Increase In ODC Activity Research Articles

TMET-32. INTRAOPERATIVE MICRODIALYSIS FOR GLIOMA METABOLIC RECONNAISSANCE AND BIOMARKER DISCOVERY

Abstract Gliomas are genomically heterogeneous tumors that may harness a convergent and therapeutically targetable set of metabolic pathways. At present, the metabolic landscape of in situ human gliomas remains incompletely characterized, hampering translational progress. To that end, we are leveraging the previously untapped potential of high molecular weight microdialysis to determine the global extracellular metabolic profiles of live human gliomas. Under an investigational device exemption, HMW microdialysis (< 100 kDa) was performed at 2.0 μL/min in an initial discovery cohort of five patients in glioma and adjacent brain during neurosurgical resection; a subsequent cohort of five patients was independently analyzed to critically evaluate results from the discovery group. Untargeted metabolomics via ultra-performance liquid chromatography-tandem mass spectrometry revealed over 300 named metabolites and five drugs from only 20 μL of microdialysate, representing a short and feasible 10 minutes of intraoperative collection time. Enrichment analysis of each patient’s tumor vs. brain ranked extracellular metabolome highlighted marked metabolic convergence within the most aggressive regions of molecular diverse tumors (FDR = 0). Pathway analysis revealed significant enrichment for large neutral amino acid pathways, including valine, leucine, and isoleucine biosynthesis (p=1.6E-9) and degradation (p=0.001) and glycine, serine, and threonine metabolism (p=4.7E-5). Notably, this amino acid signature was not as abundantly present in nonenhancing tumor when compared to enhancing tumor (Average tumor/brain: 1.9x vs. 4.3x, respectively), suggesting upregulation of neutral amino acid transporters in enhancing tumor or delivery from plasma into the CNS via a disrupted BBB. Interestingly, guanidinoacetate (GAA) was our most highly conserved and upregulated metabolite (128.9x in tumor vs. brain). Given its co-production with ornithine, the precursor to protumorigenic polyamines, we posit that GAA may serve as a biomarker of increased ODC activity in live human gliomas. In conclusion, intraoperative HMW microdialysis feasibly offers improved opportunities to perform glioma metabolic biomarker and therapeutic discovery.

Putrescine treatment reverses α-tocopherol-induced desynchronization of polyamine and retinoid metabolism during rat liver regeneration

BackgroundThe pre-treatment with α-tocopherol inhibits progression of rat liver proliferation induced by partial hepatectomy (PH), by decreasing and/or desynchronizing cyclin D1 expression and activation into the nucleus, activation and nuclear translocation of STAT-1 and -3 proteins and altering retinoid metabolism. Interactions between retinoic acid and polyamines have been reported in the PH-induced rat liver regeneration. Therefore, we evaluated the effect of low dosage of α-tocopherol on PH-induced changes in polyamine metabolism.MethodsThis study evaluated the participation of polyamine synthesis and metabolism during α-tocopherol-induced inhibition of rat liver regeneration. In PH-rats (Wistar) treated with α-tocopherol and putrescine, parameters indicative of cell proliferation, lipid peroxidation, ornithine decarboxylase expression (ODC), and polyamine levels, were determined.ResultsPre-treatment with α-tocopherol to PH-animals exerted an antioxidant effect, shifting earlier the increased ODC activity and expression, temporally affecting polyamine synthesis and ornithine metabolism. Whereas administration of putrescine induced minor changes in PH-rats, the concomitant treatment actually counteracted most of adverse actions exerted by α-tocopherol on the remnant liver, restituting its proliferative potential, without changing its antioxidant effect. Putrescine administration to these rats was also associated with lower ODC expression and activity in the proliferating liver, but the temporally shifting in the amount of liver polyamines induced by α-tocopherol, was also “synchronized” by the putrescine administration. The latter is supported by the fact that a close relationship was observed between fluctuations of polyamines and retinoids.ConclusionsPutrescine counteracted most adverse actions exerted by α-tocopherol on rat liver regeneration, restoring liver proliferative potential and restituting the decreased retinoid levels induced by α-tocopherol. Therefore interactions between polyamines and retinol, mediated by the oxidant status, should be taken into consideration in the development of new therapeutic strategies for pathologies occurring with liver cell proliferation.

