Effect of alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues.

α-Difluoromethylornithine (RMI 71.782), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (E.C. 4.1.1.17) $\text{in vitro}$, causes a rapid, long-lasting, dose-dependent decrease of ornithine decarboxylase activity in prostate and, to a lesser extent, in thymus and testis of rats when injected intraperitoneally. Repeated doses (100 mg/kg or 1 g/kg) of α-difluoromethylornithine given to rats for two weeks markedly decreased polyamine concentrations in several rat tissues and selectively slowed down growth of ventral prostate and of thymus.     

Effect on prostatic growth of 2-difluoromethylornithine, an effective inhibitor of ornithine decarboxylase.

2-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, causes marked changes in the polyamine metabolism of ventral prostate when given to adult rats in drinking water (20 g/l) for 3 consecutive days. A 90% inhibition of ornithine decarboxylase activity is accompanied by approx. 80% decreases of the concentrations of putrescine and spermidine and by a 36% decrease in spermine. Concomitantly, S-adenosylmethionine decarboxylase activity increases 7-fold and the concentration of decarboxylated S-adenosylmethionine 450-fold. When DFMO is given to immature rats for 12 consecutive days the above described changes are accompanied by a marked reduction in the age-dependent increases of the wet weight and RNA and DNA contents of the ventral prostate. In adult rats DFMO decreases the weight and RNA content of the ventral prostate within 4 days by 32% and 24% respectively and maintains them constant for the next 19 days. After 23 days of treatment, the prostatic weight is 46% of that of control animals of the same age, whereas the weights of other organs are only slightly decreased. Cytological studies carried out at this time show that DFMO reduces the size of both prostatic acini and the epithelial cells lining the acini.     

Changes in the polypeptide composition related to the growth response in chronically isoproterenol-stimulated mouse parotid glands

The administration of isoproterenol induces DNA-synthesis mitosis and growth (increase in size) responses in mouse parotid glands. Both responses were uncoupled by means of daily stimulations with isoproterenol in such a way that the DNA-synthesis mitosis response was observed during the first 4 days only, whereas the growth response was continuous since the first stimulation until about day 12. In parallel to the chronic stimulation by isoproterenol, drastic changes in the polypeptide composition of parotid glands were observed. These modifications, consisting basically of the reduction in content of a couple of major poly peptides (polypeptides A and B) together with the reciprocal massive accumulation of five new polypeptides (polypeptides C, D, E, F and G), were also progressive and continuous along the chronic stimulation by isoproterenol, even after the disappearance of the DNA-synthesis mitosis response. Thus, a relationship between specific changes in the mouse parotid content of polypeptides A, B, C, D, E, F and G and the isoproterenol-induced growth response, rather than with the DNA-synthesis mitosis response, is suggested. The correlation is firmly supported by the progressive recovery of the normal polypeptide composition upon suspending isoproterenol treatment, which allows parotid glands to return to normal size parameters.     

Changes in the polypeptide composition related to the growth response in chronically isoproterenol-stimulated mouse parotid glands

The administration of isoproterenol induces DNA-synthesis mitosis and growth (increase in size) responses in mouse parotid glands. Both responses were uncoupled by means of daily stimulations with isoproterenol in such a way that the DNA-synthesis mitosis response was observed during the first 4 days only, whereas the growth response was continuous since the first stimulation until about day 12. In parallel to the chronic stimulation by isoproterenol, drastic changes in the polypeptide composition of parotid glands were observed. These modifications, consisting basically of the reduction in content of a couple of major poly peptides (polypeptides A and B) together with the reciprocal massive accumulation of five new polypeptides (polypeptides C, D, E, F and G), were also progressive and continuous along the chronic stimulation by isoproterenol, even after the disappearance of the DNA-synthesis mitosis response. Thus, a relationship between specific changes in the mouse parotid content of polypeptides A, B, C, D, E, F and G and the isoproterenol-induced growth response, rather than with the DNA-synthesis mitosis response, is suggested. This correlation is firmly supported by the progressive recovery of the normal polypeptide composition upon suspending isoproterenol treatment, which allows parotid glands to return to normal size parameters.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

Multiple species of ornithine decarboxylase in mouse kidney. Effect of testosterone

Multiple species of ornithine decarboxylase were separated by chromatography of mouse kidney extract on DEAE-Sepharose CL-6B. The elution patterns of ornithine decarboxylase activity and immunoreactive enzyme protein in the kidneys of untreated and testosterone-treated male mice did not differ otherwise than in order of magnitude. The immunoblots of the chromatography fractions neither revealed any differences in enzyme subunit size between two experimental groups. These findings suggest that the stabilization of ornithine decarboxylase by androgens is not due to the molecular changes of enzyme protein.     

