Half-life Of Ornithine Decarboxylase Research Articles

Filarial parasites possess an antizyme but lack a functional ornithine decarboxylase

In eukaryotes, the key player in polyamine metabolism is the ornithine decarboxylase (ODC) that catalyses the first and rate limiting step in cellular polyamine synthesis. The half life of ODC is strictly regulated by the antizyme (AZ), which promotes its degradation. Older reports on the polyamine situation in filarial parasites indicate a lack of ornithine decarboxylation activity and an increased uptake of polyamines. Our in silico analysis of the Brugia malayi genome revealed only an ODC-like protein that lacks essential residues. Consequently, the recombinant protein had no enzymatic ODC activity. Furthermore, only ODC-like genes were found in the available draft genomes of other filarial parasites. In this ODC-free scenario, we set out to investigate the AZ of O. volvulus (OvAZ). The expression of the recombinant protein allowed us to analyse the localization of OvAZ in different O. volvulus stages as well as to identify it as target for the human humoral immune response. Strong immunostaining was observed in the outer zone of the uterine epithelium as well as in the uterus lumen around the periphery of the developing parasite, indicating a potential role of the OvAZ in the control of polyamine levels during embryonic development. By employing a novel in vivo method using Caenorhabditis elegans, we postulate that the OvAZ enters the secretory pathway. Even though the ODCs are absent in filarial parasites, OvAZ has the ability to bind to various ODCs, thereby demonstrating the functionality of the conserved AZ-binding domains. Finally, pull-down assays show an interaction between B. malayi AZ and the B. malayi ODC-like protein, indicating that the B. malayi ODC-like protein might function as an AZI. Taken together, our results suggest that filarial species do not possess the ODC while retaining the ODC-regulatory proteins AZ and AZI. It is tempting to speculate that both proteins are retained for the regulation of polyamine transport systems.

Increased translation efficiency and antizyme-dependent stabilization of ornithine decarboxylase in amino acid-supplemented human colon adenocarcinoma cells, Caco-2

The mechanisms of the response of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, to amino acid supplementation were studied in the human colon adenocarcinoma cell line, Caco-2. Supplementation of serum-deprived, subconfluent Caco-2 cells with any one of a series of amino acids (10 mM) resulted in increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with iso-osmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effectors and acted in a concentration-dependent manner; millimolar concentrations of most of these amino acids being sufficient to significantly increase ODC activity. In contrast, supplementation with D-methionine, L-lysine, L-aspartate or L-glutamate had little or no effect on ODC activity, whereas supplemental L-methionine, L-arginine, L-ornithine or L-cysteine was inhibitory. Polyamine assays showed that the putrescine content of cells varied in accordance with the changes in ODC activity. Western-blot and Northern-blot analyses revealed specifically increased levels of ODC protein but not mRNA, respectively, in response to supplementation with an ODC-inducing amino acid. Suppression of the increase in cycloheximide-treated cells confirmed a requirement for protein synthesis. Pulse-labelling of cells with [35S]methionine showed a 3-fold increase in the synthesis of ODC protein after 4 h of supplementation with glycine or L-serine. Supplemental glycine also augmented, reversibly, the half-life of ODC by almost 4-fold and simultaneously decreased the activity of putrescine-induced free antizyme. These results suggest that translational, but not transcriptional, regulation of ODC takes part in ODC induction by amino acids in Caco-2 cells. However, it also appears to occur in concert with decreased enzyme inactivation and/or degradation.

Increased translation efficiency and antizyme-dependent stabilization of ornithine decarboxylase in amino acid-supplemented human colon adenocarcinoma cells, Caco-2

The mechanisms of the response of ornithine decarboxylase(ODC), the rate-limiting enzyme in polyamine biosynthesis, to amino acid supplementation were studied in the human colon adenocarcinoma cell line, Caco-2. Supplementation of serum-deprived, subconfluent Caco-2 cells with any one of a series of amino acids (10 mM) resultedin increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with iso-osmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effectors and acted in a concentration-dependent manner; millimolar concentrations of most of these amino acids being sufficient to significantly increase ODC activity. In contrast, supplementation with D-methionine, L-lysine, L-aspartate or L-glutamate had little or no effect on ODC activity, whereas supplemental L-methionine, L-arginine, L-ornithine or L-cysteine was inhibitory. Polyamine assays showed that the putrescine content of cells varied in accordance with the changes in ODC activity. Western-blot and Northern-blot analyses revealed specifically increased levels of ODC protein but not mRNA,respectively, in response to supplementation with an ODC-inducing amino acid. Suppression of the increase in cycloheximide-treated cellsconfirmed a requirement for protein synthesis. Pulse-labelling of cellswith [(35)S]methionine showed a 3-fold increase in thesynthesis of ODC protein after 4 h of supplementation with glycineor L-serine. Supplemental glycine also augmented, reversibly, the half-life of ODC by almost 4-fold and simultaneously decreased the activity of putrescine-induced free antizyme. These results suggest that translational, but not transcriptional, regulation of ODC takes part in ODC induction by amino acids in Caco-2 cells. However, it also appears to occur in concert with decreased enzyme in activation and/or degradation.