Cell-cycle-dependent regulation of ornithine decarboxylase activity in the unicellular green alga Chlamydomonas reinhardtii

Polyamines play an important role in the control of cell growth and cell division. In the unicellular green alga Chlamydomonas reinhardtii as in animal cells, biosynthesis of the 3 commonly occurring polyamines (putrescine, spermidine and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. Therefore, we have investigated the regulation of ODC activity during the cell cycle of Clamydomonas reinhardtii using synchronized cultures. A 2.5-3-fold increase in ODC activity was observed during the transition to the cell division phase. This up-regulation of ODC activity was not due to an increased level of ODC-mRNA as revealed by northern-blot analyses, but correlated with an increased half-life of this particular enzyme (from 1.1 to 3.2 h). Addition of the DNA topoisomerase II inhibitor nalidixic acid during the second half of the growth period caused a transient decrease of ODC activity and a considerable delay of cell divisions. After cell division, a down-regulation of ODC activity was observed which was faster in the dark than in the light and also correlated with changes of the ODC half-life.

Hyaluronan Fragments Induce Endothelial Cell Differentiation in a CD44- and CXCL1/GRO1-dependent Manner

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.

Protein kinase Cdelta-mediated signal to ornithine decarboxylase induction is independent of skin tumor suppression.

Protein Kinase Cdelta (PKCdelta), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately eightfold) PKCdelta protein in basal epidermal cells are resistant to skin tumor formation by the 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion protocol. However, despite being resistant to skin tumor promotion by TPA, PKCdelta transgenic mice elicited a 3-4-fold increase in TPA-induced epidermal ODC activity and putrescine levels than their wild-type littermates. PKCdelta was observed to be the key component of the TPA signal transduction pathways to the induction of mouse epidermal ODC activity. To determine if TPA-induced ODC activity and associated putrescine levels in PKCdelta transgenic mice contributed to PKCdelta-mediated suppression of skin tumor promotion by TPA, the irreversible inhibitor of ODC, alpha-difluoromethylornithine (DFMO), was used. PKCdelta transgenic mice and their wild-type littermates were initiated with 100 nmol DMBA and then promoted twice weekly with 5 nmol TPA. The experimental group was given 0.5% DFMO in their drinking water, while the control group was given tap water. After 25 weeks, the number of papillomas (>2 mm) per mouse was counted. The DFMO treatment did not affect the skin tumor multiplicity of PKCdelta transgenic mice. These results indicate that PKCdelta-induced ODC activity is not involved in PKCdelta-mediated tumor suppression. Thus, the signaling pathways via PKCdelta to epidermal ODC induction and skin tumor suppression appear to be independent.

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii.

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.

Subcellular localization and light-dark control of ornithine decarboxylase in the unicellular green alga Chlamydomonas reinhardtii

The polyamines putrescine, spermidine and spermine are required for cell proliferation of both eukaryotic and prokaryotic organisms. In plant cells, putrescine, which is also the precursor of spermidine, is derived from ornithine by the action of ornithine decarboxylase (ODC, EC 4.1.1.17) or from arginine by arginine decarboxylase (ADC, EC 4.4.4.19). In the unicellular green alga Chlamydomonas reinhardtii, ODC activity exceeds ADC activity by orders of magnitude as shown in the present communication, indicating that biosynthesis of polyamines is controlled by ODC activity in this organism. ODC was localized in the cytosol and was found to consist of 50 kDa subunits as revealed by affinity labeling with [ 3 H]difluoromethylornithine and subsequent SDS-PAGE and fluorography. Its activity was much higher in light- than in dark-adapted (starved) cells. After transfer from darkness to light, a rapid increase in ODC activity was observed. This light-induced increase in ODC activity could be completely abolished by addition of cycloheximide, an inhibitor of protein biosynthesis, but not by actinomycin D inhibition of transcription. Up-regulation of ODC activity after onset of illumination could be prevented by addition of the photosystem II inhibitor 3-(3'-4'-dichlorophenyl)-1,1-dimethyl-urea. ODC activity was found to be correlated with the ATP level both in light- and dark-adapted cells. ODC half-lives of 42 and 83 min were determined for dark- and light-adapted cells, respectively.