1-Aminooxy-3-aminopropane, a new and potent inhibitor of polyamine biosynthesis that inhibits ornithine decarboxylase, adenosylmethionine decarboxylase and spermidine synthase

1-Aminooxy-3-aminopropane was shown to be a potent competitive inhibitor (Ki = 3.2 nM) of homogenous mouse kidney ornithine decarboxylase, a potent irreversible inhibitor (Ki = 50 microM) of homogeneous liver adenosylmethionine decarboxylase and a potent competitive (Ki = 2.3 microM) of homogeneous bovine brain spermidine synthase. It did not inhibit homogeneous bovine brain spermine synthase and it did not serve as a substrate for spermidine synthase. The compound did not inhibit tyrosine aminotransferase, alanine aminotransferase or aspartate aminotransferase, which are pyridoxal phosphate-containing enzymes like ornithine decarboxylase. The inactivation of adenosylmethionine decarboxylase was partially prevented by pyruvate, which is the coenzyme of adenosylmethionine decarboxylase, and by the substrate, adenosylmethionine. 1-Aminooxy-3-aminopropane at 0.5 mM concentration inhibited the growth of HL-60 promyelocytic leukemia cells and this inhibition was prevented by spermidine but not by putrescine.     

Effect of suramin (Bayer 205) on renal ornithine decarboxylase activity and polyamine concentrations in rats.

After a single high dose (20 mg/100 g i.v.) of suramin ornithine decarboxylase activity was increased rapidly in the rat kidney. Enzyme kinetic measurements indicate that on the basis of K_m values renal ornithine decarboxylases from control or suramin treated rats are indistinguishable. Renal nucleic acid and polyamine levels were also enhanced in response to suramin. Changes observed in this study are considered as biochemical signs of induced renal growth.     

Subcellular distribution of ornithine decarboxylase in rat liver and accessibility of the enzyme to α-difluoromethylornithine,an irreversible inhibitor of ornithine decarboxylase

The subcellular localisation of ornithine decarboxylase and of its synthetic irreversible inhibitor, α-difluoromethylornithine, was investigated in control rat livers and in livers of animals in which the enzyme was induced by partial hepatectomy or by treatment with dexamethasone. Ornithine decarboxylase activity was distributed in normal rat liver between the nuclear (40%, mainly nucleolar) and the cytosolic (43%) fractions. Cytosolic liver ornithine decarboxylase was markedly induced after partial hepatectomy or treatment with dexamethasone, whereas the enzyme associated with the nuclear fraction was not induced by these procedures. The irreversible inhibitor was found only in the cytosol fraction and was totally absent from the nuclear fraction.     

Cyclic AMP metabolism in mouse parotid glands. Properties of adenylate cyclase, protein kinase and phosphodiesterase.

Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands. Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity witha pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a Ka of 1.2 - 10(-7) M. Parotid cyclic AMP and cyclic GMP phosphodiesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least sixpeaks of enzyme activity in the pI range of 4-6. Previous reports of a large increase in parotid cyclic AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.     

Changes of ornithine decarboxylase activity and polyamine content upon differentiation of mouse NB-15 neuroblastoma cells

The possible functions of ornithine decarboxylase (ODC) and polyamines in the differetiation of mouse NB-15 neuroblastoma cells were investigated by examining the changes of these parameters in the differentiaton and nondifferentiating NB-15 cells over a 5-day culture period. Differentiation of NB-15 cells was induced by the addition of dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthin (IBMX) to the growth medium and was monitored by neurite outgrowth, increase of acetylcholinesterase (AChE), and R_I cAMP-binding protein. Plating of NB-15 cells in fresh serum-containing growth medium was accompanied by rapid growth and a marked increase of ODC activity, this early increase of ODC activity was attenuated, both in duration and in magnitude, in the differentiating cells. The spermidine content of the differentiating neuroblastoma cell was significantly lower than that of the nondifferentiating cells. In the fully differentiated neuroblastoma cells, the ODC activity and spermidine content were lower than that of the undifferentiated cells by approximately 15-fold and five-fold, respectively. Based on these results it is proposed that changes of polyamine metabolism may be of significance in the differentiation of mouse neuroblastoma cells.     

Effect of starvation and refeeding on polyamine concentrations and ornithine decarboxylase antizyme in mammary gland of lactating rats.

Starvation caused a marked increase in putrescine content in mammary gland of lactating rats, together with a marked decrease in activity of ornithine decarboxylase and appearance of measurable ornithine decarboxylase antizyme. 2. Refeeding for 5 h caused disappearance of free antizyme and ornithine decarboxylase activity returned to the value in fed animals. Putrescine concentration remained elevated. 3. There was no significant change in nucleic acid content of mammary gland from starved rats, but spermidine and spermine contents increased significantly. 4. Refeeding for 5 h returned the spermidine content of mammary glands to 'fed' values, and significantly decreased the content of spermine, although it did not reach control values. Thus changes in polyamine content of mammary gland in starved rats are clearly dissociated from changes in either RNA content or activities of polyamine-synthetic decarboxylases. 5. Starvation caused a fall in the content of spermidine in liver, with no change in spermine content. Refeeding for 5 h returned the spermidine content to 'fed' values.     

