Inhibition of ornithine decarboxylase by ifenprodil

The present study investigated the changes in NMDA receptor subunit proteins in diazepam-withdrawn rat cerebral cortex, using Western blotting analysis. The protein levels of the NR1 and NR2B, but not NR2A, subunits were significantly increased in diazepam-withdrawn rats compared to those in control rats. Therefore, an increase in the NR1 and NR2B subunit proteins may be responsible for both the previously observed upregulation of [3H]dizocilpine binding in the cerebral cortex and the appearance of diazepam withdrawal signs.     

Pyridoxal 5'-phosphate and the regulation of ornithine decarboxylase activity and stability

There are two forms of ornithine decarboxylase with respect to pyridoxal 5'-phosphate (pyridoxal-P) affinity in exponentially-growing Swiss 3T3 mouse fibroblasts: form I (Km approximately 10 muM) accounts for 30% of the total activity, and form II (Km approximately 0.4 muM) the remainder. Each form of the enzyme is in rapid equilibrium with ornithine and pyridoxal-P; neither form recognizes the Schiff base between ornithine and pyridoxal-P as a substrate. Total pyridoxal-P concentrations indicate that both forms may normally be at least partially active in vivo. Upon stimulation of 3T3 cells by pituitary growth factors, form I becomes undetectable within 4 h. As total activity increases over 10-fold during this time and continues to increase thereafter, a possible conversion of form I to form II could account for this increase only if the Km change reflects other changes in preexisting enzyme. The rates of cofactor dissociation are apparently the same for each form and neither rate changes with the growth state. Since rapid equilibrium kinetics apply, the forms apparently differ in their rate of cofactor association. The half-lives of the two forms in vivo are the same in unstimulated cells when measured concurrently. Also, the half-life of total activity decreases markedly upon stimulation as form II becomes dominant. These and other observations are not consistent with pyridoxal-P serving a major protective function for the enzyme in vivo.     

Feedback regulation of mammalian ornithine decarboxylase. Studies using a transient expression system

Ornithine decarboxylase catalyzes the first step in the biosynthesis of polyamines in mammalian cells. The enzyme is subject to various control mechanisms to maintain adequate intracellular levels of polyamines. Polyamines exert a strong feedback control on ornithine decarboxylase. In a recent study [van Daalen Wetters, T., Macrae, M., Brabant, M., Sittler, A. & Coffino, P. (1989) Mol. Cell. Biol. 9, 5484-5490], it was concluded that feedback control of ornithine decarboxylase is mainly, if not exclusively, a posttranslational phenomenon. The existence of a fast-acting polyamine-stimulated component of ornithine decarboxylase degradation that acts on newly synthesized monomeric forms of the enzyme was postulated. In the present study we have used a transient expression system to test this hypothesis. The expression of ornithine decarboxylase in mock-transfected COS cells varied depending on the cellular supply of polyamines as has been found in other mammalian cells. Thus, supplementing the cells with exogenous spermidine resulted in a marked decrease in ornithine decarboxylase activity, whereas depletion of intracellular polyamines, using an ornithine decarboxylase inhibitor, gave a large increase in the cellular content of the enzyme. COS cells expressing an ornithine decarboxylase mRNA devoid of its 5' non-translated region did not exhibit any feedback control of the enzyme, neither in the presence of exogenous spermidine nor when the intracellular polyamine levels were depleted to the same extent as in the mock-transfected COS cells. The results strongly suggest that the feedback control of ornithine decarboxylase is not merely a posttranslational phenomenon.     

