Initial characterization of a HTC cell variant partially resistant to the anti-proliferative effect of ornithine decarboxylase inhibitors

Under the selective pressure of -α-methylornithine (α-MeOrn), a competitive inhibitor of ornithine decarboxylase (ODC) (EC 4.1.1.17) a clone of rat hepatoma tissue culture (HTC) cells has been isolated and designated HMO_A. The growth of this clone is affected by the drug only after a lag period of three generations. The same partial resistance was observed to -α-difluoromethyl ornithine (α-DFMeOrn), an irreversible inhibitor of ODC. HMO_A cells showed elevated ODC activity with a concomitant increase of the putrescine content but no change in S-adenosyl- -methionine decarboxylase (SAM-DC) (EC 4.1.1.50) activity. Evidence is given that this overproduction of putrescine may be responsible for the partial resistance of HMO_A cells to the anti-proliferative effect of the ODC inhibitors. α-MeOrn increases ODC and SAM-DC activities and α-DFMeOrn raises SAM-DC activity in a time and cell line-dependent manner. These findings may support the concept that intracellular putrescine and spermidine play a direct or indirect regulatory role in the expression of ODC and SAM-DC. Thus, the variant cell should be useful for studies on the genetic regulation of polyamine metabolism in eukaryotic cell systems.     

Superinduction of ornithine decarboxylase by halogenated ribofuranosylbenzimidazoles.

1. The effect of dichlororibofuranosylbenzimidazole (DiCl-RB), an inhibitor of hnRNA synthesis and casein kinase-2 activity, on ornithine decarboxylase (ODC) was investigated in a difluoromethylornithine (DFMO) resistant, ODC overproducing cell line. 2. In cells growing in the absence of DFMO, DiCl-RB provoked a marked, but transient increase in ODC activity and immunoreactive ODC content. 3. The ODC response to DiCl-RB was prevented by cycloheximide and was not due to stabilization of the enzyme. 4. The dibromo derivative analogue (DiBr-RB) exerted similar effects on ODC, but was effective at lower concentrations. 5. The halogenated ribofuranosylbenzimidazoles were ineffective in cells growing in the presence of DFMO and containing higher levels of ODC protein.     

Effect of stress and sympathetic activity on rat cardiac and aortic ornithine decarboxylase activity.

Adult male rats were injected either with α- or ß-adrenergic agonists and/ or antagonists and ornithine decarboxylase (ODC) activity in the heart and aorta was measured 4 hours later. At the lower doses, isoproterenol (0.2–0.4 mg/kg) resulted in a 10-fold increase in cardiac ODC activity but caused no significant change in aortic ODC activity. In contrast, phenylephrine (1 mg/kg) caused a 4-fold increase in aorta but no change in cardiac ODC activity levels. Phenoxybenzamine pretreatment completely abolished the PE-induced increase whereas no change was seen in ISop injected animals. Similarly, pretreatment with propranolol blocked the ISop induced response on ODC activity but had no effect on the increases observed after PE. These data suggest that the sympathetic regulation of ODC activity levels is mediated primarily via the ß-receptor in the heart but through the α-receptor in the aorta.     

Relationship between ornithine decarboxylase activity and hexamethylene bisacetamide-induced differentiation of murine erythroleukemia cells

A transitory increase in ornithine decarboxylase (ODC) activity is shown not to be a prerequisite for the differentiation induced by hexamethylene bisacetamide (HMBA) in murine erythroleukemic (MEL) cells. On the contrary, conditions are described, where inhibition of the ODC activity with alpha-difluoromethyl ornithine (DFMO) stimulated the induced differentiation. Polyamine analysis demonstrated that a reduction in intracellular putrescine and spermidine occurred in MEL cells before commitment to erythrodifferentiation. The presence of DFMO increased the rapidity and the amplitude of these changes. No effect of dexamethasone on these changes in ODC activity or intracellular polyamines was observed.     

Regulation of polyamine synthesis by dietary alpha-aminoisobutyric acid and ornithine

An experiment was conducted to determine the effect of feeding ornithine in combination with alpha-aminoisobutyric acid (AIB), an inhibitor of arginase, on the regulation of polyamine synthesis in chicks. A total of 48 chicks with genetically elevated renal arginase activity was fed diets containing crystalline amino acids and 1% AIB with or without 2% ornithine. Feeding AIB reduced renal arginase activity, while renal and hepatic ornithine decarboxylase (ODC) activity increased. Feeding AIB plus ornithine caused no further reduction in renal arginase activity compared with that in chicks fed the AIB-supplemented diet. Renal and hepatic ODC activities, however, fell to below control levels. Renal, hepatic, and breast muscle ornithine concentrations increased substantially when ornithine was fed. AIB plus ornithine increased renal putrescine and spermidine concentrations. It was concluded that AIB could partially overcome the ornithine-induced inhibition of ODC activity. These findings support the hypothesis that dietary manipulation of precursor amino acids of polyamines in the presence of metabolites that induce ODC activity can influence tissue polyamine concentrations.     

