Inhibition of placental ornithine decarboxylase by DL-alpha-difluoro-methyl ornithine causes fetal growth restriction in rat

The roles of polyamines in intrauterine growth restriction (IUGR) is studied. The DL-alpha-difluoromethyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) which is a rate limiting enzyme of polyamine synthesis was administrated to pregnant rats so that we obtained rat fetuses with IUGR. The changes of maternal nutrition, damage of the placenta, and the direct effect of DFMO on the fetus were examined in this IUGR model. Administration of DFMO did not induced changes of maternal nutrition except for triglyceride and the fetal metabolic state. But the placental weight, ODC activity, and DNA in the placenta were decreased significantly. The ODC activity in the total placenta decreased to less than 10% of that of the control. Depression of ODC activity in the placenta may be the major cause of IUGR induced by DFMO administration, and polyamines play important roles to carry pregnancy.     

Increased ornithine decarboxylase activity and polyamine biosynthesis are required for optimal cytolytic T lymphocyte induction

The objective of the present investigation was to evaluate the requirement for increased ornithine decarboxylase (ODC) activity and polyamine biosynthesis in the induction of cytolytic T lymphocytes (CTL). In this regard, we have utilized alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO treatment completely abrogated Con A-induced NW T-cell ODC activity. Similarly, DFMO treatment reduced putrescine and spermidine biosynthesis 100 and 87% respectively by the end of a 48-hr incubation period. Polyamine depletion reduced the Con A-mediated polyclonal induction of CTL by 52 and 81% at 24 and 48 hr of culture, respectively. The effect of DFMO on CTL induction could be reversed by the addition of exogenous putrescine. These data indicate that the observed effects of DFMO on CTL induction were mediated through inhibition of polyamine biosynthesis. Therefore, increased ODC activity and polyamine biosynthesis are required for optimal CTL induction. Furthermore, polyamine depletion did not impair IL-2 production; however, IL-2-dependent proliferation was reduced. These data are the first to discriminate between the requirement for polyamines with regard to IL-2 responsiveness, rather than IL-2 production, during a primary T-cell mitogenic response.     

Ornithine decarboxylase induction and polyamine biosynthesis are required for the growth of interleukin-2- and interleukin-3-dependent cell lines

The objective of this study was to evaluate induction of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, and subsequent polyamine accumulation in interleukin-2 (IL-2)- and interleukin-3 (IL-3)-dependent growth. The CTLL-20 and FDC-P1 cell lines, which have been shown to be absolutely dependent on IL-2 and IL-3, respectively, were used in these studies. The CTLL-20 and FDC-P1 cells each had different temporal patterns of ODC induction following lymphokine stimulation. ODC levels increased rapidly in the FDC-P1 cells, peaking 4 hr after stimulation with IL-3. In contrast, peak ODC activity in the CTLL-20 cells occurred 18 hr following stimulation with IL-2 and reached eightfold higher levels than those observed in the FDC-P1 cells. Treatment with D,L-alpha-difluoromethylornithine X HCl X H2O (DFMO), a specific irreversible inhibitor of ODC activity, completely abrogated lymphokine-dependent ODC induction in both the CTLL-20 and FDC-P1 cell lines. Similarly, intracellular levels of the polyamines putrescine and spermidine were reduced in both cell lines following DFMO treatment. DFMO treatment reduced both IL-2- and IL-3-dependent proliferation in a dose-dependent manner. However, this inhibition could be reversed by the addition of exogenous putrescine. DFMO treatment had no effect on cell viability. Polyamine-depleted CTLL-20 and FDC-P1 cells showed decreased absorption of IL-2 and IL-3 activity, respectively. However, the addition of exogenous putrescine restored the ability of the cells to absorb the appropriate lymphokine. These data are the first to demonstrate that ODC induction and polyamine biosynthesis are required in lymphokine dependent growth.     