Mutation of aspartate-233 to valine in mouse ornithine decarboxylase reduces enzyme activity

Ornithine decarboxylase is the first and key enzyme in mammalian polyamine biosynthesis. All eukaryotic ornithine decarboxylases contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. This region contains a glycine-rich sequence typically found in a number of pyridoxal 5'-phosphate-dependent or nucleotide-binding proteins. We mutated aspartate-233 which is the only acidic residue within this region to valine. This mutation causes striking sequence similarity with the guanine nucleotide binding domain of c-H-ras. Mutated ornithine decarboxylase cDNA with a mouse mammary tumor virus long terminal repeat promoter has been transfected for stable expression into ornithine decarboxylase-deficient C55.7 cells. Ornithine decarboxylase activity of the mutated enzyme was about 20% of wild-type ornithine decarboxylase activity and it was not activated by guanosine triphosphate like the ornithine decarboxylase isoform found in some tumors and rat brain. The mutation caused an increase in K(m) value of about 20-fold both for the substrate L-ornithine and for the cofactor pyridoxal 5'-phosphate. The Ki value for the irreversible inhibitor alpha-difluoromethylornithine was also increased, whereas the half-life of the enzyme was shortened. These results suggest that the region containing aspartate-233 is essential for binding of the cofactor and thus forms part of enzymatic active site, and the mutation of aspartate-233 to valine cannot, at least alone, cause the activation of ornithine decarboxylase by guanosine triphosphate (230).     

Relative in vitro stability of ornithine decarboxylase from liver preneoplastic nodules and hepatomas

A very rapid and drastic microsome-dependent in vitro inactivation of the hydrocortisone-induced ornithine decarboxylase in rat liver was reported recently (M. F. Zuretti and E. Gravela, Biochim. Biophys. Acta. 742: 269-277, 1983). Present results show that ornithine decarboxylase from preneoplastic nodules and hepatomas, which have been induced in rats by N-2-fluorenylacetamide, is much more stable, its greater stability not being accounted for by a lower microsome-bound inactivating capacity. The possibility of a relationship between the in vitro enzyme stability and the increase of enzyme activity in neoplastic tissues is suggested.     

Mosaic pattern of ornithine transcarbamylase expression in spfash mouse liver

Mosaicism of ornithine transcarbamylase expression was immunohistochemically examined in hepatocytes of spfash heterozygous female mouse livers. An immunohistochemical method using polyclonal antibodies against ornithine transcarbamylase visualized the mosaicism in serial paraffin sections. Very complicated mosaic patterns consisting of positive and negative hepatocytes were obtained in a section of adult liver, but a computer-aided three-dimensional analysis of serial sections demonstrated that patches with complicated shapes and various sizes, which are contiguous groups of positive or negative hepatocytes and are isolated in sections, connected very well with one another. No definite orientation such as portal-central was observed in the three-dimensional images of each patch. In balanced regions of the mosaicism, most individual plates along their straight portal-central lengths did not appear to be composed of only marker-positive or marker-negative hepatocytes. By contrast, in unbalanced regions of the mosaicism, individual plates along their total portal-central lengths often consisted entirely of a major type of hepatocytes. A patch appeared to be present in more than two lobules, but each patch did not constitute a complete lobule. Complicated mosaic patterns of patches were also seen in neonatal and postnatal livers. These results suggest that, although hepatocytes proliferate and migrate extensively during development, they might allocate their daughter cells contiguously and the orientation of their allocation might be random, leading to the formation of three-dimensionally large contiguous quasiclones of hepatocytes, the shapes of which are very complicated.     

Protein-reactive metabolites of carbamazepine in mouse liver microsomes

The character of reactive metabolites formed from carbamazepine (CBZ) was sought in incubations of [14C]CBZ in hepatic microsomes prepared from adult female mice of a strain (SWV/Fnn) susceptible to CBZ-induced teratogenicity. The formation of radio-labeled protein adducts was used as an index of reactive metabolite exposure. A dependence on cytochrome P450 was shown by a requirement for NADPH and inhibition by carbon monoxide, 1-aminobenzotriazole, piperonyl butoxide, and stiripentol. The addition of ascorbic acid, caffeic acid, N-acetylcysteine, and glutathione decreased the rate of binding of the radiolabel from [14C]CBZ to microsomal protein by more than 50%. The addition of glutathione transferases diminished protein adduct formation beyond that seen with glutathione alone. Evidence for the formation of an arene oxide was sought through the use of inhibitors of epoxide hydrolases, including cyclohexene oxide, chalcone oxides (with the addition of cytosol as appropriate), and by the addition of recombinant human soluble and microsomal epoxide hydrolases and recombinant rat microsomal epoxide hydrolase. The microsomal epoxide hydrolases decreased the velocity of 14C-labeled protein adduct formation by approximately 23%, whereas inhibitors had no effect, most likely because of the low native activity of microsomal epoxide hydrolase in mice. Both DT-diaphorase and catechol-O-methyltransferase diminished 14C-labeled protein adduct formation by 54% and 45%, respectively. The data suggest that the major reactive metabolites formed from CBZ by adult female SWV/Fnn liver microsomes are quinones and arene oxides.     

