Similarities in ornithine decarboxylase regulation in intact and enucleated 3T3 cells

The role of the cytoplasm in the regulation of ornithine decarboxylase (ODC) has been examined in enucleated 3T3 cells (cytoplasts). ODC activity can be increased 15–25-fold by a cytoplasmic mechanism(s) in enucleates prepared from growing cells by treatment which lead to 50–75-fold increases in intact growing cells. Since the enzyme activity simultaneously becomes less stable in these cytoplasts as in whole cells, it appears this increase is due either to activation of pre-existing enzyme or increased synthesis. A biphasic increase during the first 20 h after stimulus is seen in cytoplasts prepared from growing cells which have been stimulated for the prior 5 h. The second increase in activity does not appear to be due to decreased degradation or conversion to a more active form. These results are analogous to those reported previously for intact growing cells in experiments which employed metabolic inhibitors, and similarly suggest that there is cytoplasmic control of ODC synthesis. Medium polyamines reduce ODC activity in cytoplasts with kinetics and characteristics similar to those previously reported for intact cells. These data are also most consistent with regulation of synthesis at the translational level.     

Spermidine mediates degradation of ornithine decarboxylase by a non-lysosomal, ubiquitin-independent mechanism

The mechanism of spermidine-induced ornithine decarboxylase (ODC, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum-free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine (Glass and Gerner: Biochem. J., 236:351-357, 1986; Sertich et al.: J. Cell Physiol., 127:114-120, 1986). Treatment of cells with 10 microM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [35S]methionine-labeled ODC and separation by two-dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine-mediated inactivation of ODC occurred in a temperature-dependent manner exhibiting pseudo-first-order kinetics over a temperature range of 22-37 degrees C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22 degrees C for 3 hours with 10 microM spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents (NH4Cl, chloroquine, antipain, leupeptin, chymostatin) had no effect on ODC degradation. ODC turnover was not dependent on ubiquitin-dependent proteolysis. Shift of ts85 cells, a temperature-sensitive mutant for ubiquitin conjugation, to 39 degrees C (nonpermissive for ubiquitin-dependent proteolysis) followed by addition of spermidine led to a rapid decline in ODC activity, with a rate similar to that seen at 32 degrees C (the permissive temperature). In contrast, spermidine-mediated ODC degradation was substantially decreased by inhibitors of protein synthesis (cycloheximide, emetine, and puromycin). These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non-lysosomal, ubiquitin-independent pathway.     

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.     

Peroxynitrite inhibits the activity of ornithine decarboxylase

Polyamines are required during cell proliferation, whereas NO has anti-proliferative properties. Ornithine decarboxylase (ODC) is a critical enzyme for the synthesis of polyamines. We tested the hypothesis that the modification of ODC by peroxynitrite (OONO-), a short-lived free radical formed from NO and superoxide produces a fall in ODC activity, and therefore polyamine synthesis and cell proliferation. The treatment of a rat recombinant ODC (rODC) with OONO- resulted in a dose-dependent inhibition of rODC activity with an IC50 of approximately 100 microM. A Western blot employing a specific antibody to nitrotyrosine revealed a dose-dependent nitration of rODC tyrosine residues. When intact IEC-6 cells were treated with ONOO-, ODC activity decreased by 49%. These data suggest a correlation between ODC activity and nitration, and a possible mechanism by which NO synthesis may modulate polyamine synthesis.     

Curcumin induces apoptosis through an ornithine decarboxylase-dependent pathway in human promyelocytic leukemia HL-60 cells

Curcumin, a well-known dietary pigment derived from the food flavoring turmeric (Curcuma longa) exhibits anti-proliferative, anti-inflammatory, and anti-oxidative activities. Recently, studies have shown that a chemopreventive effect of curcumin could be due to the hyperproduction of reactive oxygen species (ROS) inducing apoptosis in tumor cells. In our previous studies, ornithine decarboxylase (ODC) overexpression prevented tumor necrosis factor alpha (TNF-alpha)- and methotrexate-induced apoptosis via reduction of ROS. Furthermore, ODC is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. In this study, we found that enzyme activity and protein expression of ODC were reduced during curcumin treatment. Overexpression of ODC in human promyelocytic leukemia HL-60 parental cells could reduce curcumin-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsi(m)), through reducing intracellular ROS. Moreover, ODC overexpression prevented cytochrome c release and the activation of caspase-9 and caspase-3 following curcumin treatment. These results demonstrate that curcumin-induced apoptosis occurs through a mechanism of down-regulating ODC and along a ROS-dependent mitochondria-mediated pathway.     