D,L-alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, induces differentiation in MEL cells

In the present study, the effect of D,L-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), on Friend's murine erythroleukemia (MEL) cell differentiation is investigated. DFMO was able to induce differentiation of MEL cells in culture as determined by haemoglobin (Hb) content and percentage of cells synthesizing Hb detected by benzidine staining. DFMO at a concentration of 2 mM resulted in about 70% benzidine-positive cells on the fifth day. There was a time-dependent increase in the percentage of benzidine-positive cells starting from day three. However, only a 24 h presence of DFMO in the medium was required to induce differentiation suggesting that DFMO switches on a pathway during this period leading to terminal differentiation of MEL cells. DFMO induced differentiation of MEL cells was sensitive to dexamethasone and 5-bromo-2'-deoxyuridine.     

Selenium and polyamine metabolism: different effect of selenite on liver and bursa of Fabricius ornithine decarboxylase activity

1. When injected i.p., sodium selenite promoted a marked increase of rat liver ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities; when administered with the diet for 6 weeks, a less marked increase in liver ODC was observed, whereas SAMDC was not significantly changed. 2. Protein synthesis was involved in the observed modifications. The rate of ODC inactivation was also changed. 3. ODC increase was accompanied by an enhanced putrescine concentration in liver. 4. A marked increase of ODC, accompanied by an enhancement of putrescine, was promoted by selenite (i.p.) also in chicken liver, together with an enhancement of glutathione concentration. Spermidine acetyltransferase (SAT) was also increased. 5. In the bursa of Fabricius, SAT activity was also increased, whereas ODC was decreased. However the expected modifications in polyamine concentration were not observed. 6. Decrease of ODC activity in the bursa was not due to an antizyme. 7. In vitro, selenite concentrations known to inhibit cell proliferation (greater than 1 microgram/ml) inhibited both ODC and SAT activities; at lower concentration, SAT activity was enhanced.     

Effect of administration of 5-hydroxytryptophan and an inhibitor ofl-aromatic amino acid decarboxylase on glucose metabolism in rat brain

The effect of 5-hydroxytryptophan (5-HTP)—the precursor of serotonin (5-hydroxytryptamine, 5-HT)—and of an inhibitor,N-(dl-seryl)-N-(2,3,4-trihydroxybenzyl)hydrazine (Ro4-4602), ofl-aromatic amino acid decarboxylase on the metabolism of glucose to amino acids in brain tissue was investigated. Labeled glucose (20 Ci, 0.24 mg in 0.2 ml 0.9% saline) was injected intravenously into fed rats pretreated with Ro4-4602 (50 mg/kg intraperitoneally) either alone or in combination with 5-HTP (30 mg/kg intravenously) or with the appropriate vehicle. After the injection of Ro4-4602 plus 5-HTP, the concentrations of 5-HT and 5-HTP in brain were increased, but the increase of 5-HTP that Ro4-4602 slightly inhibits the reaction of decarboxylation in the brain, although at the dose used the drug is usually considered to act only peripherally. After administration of Ro4-4602 alone or combined with 5-HTP, the concentration of glucose in plasma was not significantly increased. However, the concentration of glucose in brain was markedly increased with such treatments. The administration of Ro4-4602 alone or combined with 5-HTP reduced the flux of¹⁴C from labeled glucose to amino acids in brain. The concentrations of amino acids in brain were little changed by these treatments.     

Effects of epidermal growth factor on the syntheses of DNA and polyamine in isoproterenol-stimulated murine parotid gland

The effects of epidermal growth factor (EGF) on isoproterenol (IPR)-stimulated DNA synthesis and the activities of the rate limiting enzymes of polyamine synthesis (ornithine and S-adenosylmethionine decarboxylases) in parotid glands were investigated in vitro in cultured rat parotid explants and in vivo in submandibulectomized mice (mice after bilateral removal of the submandibular and sublingual glands). When the explants were cultured on siliconized lens paper floating on chemically defined synthetic medium, IPR caused the increases of both tissue cAMP level and the two decarboxylase activities in the prereplicative period and the stimulation of DNA synthesis with similar time courses to those observed in vivo. Dibutyryl cyclic AMP (DBcAMP) also increased the enzyme activities, but not DNA synthesis. EGF (1-2 ng/ml) had little effect on the IPR- and DBcAMP-dependent increases of amylase secretion and the enzyme activities, but it markedly enhanced IPR-stimulated DNA synthesis. Moreover, increase in DNA synthesis by DBcAMP was clearly observed in the presence of EGF when the explants were treated with this nucleotide analogue only during the early prereplicative period. In in vivo experiments, IPR-dependent increase in DNA synthesis was less in submandibulectomized mice than in intact animals. This decreased response to IPR of DNA synthesis was completely reversed by administration of EGF, though EGF alone did not induce either the enzymes or DNA synthesis. In submandibulectomized mice, although increases in the enzyme activities 8 h after injection of IPR were lower and they were significantly reversed by EGF, the activities at 12 h and the changes in polyamine levels at 8 and 12 h were almost the same as those in intact mice and were not affected by EGF treatment. These results obtained in vitro and in vivo suggest that EGF participates in the maximal response of IPR-dependent DNA synthesis but is not involved in the change of polyamine synthesis induced by IPR in murine parotid glands.