The effect of colchicine on the induction of ornithine decarboxylase by 12-O-tetradecanoyl-phorbol-13-acetate

The induction of mouse epidermal ornithine decarboxylase, 1 of the earliest and largest phenotypic changes following treatment of mouse skin with the tumor-promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate, can be inhibited by prior administration of colchicine. Maximal inhibition of this enzyme induction was observed when colchicine was injected i.p. 90 or 120 min before promoter treatment, although time intervals up to 20 hr between colchicine and promoter treatment were effective. The effect of colchicine was dose dependent, with a dose as low as 25 nmoles/mouse causing an inhibition of 35%. Other microtubule-disrupting agents, vinblastine, vincristine, and Colcemid, had a similar effect on ornithine decarboxylase activity. However, beta, gamma-lumicolchicine, a photochemical derivative of colchicine with no antimitotic or microtubule-disrupting ability, and cytochalasin B, an inhibitor of microfilament-dependent processes, had no effect. N6, O2'-dibutyryl 3',5'-cyclic adenosine monophosphate, when administered just before colchicine, blocked the inhibitory action of colchicine. The results of these studies suggest that colchicine-sensitive structures, most likely containing microtubules, may be mediating elements between the binding of tumor promoters, perhaps to specific cell surface receptors, and the subsequent induction of ornithine decdaboxylase.     

Binding of paracetamol metabolites to mouse liver glutathione S-transferases

In mouse liver homogenate with an intact microsomal metabolism covalent binding of [14C]-paracetamol amounted to 1 nmol/mg protein. 65% of the total radioactivity were bound to soluble protein and 35% to microsomes. In the soluble fraction the major radioactivity peak co-chromatographed with glutathione S-transferase activity on Sephacryl S-300. Two different minor labelled fractions with apparent molecular weights of 130 000 and 25 000 daltons were also found. In a second experiment in a reconstituted system of microsomes and supernatant, 86% of the radio-activity was bound to supernatant and 14% by of microsomes. Following ion exchange chromatography of the supernatant on DEAE-Sepharose, the two major radioactivity-containing fractions coincided with GSH-S-transferase activities, but not with selenium-dependent or non-selenium-dependent glutathione peroxidase. The data show that irreversible binding of paracetamol metabolites in mouse liver occurs preferentially to GSH-S-transferases.     

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.     

Elevated expression of epidermal ornithine decarboxylase mRNA in scleroderma

Using in situ hybridization techniques, we examined the expression of ornithine decarboxylase (ODC) mRNA in the skin of five patients with systemic sclerosis (SSc) and five normal controls. Sections treated with an anti-sense probe showed concentrated grains exclusively in the epidermis of SSc patients, but not in that of normal controls. Because our subcloned anti-sense probe specifically hybridizes with ODC mRNA, these findings indicate that the expression of ODC mRNA is elevated in SSc epidermis. Possibly polyamines have an important part to play in the skin changes of SSc.     

