Absence of inactivation or phosphorylation of ornithine decarboxylase by nuclear protein kinase NII and of immunological cross-reactivity between RNA polymerase I and ornithine decarboxylase

Incubation with protein kinase NII did not result in phosphorylation or inactivation of mouse kidney ornithine decarboxylase. Partially purified ornithine decarboxylase preparations contained a protein kinase activity and stimulated the activity of RNA polymerase I. However, these properties were due to contaminating protein(s) since further purification reduced the kinase activity and removal of the ornithine decarboxylase with a specific antiserum did not abolish the ability to stimulate RNA polymerase I. Antibodies to RNA polymerase I did not interact with ornithine decarboxylase and antibodies to ornithine decarboxylase did not interact with RNA polymerase I. These results indicate that: a) mammalian ornithine decarboxylase activity is not regulated by phosphorylation by protein kinase NII or the contaminating kinase, and b) the ability of impure preparations of ornithine decarboxylase to stimulate RNA polymerase I is due to a contaminating unrelated protein.     

Proposed model of major sequential biochemical events of a trophic response.

It appears that the induction of ornithine decarboxylase regulates the rate of ribosomal RNA synthesis as well as regulating the rate of synthesis of polyamines. Further, ornithine decarboxylase, in most cases, is induced after a significant activation of cAMP-dependent protein kinase. We propose a model for the process of hypertrophy based on studies of a considerable number of mammalian growth systems. The mechanism of parallel regulation of polyamines and RNA appears to be initiated by the direct effect of ornithine decarboxylase on RNA polymerase I.     

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.     

Polyamine synthesis during lymphocyte activation : Induction of ornithine decarboxylase and S-adenosyl methionine decarboxylase

Lymphocyte stimulation by phytohaemagglutinin (PHA) is accompanied by marked increases in the activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase, two key enzymes for the synthesis of polyamines. Both enzymes increase in a biphasic manner, with the rises in S-adenosyl methionine decarboxylase preceding the increases in ornithine decarboxylase. The initial rises precede the initiation of DNA synthesis, and seem to correlate with the increased rate of ribosomal RNA synthesis. Selective inhibition of ribosomal RNA synthesis inhibits the increases in the activity of both enzymes, especially ornithine decarboxylase, more than the increase in the overall rate of protein synthesis.Both enzymes are metabolically unstable and have half-lives of less than 1 h, although the half-life of ornithine decarboxylase depends on the amino acid concentration in the culture medium. While effects of PHA on the stability of the enzymes have not been ruled out, at least part of the PHA-dependent increases in activity are due to increased synthesis or activation of the enzymes. The synthesis of S-adenosyl-methionine decarboxylase declines rapidly after inhibition of RNA synthesis, but ornithine decarboxylase activity declines at about the same rate as protein synthesis as a whole.The activities of both enzymes also increase during lymphocyte stimulation by concanavalin A, lentil extract and staphylococcal filtrate.     

Induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol 13-acetate in hamster fibroblasts. Relationship between levels of enzyme activity, immunoreactive protein, and RNA during the induction process

Phorbol ester tumor promoters and growth factors rapidly stimulate ornithine decarboxylase activity in the transformed hamster fibroblast line HE68BP. We report here a close correspondence between the time courses and magnitudes of induction of ornithine decarboxylase activity and immunoreactive ornithine decarboxylase protein following treatment of HE68BP cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or refeeding with fresh medium. Cycloheximide addition to induced cells caused a rapid fall in the levels of both ornithine decarboxylase activity and ornithine decarboxylase protein. Northern blot analysis of RNA isolated from HE68BP cells indicated that treatment with TPA and fresh medium increased the amount of two species of mRNA of lengths 2.4 and 2.1 kilobase. This increased accumulation of ornithine decarboxylase mRNA corresponded temporally to that observed at the protein level, with a 15-fold maximal induction 7 h after treatment followed by a rapid decline in hybridizable RNA. These data indicate that stimulation of ornithine decarboxylase activity by TPA or refeeding involves changes in levels of ornithine decarboxylase mRNA as well as changes in the rate of synthesis of ornithine decarboxylase protein.     