Cloning and expression of two ornithine decarboxylase forms from HMOA cells.

In HMOA cells [Mamont, Duchesne, Grove and Tardif (1978) Exp. Cell Res. 115, 387-393] the half-life of ornithine decarboxylase (ODC) is 8-14 h instead of 15 min as in the Hepatoma Tissue Culture parental cells, due to a single amino acid substitution [Miyazaki, Matsufuji, Murakami and Hayashi (1993) Eur. J. Biochem. 214, 837-844]. We demonstrate for the first time that HMOA cells possess two forms of ODC mRNA that are translated into two proteins differing greatly in turnover rates. We have cloned and transfected the cDNAs for the two ODC forms into COS-1 cells for a direct measurement of their turnover rate. The variant ODC form was much more stable than the wild-type protein, with a half-life of 14 h as compared with 2.5 h.

Leishmania donovani: Cellular control of ornithine decarboxylase in promastigotes

Ornithine decarboxylase, a key enzyme in polyamine biosynthesis, is essential for normal cell growth and proliferation. Furthermore, the inhibition of this enzyme is a potential way of controlling such growth. In order to shed light on the role of ornithine decarboxylase in regulation of Leishmania growth we examined the activity of this enzyme during the life cycle of these organisms. Exponentially growing Leishmania promastigotes were resuspended at a density of 3 × 10 6 cells/ml. 2 × 10 7 cells were withdrawn 24 hr later at different time intervals for induction studies and ornithine decarboxylase activity was measured. Ornithine decarboxylase showed a growth related pattern in L. donovani promastigotes. Induction studies showed that ornithine decarboxylase activity rapidly increased in late log phase cells when resuspended in fresh medium. A biphasic induction curve was observed similar to that observed in mammalian cells. The first peak was observed at 6 hr and the second at 16 hr. Cycloheximide and Actinomycin D inhibited induction at 16 hr by 65–68%. Polyamines at a level not inhibitory to growth (10 μM) inhibited ornithine decarboxylase induction by 30–40% late in the induction period. Putrescine and spermidine both inhibited the first peak of induction. Putrescine suppressed ornithine decarboxylase activity by 39% at 16 hr whereas spermidine by only 29%. The half life of ornithine decarboxylase in promastigote forms grown in the presence of cycloheximide was > 6 hr. These studies indicate that although the Leishmanial ornithine decarboxylase follows a similar induction pattern to that previously reported in the mammalian cells, it is less susceptible to exogenous polyamines and is comparatively stable. This lack of ornithine decarboxylase regulation and turnover may be exploitable in the development of various therapeutic agents to inhibit Leishmanial growth.

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.

Control by treatment with lithium chloride of ornithine decarboxylase in ehrlich ascites tumor cells

The activity of ornithine decarboxylase (ODC) was increased in Ehrlich ascites tumor cells by a change of the medium. This increase in the activity was inhibited by the addition of LiCl to the medium. Na + and Mg 2+ did not affect the enzyme activity. The inhibition of the enzyme activity with LiCl was not reversed by the addition of inositol or dibutyryl cyclic AMP. Total RNA was isolated from cells treated with LiCl and the relative abundance of the ODC mRNA was measured by Northern blot analysis. These levels in cells treated with LiCl were comparable to those in control cells. In the treated cells, the biological half-life of ODC was 14 min, which was the same as for the control cells. The inhibition by LiCl of ODC activity was not due to the nonspecific toxicity of LiCl. These results suggest that treatment of Ehrlich ascites tumor cells with LiCl suppressed ODC induction during translation, not during transcription or after translation.