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.     

Location of renal ornithine decarboxylase activity

Renal ornithine decarboxylase (ODC) activity was found to be more prevalent in the medulla of the normal rat kidney than in the cortex. When renal ODC activity was stimulated by ethanol, growth hormone, ACTH, or corticosterone, proportional increases were observed in both medulla and cortex. After hypophysectomy, ODC activities fell equally in both areas of the kidney. The administration of cycloheximide, which is known to cause a rebound increase after six hours in overall renal ODC activity, was followed by an increase of medullary ODC activity while cortical activity remained suppressed.     

A macromolecular inhibitor of the antizyme to ornithine decarboxylase.

A macromolecular factor that inhibits the activity of the antizyme to ornithine decarboxylase (ODC) was found in rat liver extracts. The factor, 'antizyme inhibitor', was heat-labile, non diffusable and of similar molecular size to ODC. The antizyme inhibitor re-activated ODC that had been inactivated by antizyme, apparently by replacing ODC in a complex with antizyme. Therefore the antizyme inhibitor can be used to assay the amount of inactive ODC-antizyme complex formed in vitro. When assayed by this method, the complex was shown to be eluted before ODC from a Sephadex G-100 column. Significant increase in ODC activity was observed when the antizyme inhibitor was added to crude liver extracts from rats that had been injected with 1,3-diaminopropane to cause decay of ODC activity, suggesting the presence of inactive ODC-antizyme complex in the extracts.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

Polyamine homeostasis in transgenic plants overexpressing ornithine decarboxylase includes ornithine limitation

It was reported recently that overexpression of human ornithine decarboxylase (ODC) cDNA in transgenic rice plants resulted in increased steady-state concentration of polyamines, i.e., enough biosynthetic control is invested at this step to enable adjustment of polyamine levels. To investigate critically whether constitutive overexpression of ODC is sufficient to control steady-state polyamine levels, we expressed an ODC cDNA from Datura stramonium in transgenic tobacco plants. Transgenic progeny of self-fertilised primary transformants exhibited increases in ODC activity of 25-fold in leaves and 5-fold in flower buds. However, the increase in putrescine levels was only 1.5- to 2.1-fold in leaves and 1.1- to 1.3-fold in flower buds. Emphatically, no changes to spermidine or spermine steady-state levels or to soluble or insoluble hydroxycinnamic acid-conjugated polyamines were observed. Ornithine feeding to cell suspension cultures derived from the transgenic plants indicated that putrescine accumulation was limited in part by ornithine availability. These results demonstrate that a large increase in the capacity of the tobacco plants to decarboxylate ornithine does not result in a comparable increase in the level of free or conjugated polyamines. Plant polyamine homeostatic mechanisms efficiently accommodate increased ODC activity, suggesting that polyamine biosynthetic control is invested at multiple interdependent steps.     

Cultured hepatoma cells for the study of enzyme regulation: induction of ornithine decarboxylase by insulin and asparagine

The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combination of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10(-5) M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present.     

Activities of ornithine aminotransferase and ornithine decarboxylase in chronically uremic rats

The activities of two enzymes mediating different pathways of ornithine catabolism were measured in liver and kidney of chronically uremic rats and their pair-fed controls. Two months following partial nephrectomy hepatic ornithine aminotransferase (OAT) activity tended to be lower in uremic rats and was correlated with urea clearance and with carbamoyl phosphate synthetase activity. Renal OAT activity in uremic rats was also correlated with urea clearance. When uremic rats were maintained for five months, OAT activity was significantly decreased in liver but not in kidney and the activity of ornithine decarboxylase (ODC), the enzyme regulating polyamine biosynthesis, was reduced in both liver and kidney. In cross-over experiments, evidence was obtained for a factor in uremic kidney cytosol which inhibited renal ODC activity.     

Putative ornithine decarboxylase activity in Arabidopsis thaliana: inhibition and intracellular localisation

In this work we studied putative ornithine decarboxylase activity (ODC, EC 4.1.1.17) in leaves of Arabidopsis thaliana L. (ecotype Columbia) plants at non-flowering stage (about 21 d of culture). Putative ODC activity was higher in the particulate than in the soluble fraction and activity was pH-dependent, increasing linearly with the pH. Inclusion of 10 mM arginine in the assay showed that the incidence of ornithine transcarbamoylase activity (EC 2.1.3.3) accounted for about 35% in the particulate fraction, but that its contribution was negligible in the soluble fraction. Increasing concentrations of the irreversible inhibitor α-difluoromethylornithine (DFMO) progressively inhibited putative ODC activity with a 40% inhibition at 20 mM DFMO. Taking into consideration the incidence of ornithine transcarbamoylase activity, the total inhibition of putative ODC activity was of about 75%. Fractionation experiments permitted measurement of putative ODC activity in the nuclei- and chloroplast-enriched fractions. The assays performed on membranes and stromal fractions isolated from gradient purified chloroplasts showed that the enzyme activity was associated almost totally with the plastid membranes.     