Polyamines are intracellular messengers in the beta-adrenergic regulation of Ca2+ fluxes, [Ca2+]i and membrane transport in rat heart myocytes

The beta-adrenergic agonist 1-isoproterenol (0.1 microM) evokes an acute (less than 5-10 sec) transient increase in the activity of ornithine decarboxylase (ODC), and the levels of polyamines (putrescine, spermidine, spermine) in acutely isolated rat ventricular myocytes. Isoproterenol rapidly (less than 15 sec) increases 45Ca influx and efflux, decreases [Ca2+]i, and stimulates Ca2+-dependent membrane transport (endocytosis, hexose transport, amino acid transport). The beta-adrenergic antagonist propranolol blocks isoproterenol-induced membrane transport. The ODC inhibitor alpha-difluoromethylornithine (DFMO, 5-10 mM) blocks the isoproterenol-evoked increase in ODC activity and polyamine levels and the changes in 45Ca fluxes, [Ca2+]i and membrane transport. Putrescine (0.5-1 mM) replenishes cellular polyamines and reverses the DFMO effect. These data exclude an increase in [Ca2+]i in stimulus-transport coupling, and support the hypothesis that polyamines are messengers in beta-adrenoceptor-mediated regulation of transmembrane Ca2+ fluxes, [Ca2+]i, and Ca2+-dependent membrane transport.     

On the translational control of ornithine decarboxylase expression by polyamines

Ornithine decarboxylase (ODC, EC 4.1.1.17) expression is subject to negative feedback regulation by the polyamines. The results of previous studies favor either translational or post-translational regulation. To facilitate further analysis of the mechanism by which polyamines affect ODC expression we have used a cell line (L1210-DFMOr) that overproduces ODC. This cell line was isolated by selection for resistance to the antiproliferative effect of the ODC inhibitor alpha-difluoromethylornithine (DFMO). These cells respond similarly to polyamine depletion and repletion as do their wild-type counterparts. When L1210-DFMOr cells were grown in the presence of 20 mM DFMO (i.e., when their polyamine content was reduced to an extent that still permitted a normal growth rate) ODC represented 4-5% of the soluble protein synthesized. After transfer of the cells to a medium lacking DFMO (i.e., when their polyamine pools were repleted), the rate of incorporation of [35S]methionine into ODC was one order of magnitude lower. Since this difference in incorporation of radioactivity into ODC remained the same irrespective of the pulse-label time used (between 2 and 20 min) it is likely to represent a true difference in ODC synthesis rate. Consequently, the pulse-label experiments cannot be explained by rapid degradation of the enzyme during the labeling period. The difference in ODC synthesis rate was not accompanied by a corresponding difference in the steady-state level of ODC mRNA. Analyses of the distribution of ODC mRNA in polysome profiles did not demonstrate any major difference between cells grown in the absence or presence of DFMO, even though the ODC synthesis rate differed by as much as 10-fold. However, the distribution of the ODC mRNA in the polysome profiles indicated that the message was poorly translated. Thus, most of the ODC mRNA was present in fractions containing ribosomal subunits or monosomes. Inhibition of elongation by cycloheximide treatment resulted in a shift of the ODC mRNA from the region of the gradient containing ribosomal subunits to that containing mono- and polysomes, indicating that most of the ODC mRNA was accessible to translation. Taken together these data lend support to a translational control mechanism which involves both initiation and elongation.     

Role of ornithine decarboxylase in proliferation of prolactin-dependent lymphoma cells

Mitogenic stimulation of Nb2 lymphoma cells by lactogenic hormones (prolactin, human growth hormone) caused a dramatic early increase in ornithine decarboxylase (ODC) activity that achieved a maximal level by 6-8 h. A marked increase in ODC activity was also generated when cells which had reached a growth plateau were transferred to fresh medium that did not stimulate growth. Furthermore, low concentrations of human growth hormone (20 pg/mL) elicited a proliferative response, but did not cause a detectable early increase in ODC activity. The early peak of ODC activity thus appeared not to be directly involved in mediating lactogen-stimulated growth nor was it required to support the mitogenic response. However, prolonged suppression of ODC activity by DL-alpha-difluoromethylornithine (DFMO) (200 microM) attenuated the growth of Nb2 cells (50-60% inhibition), indicating that normal cell growth was dependent on ODC and polyamine biosynthesis. Under these conditions, putrescine, the enzyme product, or the polyamines spermidine and spermine restored normal cell growth when added at a concentration of 1 microM or greater. Nb2-SP cells, variants which proliferate in the absence of prolactin, were about two times more resistant to the growth suppressive effects of DFMO than prolactin-responsive Nb2 cells.     