Effects of 4-Hydroxynonenal, A Product of Lipid Peroxidation, on Cell Proliferation and Ornithine Decarboxylase Activity

4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. Its effects on proliferation, ornithine decarboxylase (ODC) activity and DNA synthesis have been investigated in leukemic cell lines. The cells were incubated for 1 hour with different aldehyde concentrations, then washed and resuspended in medium with fresh foetal calf serum. HNE concentrations ranging from 10(-5) to 10(-6) M significantly inhibited ODC activity when induced by addition of fresh foetal calf serum both in K562 and HL-60 cells. 3H-Thymidine incorporation in K562 cells was also inhibited from 6 to 12 hours after the treatment. The same HNE concentrations did not inhibit ODC activity when added to cytosol, thus a direct action on the enzyme can be excluded. Moreover, HNE did not affect the half-life of ODC, so that a specific effect on ODC synthesis may be supposed. These data indicate a reduction of proliferative capacity of the cells and are consistent with the possibility that HNE, at concentrations close to those found in normal cells, plays a role in the control of cell proliferation.

Effect of ATP depletion and phenanthroline on the spermidine-mediated decay of ornithine decarboxylase in erythroleukemia cells

Addition of spermidine to Friend erythroleukemia cells caused a rapid decay of ornithine decarboxylase (ODC) activity and the accumulation of a ODC-antizyme complex. The induction of antizyme only partially accounted for the decrease of ODC activity by a direct inhibition of the enzyme. However, the antizyme induction was accompanied by a marked reduction of the half-life of ODC. Shift of the cells to an ATP-depleting medium prevented the spermidine-elicited decay of ODC activity as well as the accumulation of ODC-antizyme complex. However, ODC appeared to be stabilized even when ATP depletion was performed 40 min after spermidine addition, in the presence of high levels of antizyme. Similar results were obtained by treating the cells with phenanthroline, a heavy metal chelator and protease inhibitor. These findings indicate that ATP and some metalloprotease(s) may be involved in the degradation pathway of ODC, even in the presence of high levels of polyamines.

Post-translational arginylation of ornithine decarboxylase from rat hepatocytes

Ornithine decarboxylase (ODC) was purified 6500-fold from NMRI mouse kidneys under conditions designed to inhibit degradation by proteinases. The enzyme was homogeneous by SDS/polyacrylamide-gel electrophoresis, and the specific activity was among the highest reported. The yield was 70%. A monoclonal antibody against this preparation was generated and used in studies to investigate the half-life of ODC in cultured rat hepatocytes labelled with [35S]methionine. This value was 39 +/- 4 min and was unchanged when either NH4Cl (as a lysosomotropic agent) or leupeptin (as a lysosomal proteinase inhibitor) was added to the culture medium. Thus the intracellular turnover of ODC in cultured hepatocytes occurs mainly in extra-lysosomal compartments. Arginylation of rat ODC was investigated in vitro by incubation with L-[3H]arginyl-tRNA, and the incorporation of the label was compared with that of total cytosolic proteins. Arginylated ODC had a specific radioactivity 8600 times that of the bulk of cytosolic protein. Edman degradation of this ODC showed that the post-translational arginylation occurred only at the alpha-amino end of the enzyme. The inhibitor of arginyl-tRNA:protein arginyltransferase (EC 2.3.2.8), L-glutamyl-L-valyl-L-phenylalanine, increased the half-life of ODC in cultured hepatocytes from 39 min to more than 90 min. The possible significance of the preferential post-translational arginylation of ornithine decarboxylase to its rapid turnover is discussed.

Intestinal ornithine decarboxylase: half-life and regulation by putrescine

Ornithine decarboxylase (ODC) is the primary rate-limiting enzyme for polyamine synthesis. ODC levels are increased in most tissues, including the intestinal mucosa, by growth-promoting agents. This enzyme has a brief half-life of from 5 to 30 min in mammalian tissues and is regulated by its product; putrescine. The current study examines the turnover and regulation of ODC in the mucosa of the small intestine. With the use of scraped intestinal mucosa from cycloheximide-treated rats, the time course of the decline in ODC activity yielded a half-life of approximately 22 min. Labeling enzyme protein with [3H]difluoromethylornithine (DFMO) resulted in a nearly identical estimation of half-life. ODC activity of mucosa from isolated gut segments stimulated by luminal glycine (0.1-0.4 M) was enhanced 60-100% by 10 mM putrescine administered luminally. Putrescine alone had no effect on ODC. In contrast, 10(-7) M putrescine prevented 80% of the ODC activity stimulated by asparagine in IEC-6 cells (a rat intestinal crypt cell line). The half-life of ODC in unstimulated IEC-6 cells was 20 min and increased to 35 min in cells exposed to 10 mM asparagine. These data demonstrate that ODC of nonproliferating villous cells is regulated differently from the identical enzyme in proliferating crypt cells. Therefore, conclusions regarding mucosal growth should not be based totally on ODC activity from whole mucosa, since it is essentially a measure of only the enzyme present in the villous cells.