Relationships between polyamine metabolism and RNA synthesis in post-ischemic liver cell repair

In liver cells recovering from reversible ischemia the increase in RNA synthesis by isolated nuclei is preceded by activation of ornithine decarboxylase, leading in turn to an increase in putrescine concentration. Treatment of the animals with 1,3-diaminopropane and putrescine prevents ornithine decarboxylase activation but does not hinder the enhancement of RNA synthesis in post-ischemic liver nuclei; therefore, ornithine decarboxylase activation does not seem to be a necessary prerequisite for the increase in RNA synthesis. Hypophysectomy does not prevent the post-ischemic increases of ornithine decarboxylase and RNA synthesis; but pre-treatment of the animals with cycloheximide—which has a dual effect on the activity of ornithine decarboxylase—abolishes the post-ischemic enhancement of RNA synthesis. In contrast with regenerating liver, changes in ornithine decarboxylase activity and putrescine concentrations in reversible ischemia are not associated to changes in S-adenosylmethionine decarboxylase activity and in spermine and spermidine concentrations that seem to be characteristic of tissues where increases in RNA synthesis are followed by DNA synthesis and cell multiplication.     

The inhibition of the serum-stimulated increase of ornithine decarboxylase by ionophores and local anesthetics

The addition of fresh serum-containing growth medium to L1210 mouse leukemic cells in culture resulted in a 5-fold increase in ornithine decarboxylase (l-ornithine carboxy-lyase, EC 4.1.1.17) activity. The presence of microtubule disrupting agents (colchine, vinblastine) or cations (5–10 mM K⁺, Na⁺ or Mg²⁺) abolishes this increase of ornithine decarboxylase activity (Chen, K.Y., Heller, J.S. and Canellakis, E.S. (1976) Biochem. Biophys. Res. Commun. 70, 212–219). Based on these observations we proposed that fluctuation in cellular cation concentrations may act as a link between the membrane structure and ornithine decarboxylase. To test this proposal, we studied the effects of selective membrane perturbing agents such as ionophores and local anesthetics, on the serum-stimulated increase of ornithine decarboxylase activity in L1210 cells. Among the six inonophores tested, valinomycin was the most potent one, with I₅₀ value (concentration that gives 50% inhibition of orthinine decarbocylase activity) of 6·10^?9 M. Dibucaine and tetracaine were also effective inhibitors at 10^?4?10^?5 M. The I₅₀ values of valinomycin on the protein synthesis and RNA synthesis, however, were greater than 1·10^?6 M. These results substantiate the notion that ornithine decarboxylase activity can be regulated at plasma membrane level and such regulation is related to the perturbation of cellular cation pools.     

Drug-induced migration of cytoplasmic ornithine decarboxylase into rat liver nucleus in not related to increased RNA polymerase activity

1-Methyl-3-isobutylxantine (MIX) caused rapid increases in cytoplasmic and nuclear ornithine decarboxylase (ODC) activity as well as increases in RNA polymerases I and II. MIX also significantly increased labeling of nuclear proteins with [³H]-leucine while causing only a slight rise in the labeling of the cytoplasm. Cycloheximide prevented the MIX-induced increases in cytoplasmic ODC, RNA polymerases I and II, and radioactive labeling of cytoplasmic and nuclear proteins. Cycloheximide did not prevent the MIX-induced change in nuclear ODC. These data suggest that cytoplasmic ODC migrated in to the nucleus after MIX treatment but this migration was not correlated with increased RNA polymerase activity.     

Ornithine decarboxylase activity during neonatal development of the rat ventral prostate

The relationship between ornithine decarboxylase activity and growth has been examined in ventral prostates from rats aged 2–60 weeks. The incorporation of [¹²⁵I]iododeoxyuridine in vitro was used to assess DNA-synthetic activity. During 2–7 weeks post-partum ornithine decarboxylase activity/prostate and the protein content and wet weight increased exponentially. In contrast, the incorporation of [¹²⁵I]iododeoxyuridine/prostate fell during the first 4 weeks. This decline was reversed at about the time when the systemic concentrations of gonadal androgens start to rise. After 10 weeks post-partum the incorporation of [¹²⁵I]iododeoxyuridine/prostate and ornithine decarboxylase activity/prostate levelled off, whereas the DNA and protein content of the prostate contonued to increae at a slow rate.     