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.     

Phorbol myristate acetate inhibits growth in S49 cells: isolation of resistant variants

We have used S49 mouse lymphoma cells to study phorbol ester effects on growth. Treatment of wild-type (wt) cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest within 72 hr. We have selected variants that are resistant to PMA-induced growth arrest, based on a selection in the presence of 10 nM PMA. We have characterized one of these variants, termed 21.1, in detail. The 21.1 and wt cells contain similar levels of protein kinase C (PKC) as determined by [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding. Treatment of both wt and 21.1 cells with PMA results in translocation of PKC to the membrane, suggesting that the coupling between PKC and an immediate biological response is intact. PMA treatment leads to the phosphorylation of many similar proteins in wild-type and 21.1 cells. However, in the 21.1 cells there is a prominent substrate of approximately 70 kilodaltons (kD) which is no longer phosphorylated after PMA treatment. In wild-type cells ornithine decarboxylase (ODC) activity and mRNA levels are decreased within 1 hr of PMA treatment. Likewise, ODC levels are decreased in the 21.1 cells after exposure to PMA even though PMA only slightly modulates the growth of these cells. The 21.1 cells represent a unique line with a dominant phenotype in which ODC expression is uncoupled from the growth state of the cell. These cells may represent a good model system in which to examine the steps involved in phorbol ester growth regulation in S49 cells.     

The generation of oxygen radicals: a positive signal for lymphocyte activation

The induction of ornithine decarboxylase (ODC) activity in lymphocytes is associated with activation and the initiation of cellular proliferation. ODC is also an essential component in tumor promotion. Phorbol myristic acetate (PMA) is a mitogen for lymphocytes, but can also promote tumor formation. Tumor promotion is linked to the generation of free radicals induced by PMA. Modulation of intracellular glutathione is associated lymphocyte activation and in protection of cells from damage due to oxygen radicals. We examined the interaction between ODC activity and intracellular glutathione concentrations in EL4 murine lymphoblastoid cells. The intracellular glutathione concentration could be augmented in EL4 cells when cultured with the cysteine delivery agents 2-oxothiazolidine 4-carboxylate (OTC) and 2-mercaptoethanol (2-ME) and suppressed with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO). OTC and 2-ME suppressed ODC activity in fresh serum and PMA-activated EL4 cells. BSO had no effect on ODC activity of EL4 cells cultured in the presence of PMA. While both OTC and 2-ME augmented the total intracellular glutathione concentration, PMA enhanced only the level of oxidized glutathione. To determine if the mechanism by which PMA or fresh serum altered intracellular glutathione and ODC activity was through the generation of oxygen radicals, EL4 cells were cultured with free radical scavengers. The nonpermeant electron acceptor potassium ferricyanide, and the H2O2 scavenger catalase, lowered ODC activity in both serum-stimulated and PMA-activated EL4 cells. Similarly, incubation of EL4 cells with either potassium ferricyanide or catalase elevated intracellular glutathione concentrations. These data suggest that (a) modulation of intracellular glutathione in the EL4 lymphoblastoid cell line alters ODC activity induced by fresh serum and by the mitogen PMA; (b) activation of EL4 cells by PMA alone alters intracellular glutathione metabolism, which may be associated with its role as a mitogen in lymphocyte activation; and (c) the generation of free radicals in EL4 cells may play a positive role in cellular activation.     

A new perspective on ornithine decarboxylase regulation: prevention of polyamine toxicity is the overriding theme

The polyamines are essential cellular components for growth. Control of a key regulated enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), as a function of growth, is an area of intense interest. A unique regulatory property of ODC is the short half-life of the protein, which has been suggested to be an important factor in rapid activation of polyamine biosynthesis after cells are mitogenically stimulated. In this paper, it is argued that the biological significance of the short half-life of ODC is unrelated to the rate of its induction to a new steady state by growth factors, which is in fact limited by the relatively long half-life of the ODC mRNA. Instead, I suggest that the rapid turnover of ODC protein becomes of significance when cells cease growth and expeditious downregulation of the enzyme is important in preventing polyamine overproduction, which would result in cytotoxicity in the arrested cells. Although mitogenic activation of ODC expression has been studied extensively, there is very little known about the mechanisms controlling downregulation of polyamine biosynthesis during the arrest of animal cell growth. These considerations suggest that this would be a fertile area of future inquiry.     

Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP-dependent protein kinases in CHO cells

Ornithine decarboxylase (ODC) inductions by cholera toxin and by the phorbol ester tumor promoter, TPA, were compared in wild-type Chinese hamster ovary (CHO) cells and in mutant cells having altered cyclic AMP-dependent protein kinase activity. The aim of these studies was to determine whether cyclic AMP-dependent protein kinase is involved in these inductions. The time course and the magnitude of ODC inductions by either 100 ng/ml cholera toxin or 100 ng/ml TPA were similar in wild-type cells with a maximum at 3-4 hours after treatment and a return to unstimulated levels by 8 hours. Induction of ODC by cholera toxin was suppressed more than 80% in the four protein kinase mutants studied (10215, 10248, 10260, and 10265), strongly implicating a cyclic AMP-dependent kinase step in the mechanism of induction. Similar results were found with the cyclic AMP analog 8-Br-cyclic AMP and the phosphodiesterase inhibitor, methyl-isobutylxanthine. The induction of ODC by TPA, on the other hand, was only partially inhibited (approximately 50%) in three of four mutants. Lower ODC activity in two mutants stimulated by cholera toxin or TPA whose kinetics were studied in more detail could not be ascribed to a reduced affinity (Km) of ornithine for the enzyme, but appeared to be due to reduced catalytic activity (Vmax) in the extracts. These results suggest that the induction of ODC by TPA proceeds by a mechanism which is only partially dependent on an intact cyclic AMP-dependent protein kinase activity.     

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by alphaMSH and ornithine decarboxylase inhibition

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by alpha-melanocyte-stimulating hormone (alphaMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, alphaMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or alphaMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor alpha-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with alphaMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to alphaMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by alphaMSH.     

Stable ornithine decarboxylase in a rat hepatoma cell line selected for resistance to alpha-difluoromethylornithine

Ornithine decarboxylase (ODC) is extremely unstable in mammalian cells. This unusual characteristic facilitates rapid fluctuations in the activity of this enzyme in response to variations in its biosynthesis. Unfortunately, very little is known about the mechanism or regulation of this ODC-specific proteolytic pathway. This study describes the production and characterization of a variant of the rat hepatoma HTC cell line that is strikingly deficient in this pathway. This cell variant was induced by selection for growth in stepwise increasing concentrations (up to 10 mM) of the irreversible ODC inhibitor, alpha-difluoromethylornithine (DFMO). Resistance to this inhibitor appears to result from a combination of elevated (10X) ODC biosynthesis and inhibited degradation, producing greater than a 2000-fold increase in the level of ODC protein. In these variant cells (DH23b) inhibition of protein synthesis by cycloheximide did not result in rapid loss of enzyme activity or ODC protein determined by radioimmunoassay. Pulse-chase studies with [35S]methionine confirmed that this enzyme was not preferentially degraded, even when spermidine was added to the media. ODC purified from the variant cells was found to be identical to the control cell enzyme in size, isoelectric point, substrate binding kinetics, and sensitivity to the inhibitor DFMO. Also, as in the control cells, a major fraction of the ODC molecules extracted from DH23b cells was shown to be phosphorylated on a serine residue. The inability to detect physical or kinetic differences between the parent and the variant cell ODC suggests that the unusual stability of ODC in this cell is associated with a defect in a cellular mechanism for ODC-specific degradation.     

Stimulation of ornithine decarboxylase activity in chick fibroblasts by non-suppressible insulin-like activity (NSILA), insulin and serum.

A factor isolated from human serum (nonsuppressible insulin-like activity, NSILA) stimulates multiplication of serum-starved chick embryo fibroblasts and stimulates activity of ornithine decarboxylase (ODC). Physiological doses of NSILA (200 muU/ml) and pharmacological doses of insulin (200 mU/ml) stimulate ODC 4-5-fold, 10% fetal calf serum about 18-fold. Combined addition of NSILA and insulin does not result in higher activities, suggesting a common mechanism of action. The increase in cell number obtained with NSILA, insulin or serum parallels the degree of ODC stimulation. Treatment of cells with pronase also stimulates ODC activity. A sharp increase in ODC activity occurs between 2, 5 and 5.0 hours after addition of the growth factors with a peak at 4.0-4.5 hours ("activation period"). As cells leave G1 phase, ODC activity decreases rapidly. To achieve maximal activity of ODC, the growth factors have to be present during the entire "activation period." The potential to reactivate ODC decreases as cells pass through S phase. Results obtained using cycloheximide suggest that ODC is translated only in the second half of the "activation period." Data on effects of dbcAMP and dbcGMP on ODC activation by serum are discussed.     