Ornithine decarboxylase in organs of rat following chronic gamma-irradiation at low dose-rates

Purpose: To investigate the consequence of continuous low doserate exposure to gamma -rays on ornithine decarboxylase (ODC EC 4.1.1.17) activity in organs of rat. Materials and methods: Young male Wistar rats were irradiated at 1.1, 2.1 and 12.9cGy/day in the dose ranges of 9-165, 17-315 and 100-2000cGy, respectively, in a specially designed chamber. ODC activity was determined in 20000g supernatant fluid of thymus, spleen and lung by measuring the release of 14CO from l-{1-14C]ornithine. Results: Chronic gamma-irradiation modulated ODC activity. It decreased at low cumulated doses (after 8 and 15 days of exposure). At longer periods after chronic irradiation (after 45 and 90 days), ODC activity was restored up to control levels despite increasing values of cumulated doses. On day 150 a similar increase in ODC activity in spleen 2.1cGy/day and in lung at 12.9cGy/day was observed. Conclusions: These studies showed a non-monotonic pattern of the 'dose-response' curve. The results were interpreted in terms of the triggering of a homeostatic system.

The ornithine decarboxylase gene from Candida albicans. Sequence analysis and expression during dimorphism.

The gene (CaODC) coding for ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned from Candida albicans by PCR and characterized. The deduced protein contains 470 amino acids together with the substrate- and co-factor-binding sequences which define the ornithine decarboxylases of eukaryotic species. It shows a high homology with other ODCs, mainly those from Saccharomyces cerevisiae and Neurospora crassa. A putative PEST sequence, which correlates quite well with those described for other fungal ODCs, could be identified in the protein. The mRNA of the gene is 2.4 kb in size and by primer extension a long leader sequence was found at -558 bases upstream of the predicted start of translation. An identical single ODC gene was identified in three different C. albicans strains. During the dimorphic switch, a transient initial increase in ODC activity was observed. This increase was not accompanied by a rise in the levels of the transcript, suggesting that ODC activity levels may be regulated post-transcriptionally during differentiation.

Polyaminen and brain injury

The cerebral ODC/polyamine system is disturbed by brain injury. The main modifications are important increases in ODC activity and putrescine concentration, with minor variations in spermidine and spermine concentrations. A great diversity of stimuli such as cerebral ischemia or overstimulation of the central nervous system by chemical or non-chemical agents can induce polyamine disturbances. Both the contribution of polyamines to the brain damage and their involvement in the repair mechanisms triggered after brain insults have been proposed.

Ornithine decarboxylase activity in rat intestinal mucosa and liver is stimulated by central administration of 2-deoxy- d-glucose but not of 2,5-anhydro- d-mannitol