Growth regulatory effects of cyclic AMP and polyamine depletion are dissociable in cultured mouse lymphoma cells

Treatment of mouse lymphoma S49 cells with D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in S49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a S49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases.     

Polyamines and Ca2+ Mediate Hyperosmolal Opening of the Blood-Brain Barrier: In Vitro Studies in Isolated Rat Cerebral Capillaries

We recently presented evidence that the reversible opening of the blood-brain barrier (BBB) by the infusion of 1.6 M mannitol into the rat internal carotid artery is mediated by a rapid stimulation of ornithine decarboxylase (ODC) activity and putrescine synthesis in cerebral capillaries. We have now investigated this hypothesis further, using isolated rat cerebral capillaries as an in vitro model of the BBB. The ODC activity of cerebral capillary preparations was enriched up to 15-fold over that of the cerebral homogenate. Hyperosmolal mannitol in physiological buffer evoked a rapid (less than 15 s), concentration- and time-dependent increase in capillary ODC activity and an accumulation of putrescine and spermidine which was blocked by the specific ODC inhibitor, alpha-difluoromethylornithine (DFMO, 10 mM). Mannitol (1 M), as well as 2 M urea, evoked a two- to fivefold increase in the temperature-sensitive influx of 45Ca2+ and uptake of horseradish peroxidase (HRP) and 2-deoxy-D-[1-3H]glucose (DG), but not alpha-[1-14C]aminoisobutyrate, during a 2-min incubation. DFMO (10 mM) abolished 1 M mannitol-mediated stimulation of 45Ca2+ influx and uptake of HRP and DG, whereas 1 mM putrescine replenished capillary polyamines and reversed the DFMO effects. Mannitol (1 M)-induced stimulation of ODC activity and membrane transport processes was Ca2+-dependent and verapamil- and nisoldipine-sensitive. Phorbol myristate acetate (PMA, 10 nM), a protein kinase C activator, also evoked a two- to threefold stimulation of 45Ca2+ transport and HRP and DG uptake. This PMA effect was abolished by DFMO, suggesting involvement of rapid, ODC-controlled polyamine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cyclosporine and alpha-difluoromethylornithine on the growth of mouse colon cancer in vitro

The growth and survival of mouse (MC-26) colon carcinoma in vitro and in vivo are significantly reduced by inhibitors of polyamine biosynthesis. alpha-Difluoromethylornithine (DFMO), is a specific and irreversible inhibitor of ornithine decarboxylase (ODC); the rate-limiting enzyme in polyamine biosynthesis. DFMO treatment inhibits the growth of MC-26 colon cancer cells and decreases MC-26 cell survival both in vitro and in vivo. In the present study, we examined the effects of cyclosporine (CsA) on growth, survival, and polyamine levels in MC-26 colon cancer in vitro. CsA had inhibitory effects on MC-26 colon cancer growth which were similar to DFMO; these effects were blocked by the addition of the polyamine, putrescine. The combination of CsA (8.3 X 10(-4) mM) and DFMO (0.5 mM or 1.0 mM) inhibited MC-26 cell survival to a greater extent than either agent alone. These results suggest that CsA given in combination with other agents which inhibit polyamine synthesis may be useful for the treatment of colon cancer.     