Regulation of Saccharomyces cerevisiae ornithine decarboxylase expression in response to polyamine

The mechanism of yeast ornithine decarboxylase (ODC) regulation in response to polyamines was examined. ODC catalyzes the first step of polyamine biosynthesis, the conversion of ornithine to putrescine. ODC activity was modulated approximately 200-fold by varying the availability of polyamines. Variations in ODC activity were associated with parallel changes in the amount of active enzyme molecules. Regulation of ODC activity did not appear to be mediated by changes in the stability of the enzyme. The half-life of ODC was approximately 75 min and was unaffected by the availability of polyamines. Polyamines had no demonstrable effect on the transcriptional or translational stages of ODC expression. The amount of ODC mRNA and the number of ribosomes associated with the mRNA were constant despite the variations in ODC activity. Unlike translationally controlled genes, the regulation of ODC expression did not require either the 5'- or the 3'-untranslated regions of the ODC gene transcript. While gene dosage experiments indicated that negative trans-acting factors are involved in control of ODC expression, these implied factors did not appear to affect translation of ODC mRNA. The results are interpreted to suggest that polyamines regulate ODC expression at a post-translational step prior to assembly of the active form of the enzyme.

Trypanosoma brucei brucei: Regulation of ornithine decarboxylase in procyclic forms and trypomastigotes

Ornithine decarboxylase (ODC) activity was measured in procyclic forms of Trypanosoma brucei brucei grown in semidefined medium. ODC activity rapidly increased in late log-phase cells which were resuspended in fresh medium. A biphasic induction curve similar to that observed in mammalian cells was observed over an 18-hr period. ODC activity increased 4.5-to 25-fold over control levels measured at zero time. Actinomycin D and cycloheximide inhibited induction by >90%. Polyamines at a level not inhibitory to growth (10 μ M) inhibited ODC induction, but only by 30–50%, late in the induction period. Putrescine inhibited the first peak of induction and suppressed activity at 14 hr by 75%. Polyamine analogs such as bis(ethyl)spermidine were not effective suppressors of ODC activity. The half-life of ODC in procylic forms grown in the presence of cycloheximide was >6 hr, while that of bloodstream trypomastigotes in mice treated with cycloheximide was 5 hr. A single dose of the ODC inhibitor dl-α-difluoromethylornithine given to infected rats or mice suppressed trypanosome ODC activity >90% for more than 7 hr. These studies indicate that although trypanosome ODC increases rapidly under log growth conditions, it is less susceptible to fluctuation and external control than the enzyme from mammalian sources. The latter may be a factor in the clinical efficacy of ODC inhibitors.

Stabilization of ornithine decarboxylase in mouse liver and lung by methylglyoxal bis(cyclohexylamidinohydrazone)

The intraperitoneal injection of methylglyoxal bis(cyclohexylamidinohydrazone) (MGBC), an inhibitor of S-adenosylmethionine decarboxylase and spermidine synthase, markedly increased (7-fold of the basal level at 4 hr) ornithine decarboxylase (ODC) activity in normal mouse liver. ODC activity was also increased 2.5-fold over the basal level in mouse lung at 6 hr after the injection. The effect of MGBC on ODC activity occurred in a dose-dependent manner. Measurement of the apparent half-life of ODC induced in the liver and lung by MGBC treatment revealed a clear decrease in the decay rate of the enzyme in both the tissues. Activities of S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine/spermine N 1-acetyltransferase (SAT) were not increased by the intraperitoneal injection of MGBC. There was a large rise in putrescine and a fall in spermidine and spermine in the liver and lung except for brain within an 8 hr period in response to MGBC, suggesting that these changes resulted from the stabilization of ODC and inhibitions of AdoMetDC and spermidine synthase.

Ornithine decarboxylase properties: is there a role for a microsome-bound inactivating activity?