It has been shown that 2-deoxy- d-glucose (2-DG) inhibits glucose utilization and elicits feeding through the lateral hypothalamus. In contrast, 2,5-anhydro- d-mannitol (2,5-AM), blocking glycogenolysis and/or gluconeogenesis, elicits feeding through the ventromedial hypothalamus. The aim of the present study was to determine whether ornithine decarboxylase (0130 activity in the rat small intestine is stimulated by infusion into the third ventricle of 2-DG or of 2,5-AM. Under anesthesia, a cannula was implanted into the third ventricle one week before the experiment. Each rat was infused with 6, 12, and 24 μmol 2-DG or 2,5-AM into the third ventricle without disturbing the behavior. Ingestive behavior was observed for one h after the infusion. ODC activity in the intestinal mucosa and the liver was measured 2 h after the infusion. Additionally, ODC activity was measured in vagotomized rats. Both test solutions elicit feeding at 24 μmol/rat. Infusion of 2-DG into the cerebroventricle significantly increased ODC activity in the duodenal and jejunal mucosa and the liver. In contrast to 2-DG, infusion of 2,5-AM did not increase ODC activity in the intestinal mucosa or liver. Truncal vagotomy attenuated the increase of ODC activity in the intestinal mucosa and liver induced by 2-DG. The present study showed that 2-DG, but not 2,5-AM, increased ODC activity in the peripheral organs, indicating that glucose-metabolism at specific sites of the central nervous system, including the lateral hypothalamus, is important for stimulatory signals to ODC activity. It is also indicated that the stimulatory signals from the central nervous system are mediated, at least in part, via the efferent vagal nerve.

Transcription-dependent and -independent regulation of hepatic ornithine decarboxylase activity by CNS beta-endorphin in rat pups.

We have previously shown that intracerebroventricular administration of relatively low doses of beta-endorphin suppresses basal levels of hepatic ODC activity as well as tissue ODC responsiveness to administered insulin in developing rats. Using Northern blotting analysis, the current studies examine whether these effects of CNS beta-endorphin may be mediated by changes in ODC gene expression. Subcutaneous administration of insulin (20 IU/kg body weight) rapidly and profoundly increased liver ODC activity. The time course of the response was characterized by proportionally increased levels of ODC mRNA, suggesting that insulin-induced stimulation of ODC activity is due to an increased transcription of ODC mRNA. Pretreatment with actinomycin D (2 mg/kg body weight, intraperitoneally) completely prevented the insulin-induced increase in ODC activity, confirming the requirement for the de novo synthesis of ODC mRNA for the effect. More importantly, intracerebroventricular but not subcutaneous injection of beta-endorphin (1 microgram) markedly diminished the stimulatory effect of insulin on hepatic ODC mRNA accumulation. The time course and magnitude of the inhibition of mRNA accumulation essentially mirrored that of the peptide on ODC activity. On the other hand, contrary to the inhibitory effect of beta-endorphin on basal ODC activity, the peptide did not lower basal ODC mRNA levels when given alone. Taken together, the results from these studies provide evidence for the existence of at least two separate mechanisms through which CNS beta-endorphin might downregulate ODC activity in peripheral organs of rat pups. The peptide can suppress insulin-induced ODC activity in the liver tissue by decreasing the rate of transcription of the ODC gene, whereas the inhibition of basal ODC activity appears to involve posttranscriptional mechanisms.

Identification and evaluation of the role of endogenous tyrosine kinases in azoxymethane induction of proliferative processes in the colonic mucosa of rats