Involvement of the ornithine decarboxylase pathway in macrophage collagenase production

Definition of the cellular events involved in the production of collagenase by macrophages following activation has revealed prostaglandin E2 (PGE2)- and cAMP-dependent steps. Since ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is regulated by cAMP and is associated with certain aspects of protein synthesis, the potential role of this enzyme and its polyamine product, putrescine, in collagenase synthesis was examined. Lipopolysaccharide (LPS) activation of macrophages resulted in a maximal ODC response after 6 to 9 h with a 10- to 12-fold elevation in enzyme activity. This elevation in ODC appeared to be regulated by PGE2 since indomethacin inhibited LPS-induced macrophage ODC levels by 70%. Associated with the indomethacin-mediated inhibition of ODC was a loss of collagenase synthesis. Furthermore, partial restoration of collagenase production in indomethacin-inhibited cultures could be achieved by the addition of putrescine. In additional studies alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, also inhibited collagenase production when added to LPS-treated macrophages. This inhibition by DFMO could be reversed by the exogenous addition of putrescine. These findings demonstrate that the ODC pathway is an important intracellular component in the sequence of events that lead to macrophage collagenase synthesis.     

Polyamine transport systems in the LLC-PK1 renal epithelial established cell line

LLC-PK1 cells were brought to a quiescent state by treatment with DL-2-difluoromethylornithine (DFMO), a specific inhibitor of L-ornithine decarboxylase (ODC). The inhibition of ODC, which is the key enzyme for polyamine synthesis, strongly reduced the cellular content of putrescine and spermidine. The cells resumed DNA-synthesis followed by mitosis when exogenous putrescine was added. DFMO treatment strongly stimulated the putrescine uptake capability. A kinetic analysis of the initial uptake rates revealed a saturable Na+-dependent and a saturable Na+-independent pathway on top of non-saturable diffusion. The stimulation by DFMO was exclusively due to an effect on the Vmax values of the saturable pathways. The Na+-dependent transporter had a higher affinity for putrescine (apparent Km = 4.7 +/- 0.7 microM) than the Na+-independent transporter (apparent Km = 29.8 +/- 3.5 microM). As a consequence, although the latter transporter had a higher Vmax, the Na+-dependent transport was more important at a physiological putrescine concentration. Putrescine uptake by both transporters was inhibited with similar relative affinities by spermidine, spermine as well as by the antileukemic agent, methylglyoxal bis(guanylhydrazone), but not by amino acids. The activity of the Na+-dependent transporter was very much dependent on SH-group reagents, whereas the Na+-independent transporter was not affected. Both transporters were inhibited by metabolic inhibitors and by ionophores but the Na+-dependent transporter was affected to a greater extent. For both transporters there was a down-regulation in response to exogenous putrescine. This suggests that the polyamine transporters in LLC-PK1 are adaptively regulated and may contribute to the regulation of the cellular polyamine level and cellular proliferation.     

An evaluation of the involvement of polyamines in modulating MCF-7 human breast cancer cell proliferation and progesterone receptor levels by estrogen and antiestrogen

The present studies were undertaken to determine the importance of the polyamine biosynthetic pathway in cellular proliferation and hormone-regulated progesterone receptor synthesis in estrogen receptor-containing breast cancer cells. Treatment of MCF-7 cells with difluoromethylornithine (DFMO), the irreversible inhibitor of the enzyme ornithine decarboxylase (ODC), prevented estradiol-induced cell proliferation in a dose-dependent fashion. DFMO inhibition of estradiol-induced cell proliferation was completely recoverable by the addition of exogenous putrescine while putrescine alone did not stimulate proliferation of control cells. ODC activity was 4-fold greater in estrogen-treated cells and DFMO (5 mM) fully inhibited ODC activity. DFMO was able to suppress only slightly further the proliferation of antiestrogen (tamoxifen) treated cells and putrescine was able to recover this DFMO inhibition. In contrast to the suppressive effect of DFMO on cell proliferation, DFMO had no effect on the ability of estrogen to stimulate increased (4-fold elevated) levels of progesterone receptor. Hence, while ODC activity appears important for estrogen-induced cell proliferation, inhibition of the activity of this enzyme has no effect on the ability of estradiol to increase cellular progesterone receptor content.     