Promotion of rat hepatocarcinogenesis by dimethylarsinic acid: association with elevated ornithine decarboxylase activity and formation of 8-hydroxydeoxyguanosine in the liver

Arsenicals are epidemiologically significant chemicals in relation to induction of liver cancer in man. In the present study, we investigated the dose-dependent promotion potential of dimethylarsinic acid (DMAA), a major metabolite of inorganic arsenicals in mammals, in a rat liver carcinogenesis model. In experiment 1, glutathione-S-transferase placental form (GST-P)-positive foci, putative preneoplastic lesions, were employed as endpoints of a liver medium-term bioassay for carcinogens (Ito test). Starting 2 weeks after initiation with diethylnitrosamine, male F344 rats were treated with 0, 25, 50 or 100 ppm of DMAA in the drinking water for 6 weeks. All animals underwent two-thirds partial hepatectomy at week 3 after initiation. Examination of liver sections after termination at 8 weeks revealed dose-dependent increases in the numbers and areas of GST-P-positive foci in DMAA-treated rats as compared with controls. In experiment 2, ornithine decarboxylase activity, which is a biomarker of cell proliferation, was found to be significantly increased in the livers of rats treated with DMAA. In experiment 3, formation of 8-hydroxydeoxyguanosine, which is a marker of oxygen radical-mediated DNA damage, was significantly increased after administration of DMAA. These results indicate that DMAA has the potential to promote rat liver carcinogenesis, possibly via a mechanism involving stimulation of cell proliferation and DNA damage caused by oxygen radicals.     

Polyamines and regulation of spermatogenesis: selective stimulation of late spermatogonia in transgenic mice overexpressing the human ornithine decarboxylase gene

Polyamines are believed to participate in the induction of cell growth, differentiation, and proliferation, but their role in spermatogenesis has remained obscure. Two transgenic mouse lines (K2 and K15) that overexpress the human ornithine decarboxylase (ODC) gene coding for a rate-controlling enzyme in polyamine biosynthesis and, hence, contain high levels of tissue putrescine have been used to study the stage-specific role of ODC in spermatogenesis. In K2 mice with 30-fold testicular ODC overexpression, [3H]thymidine incorporation at stages I-VI of the cycle of the seminiferous epithelium was significantly above the control level. This may reflect a specific stimulation of DNA synthesis in type A4, intermediate, and type B spermatogonia. The K15 mice that have about 70-fold ODC overexpression showed an elevation of DNA synthesis only at stage V of the cycle, suggesting a specific dependence of type B spermatogonia on putrescine. In K15 mice, [3H]thymidine incorporation of stage VIII tubule segments was decreased, suggesting that excess amounts of putrescine selectively inhibit meiotic DNA synthesis. We propose that putrescine has strictly selective local stimulatory and inhibitory actions during spermatogenic DNA synthesis, and that its excess amounts ultimately may lead to decreased fertility.     

Rapid spectrophotometric method for determining ornithine decarboxylase using 2,4-dinitrofluorobenzene

A sensitive spectrophotometric micromethod was developed for estimation of ornithine decarboxylase (ODC) activity in animal tissues. Distinct increase in the enzymatic activity was found in regenerating and malignant liver tissues as well as in hepatomas G-27 and G-46 as compared with the corresponding tissues of control animals. The linear relationship between the optic density and content of dinitrophenyl putrescine was maintained over the range of 0 to 150 nmol per a sample. The ODC pH optimum was observed within the zone pH 6.4-6.8; it did not depend on the nature of the buffer, used for tissue homogenization. The recovery of putrescine added into the sample was equal to 102.97 +/- 2.22%. The method had an error less than 3%, its sensitivity was 0.1 nmol/sample and the reproducibility was about 100%.     

Effect of haloperidol on adrenal ornithine decarboxylase activity of the rat

The administration of the dopamine antagonist haloperidol (HLP) to rats produced a temporary increase in adrenomedullary and cortical ornithine decarboxylase (ODC) activity. The time-course of stimulation of ODC activity by HLP showed different patterns in both structures. Medullary ODC activity was highest at 2.5 h, decreasing at later times; cortical ODC activity was not affected by the drug at 2.5 h, but then increased up to at least 6.5 h. The medullary increase observed at 2.5 h was dose-related and could be prevented by splanchnicotomy. Hypophysectomized rats, on the contrary, showed an enhanced response to HLP. The results suggest that haloperidol-induced increase of adrenomedullary ODC activity is caused by a reflex increase in preganglionic nerve activity, and that the pituitary gland can modulate this response. Cortical ODC response to HLP, as previously demonstrated, is mediated entirely by the hypophysis.