Although tyrosine kinases (Tyr-k) are known to play a role in regulating proliferation of normal, preneoplastic and neoplastic cells, little is known about the identity of different species of Tyr-k involved in this process. Utilizing a non-denaturing polyacrylamide gel electrophoresis system, in which the separated proteins from tissue extracts are assayed directly for Tyr-k, we attempted to identify the species of Tyr-k that may be involved in azoxymethane (AOM) induction of colonic mucosal ornithine decar☐ylase (ODC) activity, an enzyme whose activity is known to rise in rapidly proliferating cells. We have observed that 5 days after a single injection of the colonic carcinogen AOM (20 mg/kg body wt.) to 3–4-month old rats, a significant 230% rise in colonic mucosal proliferative activity (as evidenced by 5-bromo-2′-deoxyuridine (BrdU) immunoreactivity) was also accompanied by a 550% increase in ODC activity. This was also associated with a marked rise (140–240%) in the relative activity of Tyr-k of three mucosal proteins with M r of 165, 145 and 125 kDa. Since the molecular mass of one of the Tyr-k (165 kDa) corresponded to that of EGF-receptor (EGF-R), this led us to examine the role of EGF-R Tyr-k in AOM induction of colonic mucosal ODC. We observed that a 320% increase in mucosal ODC activity, 5 days after AOM injection, was accompanied by over 200% rise in Tyr-k activity of EGF-R. Daily injection of tyrphostin (300 μg/kg body wt.), a Tyr-k inhibitor with a higher specificity for EGF-R Tyr-k, significantly attenuated AOM-induced stimulation of both ODC and Tyr-k activity of EGF-R. Administration of AOM also stimulated the rate of synthesis and secretion of TGF-α in isolated colonocytes. In addition, the levels of TGF-α and its mRNA in the colonic mucosa were also found to be 100% and 250% higher, respectively, in AOM-treated rats when compared with the controls. We suggest that (a) activation of intrinsic Tyr-k of EGF-R is an important event in AOM induction of colonic mucosal proliferative processes, and (b) this activation is thought to be mediated by TGF-α through an autocrine mechanism.

Tumor‐promoting activity of 2,4‐dinitrofluorobenzene

The aim of this study was to determine whether the skin-sensitizing agent 2,4-dinitrofluorobenzene (DNFB) would elicit the same morphological and biochemical events that are characteristic of 12-O-tetradecanoylphorbol-13-acetate (TPA). While single applications of 0.1% or 0.2% DNFB produced only mild epidermal hyperplasia, multiple applications produced pronounced hyperplasia. Compared with TPA, a single application of DNFB produced small increases in ODC activity, although a second DNFB treatment produced a greater response. Both DNFB and TPA caused marked induction of ODC, c-fos and c-jun mRNA. Vascular permeability increased significantly in response to DNFB, such that after 15 hr the response was quantitatively the same as for TPA. Repeated TPA produced the same response as a single application, but repeated DNFB resulted in a response that was half that of TPA. In contrast to TPA, DNFB failed to activate partially purified protein kinase C (PKC), although it did cause transient down-regulation of activity 15 hr after treatment. The ability of DNFB to induce ODC activity, however, was unaffected by prior down-regulation of PKC. DNFB was also shown to promote tumors in initiated SSIN mice. Twice-weekly applications of 0.1% or 0.2% DNFB resulted in approximately 65% and 85% of the mice developing an average of 2.0 or 3.2 tumors each, respectively. These results demonstrate that DNFB elicits many of the same changes as TPA and that it does so in a PKC-independent manner.

Regulation of spermidine transport in l1210 cells

1. 1. 1 mM 2-amino isobutyric add (AIB), glutamine or asparagine when preincubated for 3 hr with L1210 cells promoted a marked increase in the rate of spermidine uptake. 2. 2. Cycloheximide also increased the transport rate and completely prevented the increase due to AIB. 3. 3. Trifluoperazine and iso-H7 inhibited the uptake of spermidine, much less the uptake of AIB. 4. 4. Adenosine promoted an increase in the uptake of AIB, a decrease in that of spermidine. 5. 5. Hypotonic stress also increased the rate of spermidine transport. This modification was only partially prevented by cycloheximide. 6. 6. Okadaic arid had no effect on this increase, whereas it prevented the increase of ODC activity.

In vivo effect of Berenil on rat liver polyamine metabolism

1. 1. Berenil, administered to rats in vivo, promoted a decrease in liver SAMDC activity, but an increase in ODC and SAT activity. 2. 2. Its effect on ODC was completely prevented by cycloheximide, that on SAT only partially. 3. 3. Berenil had no effect on ODC activity in adrenalectomized rats. Adrenergic antagonists counteracted the effect of Berenil on ODC activity. 4. 4. Polyamine content was increased. The maximum modification was observed for putrescine and N 1-acetylspermidine.