Inhibition of ornithine decarboxylase by retinoic acid and difluoromethylornithine in relation to their effects on differentiation and proliferation

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.     

Consequences of aberrant ornithine decarboxylase regulation in rat hepatoma cells

DH23A cells, an α-difluoromethylornithine (DFMO)–resistant variant of rat hepatoma tissue culture cells (HTC), contain high levels of very stable ornithine decarboxylase (ODC). In the absence of DFMO, the high ODC activity results in a large accumulation of endogenous putrescine. Concomitant with the putrescine increase is a period of cytostasis and a subsequent loss of viable cells. In contrast, HTC cells with a moderate polyamine content can be maintained in exponential growth. This suggests that a moderate polyamine concentration is necessary for both optimal cell growth and survival. The cytoxicity observed in the DH23A cells is apparently not due to byproducts of polyamine oxidation or alterations in steady state intracellular pH or free [Ca²⁺]. It is possible to mimic the effects of high levels of stable ODC by treatment of cells with exogenous putrescine in the presence of DFMO. This suggests that overaccumulation of putrescine is the causative agent in the observed cytotoxicity, although the mechanism is unclear. These data support the hypothesis that downregulation of ODC may be necessary to prevent accumulation of cytotoxic concentrations of the polyamines. © 1994 Wiley-Liss, Inc.     

Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells

The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by α-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.     

Ehrlich ascites tumour cells become refractory to alpha-difluoromethylornithine at a certain stage of growth

When Ehrlich ascites tumour cells are induced to proliferate by serum stimulation, the ornithine decarboxylase (ODC) activity increases rapidly and reaches two to three peaks during the first 24 h. Inhibition of the first peak in ODC activity (occurring at 4 h) by adding alpha-difluoromethylornithine (DFMO) within 2 h of serum stimulation, results in maximal growth inhibition. Under these conditions, similar degrees of polyamine depletion are achieved. When DFMO is added 3 h after seeding, however, enough polyamines have already accumulated during the initial burst in ODC activity to reduce the antiproliferative effect of the drug. The antiproliferative effect is further reduced when DFMO is added 6 h after seeding. When DFMO is added 23 h after seeding, i.e. after maximal accumulation of polyamines, there is no inhibition of cell proliferation. These findings are important to consider both when designing experimental as well as clinical regimens for this drug.     

Thyroid function and polyamines. III. Changes in ornithine decarboxylase activity and polyamine contents in the rat thyroid during hyperplasia and involution

Changes in ornithine decarboxylase (ODC) activity and in polyamine contents of the rat thyroid were studied under various experimental conditions. Methylthiouracil (MTU) treatment produced several-fold increases in the thyroid ODC activity and in the content of putrescine, spermidine and spermine within a week. While serum thyrotropin (TSH) levels increased gradually up to 3 weeks, the content of both putrescine and spermidine tended to reach a plateau after 2 weeks of the goitrogen treatment; spermine content continued to increase progressively for 3 weeks. Discontinuance of MTU at 7 days resulted in a rapid decline in the elevated thyroid ODC activity, followed by a diminution of putrescine, spermidine and RNA contents. Thyroidal putrescine, spermidine and RNA responded more sensitively to both introduction and withdrawal of TSH stimulation than thyroidal spermine and DNA. Excess iodide, having no effect on the basal level of thyroid ODC, suppressed the MTU-induced increase in this enzyme activity without affecting circulating TSH, thyroxine (T4) and triiodothyronine (T3) levels. There was a significant negative correlation between the ODC activity and intrathyroidal concentration of iodine in MTU-pretreated rats. Theophylline increased the thyroid weight and ODC activity when given to rats fed with a subeffective dose of MTU. Analyses of serum TSH, T4, T3 and of thyroidal iodine revealed that TSH-induced thyroid ODC activity was suppressed by increased circulating thyroid hormones and/or intrathyroidal iodine. Furthermore, it was suggested that thyroid hormones and excess iodide acted directly on the thyroid to alter polyamine biosynthesis, possibly by changing the responsiveness of the gland to TSH.