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.     

Relationship between RNA polymerase I activity and ornithine decarboxylase in rat liver tissues.

In order to examine the relationship between RNA polymerase I and ornithine decarboxylase (ODC), three lines of experiments were performed, with the following results. The glucocorticoid-induced increase of RNA polymerase I in rat liver nuclei was not abolished by administration of inhibitors of ODC synthesis and activity, namely 1,3-diaminopropane and 2-difluoromethylornithine respectively. Anti-ODC antibody did not cross-react with RNA polymerase I solubilized from rat liver nucleoli, indicating the absence of a common protein sequence in these enzymes. The ODC preparation which was treated with transglutaminase in the presence of putrescine could not stimulate the activity of RNA polymerase I in nuclei of liver and prostate. All these results suggest that the increases in ODC protein or activity are not a prerequisite to the increase in RNA polymerase I after hormonal or physiological stimuli, but rather that the increases in both enzymes are separate responses to the primary stimuli.     

Effect of lithium chloride on ornithine decarboxylase activity in rat adrenal.

The effects of lithium chloride on ornithine decarboxylase (ODC) activity were compared in the adrenal and kidney of control (saline treated) and prolactin-treated rats. ODC activity was decreased in kidney of both groups of animals, the magnitude of the effect of lithium in the hormone-treated group varying with the time of administering the lithium relative to prolactin. The response in the adrenal was quite different. Following treatment with LiCl, there was a gradual increase in ODC activity from a low of 10-35 pmol CO2 x 30 min-1.mg protein-1 in control animals to values 20- to 30-fold greater at 5 h. In rats treated simultaneously with LiCl and prolactin, ODC activity was greater at 5 h than that observed in animals receiving either compound alone, indicating that their effects were additive. When LiCl was given 4 h after prolactin, i.e., 1 h before sacrifice, ODC activity decreased to a very low level at 5 h, as in other tissues. The increase in ODC activity in the adrenal following LiCl is of the same magnitude as the changes observed in tissues stimulated to undergo alterations in proliferation, differentiation, or metabolic or membrane activity by hormones and other external stimuli.     

Effects of putrescine on synthesis and degradation of ornithine decarboxylase in primary cultured hepatocytes

Changes in both synthesis rate and degradation rate of ornithine decarboxylase (ODC) were pursued in primary cultures of adult rat hepatocytes during the process of ODC induction caused by asparagine and glucagon and also during the process of rapid ODC decay caused by putrescine. The synthesis rate of ODC was determined by [35S]methionine incorporation into the enzyme, which was separated afterwards by immunoprecipitation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The degradation rate of ODC was determined by following the decay of prelabeled ODC. The enzyme induction caused by asparagine (10 mM) and glucagon (1 microM) was due both to an increase in the synthesis rate and to a decrease in the degradation rate. Addition of 10 mM putrescine caused a rapid decay of ODC activity, which was faster than ODC decay in the presence of cycloheximide. This rapid decay in ODC activity was accompanied by slightly slower decay in ODC protein, which was due both to partial suppression of ODC synthesis and to several fold acceleration of ODC degradation.     

Degrons of yeast and mammalian ornithine decarboxylase enzymes make potent combination for regulated targeted protein degradation

Elevation of polyamine levels in eukaryotes leads to rapid degradation of ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis pathway. ODC in yeast (yODC) has two domains, the Nα/β domain consisting of α/β barrel domain (α/β) preceded by an overhang of 50 residues at its N-terminus (N50) and β sheet domain at its C-terminus. Two degradation determinant signals or degrons in yODC sequence, namely the N50 and the antizyme-binding element (AzBE) housed in the α/β domain, are responsible for its degradation by proteasomes. Antizyme (Az) induced under polyamine excess binds to AzBE and delivers ODC to proteasome, while the N50 threads the protein into proteasome. It was previously reported by us that the peptide Nα/β of yODC acts as an independent transplantable degron, whose action can be modulated with the help of antizyme by varying polyamine levels. Mammalian ODC (mODC), in spite of its 40% sequence homology with yODC, is devoid of N50 of yODC and instead sports a C-terminal tail of 37 residues (CmODC). CmODC acts as an independent transplantable degron with no equivalent in yODC. The present study investigates the merits of employing the two degrons Nα/β and CmODC together for targeted protein degradation by expressing them in a chimeric fusion with green fluorescent protein (GFP). Our results establish that under the regulation of antizyme, the signals Nα/β and CmODC acting together enhance degradation better than either degron in isolation. The combination of Nα/β and CmODC can be employed to study the function of novel proteins through their rapid removal.