Okadaic acid inhibits insulin stimulation of both ornithine decarboxylase and spermidine transport in hepatocyte cultures

1. 1. Okadaic acid inhibited basal ODC activity in rat hepatocytes in culture and prevented any increase in ODC activity and in the rate of spermidine uptake promoted by both insulin and hypotonicity. 2. 2. The increase promoted by AIB was not counteracted by okadaic acid.

Effects of gastrin and difluoromethylornithine on growth of human colon cancer.

The effect of difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase activity, was evaluated in vivo and in vitro on the growth of a gastrin-sensitive human colon carcinoma (WiDr). In vivo, mice bearing the tumor treated with pentagastrin had larger tumors with higher ornithine decarboxylase activity and polyamine content (P < 0.05) than mice not treated with pentagastrin. Difluoromethylornithine treatment significantly decreased ornithine decarboxylase in both the pentagastrin-treated and the untreated animals; however, DFMO had no effect on tumor volume, weight, protein, or DNA content. In cell culture, gastrin treatment increased WiDr cell number and [3H]thymidine incorporation in the presence or absence of serum. In serum-free conditions, however, gastrin stimulated cell growth without concomitantly increasing ODC activity. DFMO, on the other hand, decreased both ODC activity and growth. These studies suggest that the trophic effect of gastrin on WiDr human colon cancer is independent of ODC activity. Since gastrin treatment increased ODC activity in vivo, gastrin may interact in vitro with other factors present in serum that can alter ODC activity.

Independent actions of asparagine and high levels of free Ca 2+ in the induction of ornithine decarboxylase

During growth stimulation of cells, Ca 2+ and amino acids of the A, ASC and N transport systems are important for the induction of ornithine decarboxylase (ODC, l-ornithine carboxylyase, EC 4.1.1.17). In order to clarify the relationship between Ca 2+ and amino acids, we studied the induction of ODC by asparagine under three different Ca 2+ states in H-35 rat hepatoma cells. First, in normal cells, extracellular Ca 2+ above 0.1 mM and 10 mM asparagine separately stimulated ODC activity and their effects were approximately additive. In these normal cells, asparagine could act in the absence of medium Ca 2+. TMB-8, a sequestered-Ca 2+ release antagonist, had no effect on ODC induction whilst the asparagine action is sensitive to treatment with W 7, a Ca-calmodulin antagonist, or lanthanum, a Ca 2+ antagonist. Secondly, in cells treated with 0.5 mM EGTA in Ca 2+-free medium, the asparagine action on ODC induction was blocked but the inhibition could be reversed by the addition of Ca 2+ to the medium. Thirdly, ionomycin treatment in the absence of medium Ca 2+ did not block the asparagine effect. Furthermore, in ionomycin-treated cells, the presence of high levels of medium Ca 2+ increased ODC activity, but this increase was additive to, and could not replace, the action of asparagine. Our results indicate that the asparagine action does not depend on an increase of intracellular free-Ca 2+.

The induction of ornithine decarboxylase by ornithine takes place at post-transcriptional level in perifused Ehrlich carcinoma cells

The increase in ODC activity during perifusion of Ehrlich carcinoma cells with 0.5 mM ornithine correlates with an increase in ‘de novo’ synthetized ODC protein. ODC synthesis was followed by immunoprecipitation of equal quantities of 35S-labelled proteins after 10, 20 and 30 min of labelling. In addition, the rate of ‘de novo’ protein synthesis is very much elevated in cells perifused with saline buffer supplemented with 0.5 mM ornithine than in cells perifused with the saline buffer only. In spite of the higher specific ODC activity observed in cells perifused with saline buffer plus 0.5 mM ornithine respect to cells perifused with only saline buffer for 3.5 h, no elevation in ODC mRNA was observed when the cells were perifused in the presence of 0.5 mM ornithine.