Dissociation of RNA synthesis from the calcium requirement for serum-increased ornithine decarboxylase activity in rat glioma cells

When C6-2B rat glioma cells were stimulated with calf serum in the presence of calcium, ornithine decarboxylase activity increased maximally in 6-8 h after an initial 2-3 h lag period wherein RNA synthesis occurred. The increase of ornithine decarboxylase activity in serum-stimulated C6-2B cells was prevented by the calcium chelator EGTA, but EGTA had no effect upon RNA synthesis as judged by [3H]uridine incorporation into RNA. In addition, the calcium requirement for increased ornithine decarboxylase activity was temporally distal to the lag period. EGTA appeared to inhibit the synthesis of ornithine decarboxylase, because the half-life values of ornithine decarboxylase activity were similar (37-47 min) in the presence of EGTA or protein synthesis inhibitors such as cycloheximide or emetine. Also, calcium readdition rapidly reversed EGTA inhibition of ornithine decarboxylase activity by a mechanism which could be blocked by cycloheximide.     

Regulations of thyroid decarboxylase by the polyamines Induction of a protein inhibitor of ornithine decarboxylase by the end-products of the reaction

When spermdine, putrescine or 1,3-diaminopropane was injected (12.5 μmol/100 g body weight) into rats i h before thyrotropin, ornithine decarboxylase activity was increased by 75–150% over control levels. However, when 75 μmol polyamine/100 g body weight was injected, thyrotropin-activated activity was inhibited by 70–95%. Multiple polyamine injections inhibited goitrogen-induced activity and gland weight increase by approx. 35%.The polyamines also inhibited thyrotrophin-activated rat thyroid ornithine decarboxylase in vitro in a dose-related fashion, with 50% inhibition occurring at 2–5 · 10⁻⁴ M. The inhibition was not due to a direct effect on the enzyme. No stimulation was seen with low concentration of polyamine. The polyamines had no effect on in vitro thyroid protein/RNA synthesis or glucose oxidation but had a biphasic effect on plasma membrane adenylate cyclase activity.A protein inhibitor to thyroid ornithine decarboxylase was generated in vivo by multiple injections of the polyamines into rats, and in vitro by incubating bovine thyroid slices with 2–10 mM polyamine. The inhibitor was non-dialyzable, destroyed by boiling, and its formation was blocked in a dose-related fashion by cycloheximide.We conclude that: (1) thyroid ornithine decarboxylase is subject not only to positive control, but is also negatively regulated by its end-products, the polyamines, which induce a protein inhibitor to ornithine decarboxylase; (2) since gland growth is also inhibited under these conditions, the polyamine effect on thyroid ornithine decarboxylase may be biologically significant.     

Studies on the role of protein synthesis and of sodium on the regulation of ornithine decarboxylase activity

The minimum requirements for eliciting or enhancing ornithine decarboxylase activity (EC. 4.1.1.17); L-ornithine carboxylase) in neuroblastoma cells incubated in salts-glucose solutions have been investigated. These incubation conditions permit the study of changes in ornithine decarboxylase activity independently of the growth-associated reactions that occur in cell culture media (Chen, K.Y. and Canellakis, E.S. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3791–3795). Ornithine decarboxylase activity can be elicited by a variety of asparagine and other amino acid analogs, including α-aminoisobutyric acid, that cannot participate in protein synthesis. Of the eleven asparagine analogs tested. $\text{α-N-}\text{CH}{}_3\text{-}\text{DL}\text{-asparagine}$ is the most potent in eliciting ornithine decarboxylase activity and is equivalent to asparagine in this regard. Inclusion of polar groups into the asparagine molecule results in the loss of its ability to elicit ornithine decarboxylase activity. With the use of these analogs and of analogs of other amino acids it is shown that the rapid fall in ornithine decarboxylase activity that is noted following cycloheximide treatment may not be a consequence of the inhibition of protein synthesis. The rapid fall in ornithine decarboxylase activity is primarily due to the removal of the agent that elicits and stabilizes its activity. These results, the finding that α-amminoisobutyric acid stimulates ornithine decarboxylase activity and that sodium is required for the stimulation of ornithine decarboxylase activity are discussed in relation to the ‘A’ amino acid transport system.     