Accumulation of ornithine decarboxylase-antizyme complex in HMOA cells.

A new method was developed for the assay of ornithine decarboxylase (ODC)-antizyme complex, in which alpha-difluoromethylornithine (DFMO)-inactivated ODC was used to release active ODC competitively from the complex. ODC-antizyme complex was present in the extracts of hepatoma tissue-culture (HTC) cells and of ODC-stabilized variant HMOA cells, in much larger amounts in the latter. Cellular amounts of the complex fluctuated after a change of medium in a similar manner in HTC and HMOA cells, increasing during the period of ODC decay. After treatment with cycloheximide, the decay of ODC-antizyme complex in HMOA cells was more rapid than the decay of free ODC, but it was much slower than the decay of free ODC or complexed ODC in HTC cells. Administration of putrescine caused a rapid increase in the amount of ODC-antizyme complex in both HTC and HMOA cells, but nevertheless the decay of total ODC (free ODC plus ODC-antizyme complex) was more rapid with putrescine than with cycloheximide. These results suggested the possibility that ODC is degraded through complex-formation with antizyme. In contrast with complexed antizyme, free antizyme was not stabilized in HMOA cells.     

Ornithine decarboxylase prevents dibenzoylmethane-induced apoptosis through repressing reactive oxygen species generation

Dibenzoylmethane (DBM) belongs to the flavonoid family and is a minor constituent of the root extract of licorice and the β-diketone analogue of curcumin. It exhibits antimutagenic, anticancer, and chemopreventive effects. Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway, plays an important role in growth, proliferation, and transformation. Our previous studies showed ODC overexpression prevented etoposide-, paclitaxel-, and cisplatin-induced apoptosis. Here, we investigated one mechanism of DBM-induced apoptosis and the antiapoptotic effects of ODC during DBM treatment. We found that DBM induced apoptosis, promoted reactive oxygen species (ROS) generation, and disrupted the mitochondrial membrane potential (Δψ(m). N-acetylcysteine, a ROS scavenger, reduced DBM-induced apoptosis, which led to the loss of Δψ(m) due to reduced ROS. Overexpression of ODC in parental cells had the same effects as the ROS scavenger. The results demonstrated that DBM-induced apoptosis was a ROS-dependent pathway and ODC overexpression blocked DBM-induced apoptosis by inhibiting intracellular ROS production.     

Evidence that AMP-activated protein kinase can negatively modulate Ornithine decarboxylase activity in cardiac myoblasts

The responses of AMP-activated protein kinase (AMPK) and Ornithine decarboxylase (ODC) to isoproterenol have been examined in H9c2 cardiomyoblasts, AMPK represents the link between cell growth and energy availability whereas ODC, the key enzyme in polyamine biosynthesis, is essential for all growth processes and it is thought to have a role in the development of cardiac hypertrophy. Isoproterenol rapidly induced ODC activity in H9c2 cardiomyoblasts by promoting the synthesis of the enzyme protein and this effect was counteracted by inhibitors of the PI3K/Akt pathway. The increase in enzyme activity became significant between 15 and 30min after the treatment. At the same time, isoproterenol stimulated the phosphorylation of AMPKα catalytic subunits (Thr172), that was associated to an increase in acetyl coenzyme A carboxylase (Ser72) phosphorylation. Downregulation of both α1 and α2 isoforms of the AMPK catalytic subunit by siRNA to knockdown AMPK enzymatic activity, led to superinduction of ODC in isoproterenol-treated cardiomyoblasts. Downregulation of AMPKα increased ODC activity even in cells treated with other adrenergic agonists and in control cells. Analogue results were obtained in SH-SY5Y neuroblastoma cells transfected with a shRNA construct against AMPKα. In conclusion, isoproterenol quickly activates in H9c2 cardiomyoblasts two events that seem to contrast one another. The first one, an increase in ODC activity, is linked to cell growth, whereas the second, AMPK activation, is a homeostatic mechanism that negatively modulates the first. The modulation of ODC activity by AMPK represents a mechanism that may contribute to control cell growth processes.