increase in the hippocampal SOD activity 24 and 48 h after global cerebral ischemia followed by a decrease in the enzymatic activity 168 h after ischemic insult. In the ischemic rats treated with HBO the level of hippocampal SOD activity was significantly higher after 168 h of reperfusion in comparison to the ischemic, non HBO-treated animals. In addition, it was found that global cerebral ischemia induced a statistically significant decrease of the hippocampal Na⁺,K⁺-ATPase activity starting from 1 to 168 h of reperfusion. Maximal enzymatic inhibition was obtained 24 h after the ischemic damage. Decline in Na⁺,K⁺-ATPase activity was prevented in the animals exposed to HBO treatment within the first 24 h of reperfusion. Our results suggest that global cerebral ischemia induces significant alterations in the hippocampal SOD and Na⁺,K⁺-ATPase activities during different periods of reperfusion. Enhanced SOD activity and preserved Na⁺,K⁺-ATPase activity within particular periods of reperfusion, could be indicators of a possible benefitial role of HBO treatment in severe brain ischemia.     

Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats

Accumulating evidence implicates activation (phosphorylation) of mitogen-activated protein kinases (MAPK) during nonlethal ischemic preconditioning in the protection of hippocampal CA1 neuron against subsequent ischemic events. In this paper, we undertook to identify the role of extracellular signal regulated kinase (ERK) 5 in cerebral ischemic preconditioning (CIP). Three minutes of ischemia was induced as preconditioning stimulus. Three days later, 6 min of ischemia was induced. The levels of ERK5 protein expression and its activation were detected with or without the CIP in hippocampal CA1 and the dentate gyrus (DG) regions. Our results showed that ERK5 was activated selectively in hippocampal CA1 region with, but not without, the ischemic preconditioning. Notably, during the later phase of reperfusion, the rise in ERK5 activation was strong and persistent with a peak occurring at the third day. The activation peak was effectively prevented and ERK5 protein expression was significantly decreased by intracerebroventricular infusion of ERK5 antisense oligonucleotide (every 24 h for 3 days before the preconditioning), but not by sense oligonucleotide or vehicle. Subsequently, the CA1 neuronal loss was largely elevated. Moreover, both MK801 (10 microM), an antagonist of NMDA receptor, and EGTA (100 mM, but neither 50 nor 150 mM), an extracellular Ca2+ chelator, not only effectively inhibited the ERK5 activation but also markedly abolished CIP-induced survival of the CA1 neurons. These results suggested that activation of the ERK5 pathway by CIP was at least partly dependent on moderate Ca2+ influx via NMDA receptor, which might contribute to ischemic tolerance in hippocampal CA1 region of rats.     

Plasminogen activator production in a rat model of Pneumocystis carinii pneumonia

Several studies have indicated that the serine protease urokinase-plasminogen-activator (uPA) is an important factor in host defense against pulmonary pathogens. To gain a better insight into the role of uPA in Pneumocystis carinii (P. carinii) pneumonia (PCP), we evaluated PA production in alveolar macrophages (AMs) obtained from rats with steroid-induced PCP. Treatment with cortisone acetate favored PCP in 91% of rats. In the bronchoalveolar lavage (BAL) samples of immunosuppressed rats both with and without PCP, we observed a decrease in uPA activity as well as a decrease in cell number. Urokinase-PA production by AMs was reduced in rats treated with cortisone alone. However, an increase in cell-associated uPA was observed in rats with PCP. This increase appears to be produced in response to P carinii infection. In fact, when AMs obtained from untreated healthy or immunosuppressed uninfected rats were challenged with P carinii, a significant increase in PA activity in cell lysates was observed, though a lower response was obtained in cortisone-treated animals. Our results suggest that healthy AMs respond to the presence of P carinii with an increase in uPA production and that this response in immunodepressed rat-AMs is partially impaired.     