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activtivity in rat kidney.Adminsitration of diaminopropane 60 min before partial hepatectomy only marginally inhibited orthine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant.An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed $\text{S-}\text{adenosyl}\text{-}\text{l}\text{-}\text{methionine}$ decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy.Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals.Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life.Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [¹⁴C] methionine 4 h after hepatectomy or after administration of porcine growth hormone.Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornitine decarboxylase activity and spemedicine synthesis.     

Stimulation of ornithine decarboxylase synthesis in the rat thyroid

High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with ¹²⁵I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids $\text{in vitro}$. ¹²⁵I-labeled antibody incorporation into the enzyme antibody complex after induction $\text{in vitro}$ showed an increase which paralleled the increase in enzymatic activity and thus suggested $\text{de novo}$ synthesis of thyroid enzyme protein.     

Effect of androgen on ornithine decarboxylase activity in androgen-dependent mouse mammary tumor (Shionogi Carcinoma 115) and its androgen-independent subline (CS 2)

The activity of ornithine decarboxylase in androgen-dependent mouse mammary tumor (Shionogi Carcinoma 115) was reduced to 25% by castration of tumor-bearing mice and restored to the normal level 12 h after administration of testosterone or 5 alpha-dihydrotestosterone. Administration of estradiol-17 beta to the tumor-bearing castrated mice also stimulated the enzyme activity while progesterone and cortisol had little effect. On the other hand, the enzyme activity was affected by neither castration nor androgen injection to CS 2, which is a subline of SC 115 and completely independent of androgen for growth. The inhibition of ornithine decarboxylase activity in SC 115 by injecting alpha-difluoromethylornithine did not affect the enhancement of RNA polymerase I activity by androgen, showing independent elevation of the levels of the two enzymes by androgen.     

Multihormonal regulation of ornithine decarboxylase activity in highly differentiated porcine granulosa cells in vitro

Purified luteinizing hormone, but not follicle-stimulating hormone, elicited time- and dose-dependent stimulation of the cytosolic enzyme, ornithine decarboxylase, in highly differentiated, porcine granulosa cells maintained in vitro in chemically defined medium. Enzymic induction was susceptible to inhibitors of protein and RNA synthesis, and was suppressed by selective direct and indirect inhibitors of ornithine decarboxylase. Physiologic concentrations of prostaglandin E₂ and L-epinephrine also enhanced enzymic activity in a dose-dependent and saturable manner. Systematic comparison of the hormonal induction of ornithine decarboxylase in highly differentiated versus poorly differentiated granulosa cells revealed distinctive patterns of enzymic responsivity in relation to the degree of cytodifferentiation attained in vivo. This in vitro model is likely to permit further detailed examination of the molecular mechanisms subserving the hormonal control of ovarian ornithine decarboxylase activity in spontaneously differentiated granulosa cells maintained under chemically defined conditions in vitro.     

Effect of sodium butyrate on induction of ornithine decarboxylase activity in phytohemagglutinin-stimulated lymphocytes

Effect of sodium butyrate on DNA synthesis and the induction of ornithine decarboxylase (EC 4.1.1.17), a rate-limiting enzyme of polyamine biosynthesis, was studied in phytohemagglutinin(PHA)-stimulated bovine lymphocytes. Millimolar concentrations of butyrate completely inhibited the incorporation of [³H] thymidine into the acid-insoluble fraction and reversibly suppressed the induction of ornithine decarboxylase. Other shortchain fatty acids were much less active than butyrate. These results suggest that the suppression of ornithine decarboxylase activity may be one of the reasons for the inhibition of DNA synthesis with butyrate in bovine lymphocytes, because our previous experimental results have shown that the induction of ornithine decarboxylase closely correlates with the DNA synthesis in growth-stimulated cells.     

Hydroxyurea inhibits ODC induction, but not the G1 to S phase transition.

Chinese hamster ovary cells, selected in mitosis and plated into medium containing hydroxyurea, can progress through G₁ and enter S phase although bulk DNA synthesis is prevented. As the cells progress through G₁ in the presence of hydroxyurea, ornithine decarboxylase activity remains low while general protein synthesis appears unaffected. After hydroxyurea is removed, ornithine decarboxylase activity increases, but only after approximately 20% of the DNA has been replicated. These results suggest that ornithine decarboxylase induction is not essential for cellular progression into S phase but is required for the completion of DNA synthesis.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.