Liver microsomes have a strong ornithine decarboxylase (ODC) inactivating capacity in vitro. The present results suggest that this may be involved in regulation of ODC activity in vivo: (1) the ODC inactivating capacity of microsomes appears susceptible to in vivo modulation: a single administration of thioacetamide, which induces ODC, also causes a significant increase in the inactivating capacity of the microsomes; (2) under conditions leading to increased microsome-bound ODC-inactivating capacity (e.g. liver from thioacetamide-treated rates versus regenerating liver) ODC displays a greater thermal lability and inactivability in vitro. A possible involvement of this microsomal activity in an autoregulatory pathway of ODC is suggested by the fact that it is induced by the administration of polyamines. However, inhibition of ODC activity by alpha-difluoromethylornithine does not prevent the increase of the microsomal activity caused by thioacetamide. Thus, polyamine biosynthesis does not appear to be an absolute requirement for induction of the microsomal ODC-inactivating capacity. The apparent half-life of ODC in vivo, as evaluated after cycloheximide administration, does not appear to correlate with the microsomal ODC-inactivating capacity content and the stability properties of ODC in vitro.

Regulation of ovarian ornithine decarboxylase by human chorionic gonadotrophin.

Treatment of 29-day-old female Sprague-Dawley rats with human chorionic gonadotrophin (hCG) produced a large and rapid increase in the activity of ornithine decarboxylase. Measurements that use a specific radioimmunoassay showed that the increased activity could be accounted for by a parallel change in the amount of ornithine decarboxylase protein. The increased protein content was caused by an increased rate of synthesis, since the half-life of ornithine decarboxylase was not changed by the hormone treatment. The content of mRNA for ornithine decarboxylase was determined by hybridization with a cDNA probe, and it was found that the increased amount of protein was correlated with a change in the amount of mRNA. These results indicate that treatment with hCG induces ornithine decarboxylase in the rat ovary by increasing the production or the stability of the mRNA for this enzyme. The increased amount of ornithine decarboxylase led to an increase in putrescine in the ovary but did not increase the content of the polyamines spermidine and spermine. These findings show that, despite its rapid and large scale induction, ornithine decarboxylase is not the rate-limiting step that determines the content of these polyamines in this tissue. They also suggest that putrescine itself may play an important role in the ovary.

A human neuroblastoma cell line with a stable ornithine decarboxylase in vivo and in vitro

A human neuroblastoma cell line (Paju) grew in 10 mM difluoromethyl-ornithine, which at this concentration normally stops the growth of all mammalian cells. Ornithine decarboxylase from Paju was resistant to inhibition in vitro by difluoromethylornithine, and required 10 uM of the compound for 50% inhibition, whereas ornithine decarboxylase from SH-SY5Y cells (another human neuroblastoma) and from rat liver needed only 0.5 uM difluoromethylornithine. Paju ornithine decarboxylase also exhibited a long half-life (over eight hours) in vivo. The half-life of immunoreactive protein was significantly longer than that of the activity. The long half-life of ornithine decarboxylase in Paju cells leads to its accumulation to a specific activity of 2000 nmol/mg of protein per 30 min during rapid growth (the corresponding activity in SH-SY5Y cells was about 2.5). When partially purified ornithine decarboxylase from Paju cells was incubated with rat liver microsomes it was inactivated with a half-life of 75 min. This inactivation was accompanied by a fall in the amount of immunoreactive protein. In the same inactivating system partially purified SH-SY5Y ornithine decarboxylase had a half-life of 38 min and its half-life in vivo was 50 min. The corresponding values for rat liver ornithine decarboxylase were 45 min and 40 min, respectively. Rat liver microsomes also inactivated rat liver adenosylmethionine decarboxylase. These results suggest that Paju ornithine decarboxylase has an altered molecular conformation, rendering it resistant to (i) difluoromethylornithine and (ii) proteolytic degradation both in vivo and in vitro.

Age-related changes in inducible mouse liver enzymes: Ornithine decarboxylase and tyrosine aminotransferase

The time required to induce two inducible hepatic enzymes, ornithine decarboxylase (ODC) and tyrosine aminotransferase (TAT), by growth hormone and dexamethasone, respectively, increases with age. Specific activity at the peak of induction is the same for all ages. On the other hand, for basal TAT the specific activity per unit of TAT antigen was found to decrease considerably with age. The half-life of ODC was determined after cycloheximide administration. The apparent half-life at the peak of ODC induction increases from 15 minutes in 3–4-month-old mice to 30 minutes in 24-month-old animals. Loss of efficiency in the protein degradation system is implicated in this phenomenon as no apparent differences could be observed in the susceptibility of ODC and TAT from young or old mice to chymotrypsin. ODC and TAT are activated by temperatures of up to 37 °C and 50 °C, respectively. ODC is inactivated at 50 °C while TAT is inactivated at 76 °C. “Young” ODC and TAT are more readily activated or inactivated by heating than the “old” enzymes.