Effect of Glutamine Synthesis Inhibition with Methionine Sulfoximine on the Nitric Oxide–Cyclic GMP Pathway in the Rat Striatum Treated Acutely with Ammonia: A Microdialysis Study

Ammonia neurotoxicity is associated with overactivation of N-methyl-d-aspartate (NMDA) receptors leading to enhanced nitric oxide and cyclic GMP synthesis and to accumulation of reactive oxygen and nitrogen species. Ammonia is detoxified in the brain via synthesis of glutamine, which if accumulated in excess contributes to astrocytic swelling, mitochondrial dysfunction and cerebral edema. This study was aimed at testing the hypothesis that the activity of the NMDA/NO/cGMP pathway is controlled by the ammonia-induced production of Gln in the brain. Ammonium chloride (final concentration 5 mM), infused for 40 min to the rat striatum via a microdialysis probe, caused a significant increase in Gln (by 40%), NO oxidation products (nitrite+nitrate = NOx) (by 35%) and cGMP (by 50%) concentration in the microdialysate. A Gln synthetase inhibitor, methionine sulfoximine (MSO, 5 mM), added directly to the microdialysate, completely prevented ammonia-mediated production of Gln, and paradoxically, it increased ammonia-mediated production of NOx and cGMP by 230% and 250%, respectively. Of note, MSO given alone significantly reduced basal Gln concentration in the rat striatum, had no effect on the basal NOx concentration, and attenuated basal concentration of cGMP in the microdialysate by 50%. The results of the present study suggest that Gln, at physiological concentrations, may ameliorate excessive activation of the NO–cGMP pathway by neurotoxic concentrations of ammonia. However, in view of potential direct interference of MSO with the pathway, exogenously added Gln and less toxic modulators of Gln content and/or transport will have to be employed in further studies on the underlying mechanisms. Special issue article in honor of Dr. Frode Fonnum. Wojciech Hilgier and Michal Węgrzynowicz contributed equally to this work.     

(−)-Epigallocatechin Gallate Protects Against Cerebral Ischemia-Induced Oxidative Stress via Nrf2/ARE Signaling

(?)-Epigallocatechin gallate (EGCG) has recently been shown to exert neuroprotection in a variety of neurological diseases; however, its role and the underlying mechanisms in cerebral ischemic injury are not fully understood. This study was conducted to investigate the potential neuroprotective effects of EGCG and the possible role of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway in the putative neuroprotection against experimental stroke in rats. The results revealed that EGCG exhibit significant neuroprotection, as evidenced by reduced infarction size and the decrease in transferase dUTP nick end labeling-positive neurons. Furthermore, EGCG also enhanced levels of Nrf2 and its downstream ARE pathway genes such as heme oxygenase-1, glutamate-cysteine ligase modulatory subunit and glutamate-cysteine ligase regulatory subunit, as compared to control groups. In accordance with its induction of Nrf2 activation, EGCG exerted a robust attenuation of reactive oxygen species generation and an increase in glutathione content in ischemic cortex. Taken together, these results demonstrated that EGCG exerted significant antioxidant and neuroprotective effects following focal cerebral ischemia, possibly through the activation of the Nrf2/ARE signaling pathway.     

S-Adenosyl-l-homocysteine in brain

Administration of methionine sulfoximine (MSO) to rats and mice significantly decreased cerebral levels ofS-adenosyl-l-homocysteine (AdoHcy). Concurrent administration of methionine prevented this decrease and, when methionine was given alone, significantly elevated AdoHcy levels resulted in both species. Regionally, AdoHcy levels varied from 20 nmol/g in rat cerebellum and spinal cord to about 60 nmol/g in hypothalamus and midbrain. MSO decreased AdoHcy in all regions tested except striatum, midbrain, and spinal cord. AdoMet/AdoHcy ratios (methylation index) varied from 0.48 in hypothalamus to 2.4 in cerebellum, and MSO administration decreased these ratios in all regions except hypothalamus. AdoHcy hydrolase activity was lowest in hypothalamus, highest in brainstem and, generally, varied inversely with regional AdoHcy levels. MSO decreased AdoHcy hydrolase activity in all regions except hypothalamus and spinal cord. Cycloleucine administration resulted in significantly decreased levels of mouse brain AdoHcy, whereas the administration of dihydroxyphenylalanine (DOPA) failed to affect AdoHcy levels. It is concluded that (a) cerebral AdoHcy levels are more tightly regulated than are those of AdoMet after MSO administration, (b) slight fluctuations of AdoHcy levels may be important in regulating AdoHcy hydrolase activity and hence AdoHcy catabolism in vivo, (c) the AdoMet/AdoHcy ratio reflects the absolute AdoMet concentration rather than the transmethylation flux, (d) the decreased AdoMet levels in midbrain, cortex, and striatum after MSO with no corresponding decrease in AdoHcy suggest an enhanced AdoMet utilization, hence an increased transmethylation in the MSO preconvulsant state.Supported by USPHS, NINCDS grant NS-06294.     

Endobain E, a brain endogenous factor, is present and modulates NMDA receptor in ischemic conditions

We have isolated from rat cerebral cortex an endogenous Na(+), K(+)-ATPase inhibitor, termed endobain E, which modulates glutamatergic N-methyl-d-aspartate (NMDA) receptor. This endogenous factor allosterically decreases [(3)H]dizocilpine binding to NMDA receptor, most likely acting as a weak channel blocker. In the present study we investigated whether endobain E is present in the cerebral cortex of rats subjected to ischemia and modulates NMDA receptor exposed to the same conditions. Ischemia-reperfusion was carried out by bilateral occlusion of common carotid arteries followed by a 15-min reperfusion period. Elution profile of brain soluble fraction showed that endobain E is present in cerebral cortex of ischemia-reperfusion rats. On assaying its effect on synaptosomal membrane Na(+), K(+)-ATPase activity and [(3)H]dizocilpine binding to cerebral cortex membranes prepared from animals without treatment, it was found that the endogenous modulator isolated from ischemia-reperfusion rats was able to inhibit both enzyme activity and ligand binding. On the other hand, endobain E prepared from rats without treatment also decreased binding to cerebral cortex or hippocampal membranes obtained from animals exposed to ischemia-reperfusion. Since ischemia decreases tissue pH and NMDA receptor activity varies according to proton concentration, pH influence on endobain E effect was tested. Endobain E ( approximately 80 mg original tissue) decreased [(3)H]dizocilpine binding 25% at pH 7.4 or 8.0 but 90% at pH 6.5. These results demonstrate that endobain E is present and also able to modulate NMDA receptor in the short-term period that follows cerebral ischemia and that its effect depends on proton concentration, suggesting greater NMDA receptor modulation by endobain E at low pH, typical of ischemic tissues.     

Phenotypic features of breast cancer cells overexpressing ornithine-decarboxylase

Polyamines (PA) have been shown to be critical mediators of estradiol-induced breast cancer cell proliferation. This finding suggests that constitutive activation of the PA pathway may promote tumor progression, possibly leading to hormone independence. To test this hypothesis, we transfected hormone-responsive MCF-7 breast cancer cells with a complementary DNA coding for ornithine-decarboxylase (ODC), the first rate-limiting enzyme in PA biosynthesis. Marked ODC over-expression observed in stably transfected clones was associated with a selective increase in cellular putrescine content, while spermidine and spermine levels were not altered. ODC-overexpressing MCF-7 cells were resistant to the antiproliferative effects of low but not high concentrations of the enzyme inhibitor, α-difluoromethylornithine. In agreement with our hypothesis, sensitivity to the growth-promoting action of estradiol was reduced by approximately one third (P < 0.001) in ODC-overexpressing MCF-7 cells compared with vector-only transfected clones. Basal growth under anchorage-dependent conditions was only marginally increased by ODC overexpression (P = 0.048), while clonogenicity in soft agar was actually reduced. These data suggest that activation of PA biosynthesis may contribute in part to the acquisition of estrogen independence by breast cancer cells. Since only putrescine content was increased as a result of ODC overexpression, these data may underestimate the overall influence of the PA pathway on breast cancer phenotype. © 1995 Wiley-Liss, Inc.