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.     

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.     

Aminooxypropylamine--an effective inhibitor of ornithine decarboxylase in vitro and in vivo

Hydroxylamine-containing analogues of putrescine and cadaverine have been found effective in inhibiting the mouse liver ornithine decarboxylase, the best among synthesized were 1-aminooxy-3-aminopropane (I50 2.10(-8) M) and 1-aminooxy-4-aminobutane (I50 2.10(-7) M). The inhibitory effect of these substances on the mouse liver ornithine-transaminase and S-adenosylmethionine decarboxylase from E. coli was displayed at concentrations higher by several orders of magnitude, that demonstrated the specificity of the compounds of this type. 1-Aminooxy-3-aminopropane in experiments in vivo suppressed the ornithine decarboxylase activity in mouse liver at 16 mg/kg by 75%, the toxic effect being insignificant.     

In vitro effects of the polyamine biosynthesis inhibitor, α-difluoromethylornithine, on ornithine decarboxylase activities from three plant pathogenic fungi

The effects of α-difluoromethylornithine (DFMO) on in vitro ornithine decarboxylase (ODC) activities from three plant pathogenic fungi, Pyrenophora avenae, Pyricularia oryzae and Uromyces viciae-fabae , were studied. DFMO concentrations from 0·01 to 1·0 mmol/l produced no significant effects on ODC activities from the three fungi. However, increasing the DFMO concentration to 5 mmol/l produced a substantial reduction in in vitro ODC activity from Pyre, avenae. The ODC inhibitor, α-monofluoromethylornithine (2 mmol/l), significantly reduced in vitro ODC activity from Pyre. avenae , whereas RR-methyl acetylenic putrescine, an ODC inhibitor based on putrescine, produced no significant effect on the fungal enzyme.     

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.     

Effect of 1,3-diaminopropan-2-o1, an inhibitor of ornithine decarboxylase, on the metabolic response of liver to insulin

The effect of diaminopropanol, an inhibitor of polyamine synthesis, on the metabolic response of liver to insulin was studied in streptozotocin-diabetic rats. Insulin elicited a prompt and very marked increase in ornithine and S-adenosylmethionine decarboxylase activities and in putrescine concentration. Pretreatment of rats with diaminopropanol prevented the increase in the decarboxylases and resulted in decreased spermidine and spermine content of liver. The insulin-induced increase in glycogen content was depressed by 50% and the increase in the rate of lipogenesis in vivo was completely prevented by prior injection of diaminopropanol. These studies implicate altered polyamine metabolism in the metabolic response of liver of streptozotocin-diabetic rats to insulin.     

Human ornithine decarboxylase paralogue (ODCp) is an antizyme inhibitor but not an arginine decarboxylase

ODC (ornithine decarboxylase), the rate-limiting enzyme in polyamine biosynthesis, is regulated by specific inhibitors, AZs (antizymes), which in turn are inhibited by AZI (AZ inhibitor). We originally identified and cloned the cDNA for a novel human ODC-like protein called ODCp (ODC paralogue). Since ODCp was devoid of ODC catalytic activity, we proposed that ODCp is a novel form of AZI. ODCp has subsequently been suggested to function either as mammalian ADC (arginine decarboxylase) or as AZI in mice. Here, we report that human ODCp is a novel AZI (AZIN2). By using yeast two-hybrid screening and in vitro binding assay, we show that ODCp binds AZ1-3. Measurements of the ODC activity and ODC degradation assay reveal that ODCp inhibits AZ1 function as efficiently as AZI both in vitro and in vivo. We further demonstrate that the degradation of ODCp is ubiquitin-dependent and AZ1-independent similar to the degradation of AZI. We also show that human ODCp has no intrinsic ADC activity.     

The inhibition of induction of microsomal monooxygenase activity by 1,3-diamino-2-propanol, an inhibitor of ornithine decarboxylase.

The p.o. administration of 1,3-diamino-2-propanol, an indirect inhibitor of ornithine decarboxylase, to rats and mice inhibited in a dose-dependent manner the induction of cytochrome P-450, benzo(a)pyrene hydroxylase, and 7-ethoxycoumarin O-deethylase by phenobarbital, β-naphtoflavone or Clophen C, a mixture of polychlorinated biphenyls. The results are consistent with the hypothesis that the induction of ornithine decarboxylase is a necessary step in the induction of microsomal monooxygenases.     

A macromolecular inhibitor of the antizyme to ornithine decarboxylase.

A macromolecular factor that inhibits the activity of the antizyme to ornithine decarboxylase (ODC) was found in rat liver extracts. The factor, 'antizyme inhibitor', was heat-labile, non diffusable and of similar molecular size to ODC. The antizyme inhibitor re-activated ODC that had been inactivated by antizyme, apparently by replacing ODC in a complex with antizyme. Therefore the antizyme inhibitor can be used to assay the amount of inactive ODC-antizyme complex formed in vitro. When assayed by this method, the complex was shown to be eluted before ODC from a Sephadex G-100 column. Significant increase in ODC activity was observed when the antizyme inhibitor was added to crude liver extracts from rats that had been injected with 1,3-diaminopropane to cause decay of ODC activity, suggesting the presence of inactive ODC-antizyme complex in the extracts.     

High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads

High activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, is typically present in rapidly proliferating normal and malignant cells. The mitotically inactive steroidogenic cells in rodent testis and ovaries, however, also display high ODC activity. The activity of ODC in these cells responds to luteinizing hormone, and inhibition of ODC reduces the production of steroid hormones. Polyamines and ODC also control proliferation of germ cells and spermiogenesis. The activity of ODC, especially in proliferating cells, is regulated by antizyme inhibitor (AZIN). This protein displaces ODC from a complex with its inhibitor, antizyme. We have previously identified and cloned a second AZIN, i.e. antizyme inhibitor 2 (AZIN2), which has the highest levels of expression in brain and in testis. In the present study, we have used immunohistochemistry and in situ hybridization to localize the expression of AZIN2 in human gonads. We found a robust expression of AZIN2 in steroidogenic cells: testicular Leydig cells and Leydig cell tumors, in ovarian luteinized cells lining corpus luteum cysts, and in hilus cells. The results suggest that AZIN2 is not primarily involved in regulating the proliferation of the germinal epithelium, indicating a different role for AZIN1 and AZIN2 in the regulation of ODC. The localization of AZIN2 implies possible involvement in the gonadal synthesis and/or release of steroid hormones.     

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.     

Gene expression of ornithine decarboxylase in L1210 leukaemia cells exposed to DL-2-difluoromethylornithine in the presence of cadaverine.

Cultured mouse L1210 leukaemia cells treated with DL-2-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), in the presence of micromolar concentrations of cadaverine, started to overproduce ornithine decarboxylase after an exposure of several weeks. The more than 60-fold excess of the enzyme protein in the drug-treated cells apparently resulted from a strikingly enhanced accumulation of mRNA for the enzyme associated with only a modest (about 2-fold) gene amplification.     

An effective inhibitor of S-adenosylmethionine decarboxylase

New analogues of cAMP, namely 2'-phosphate of cAMP and its esters, were obtained by direct phosphorylation of N6-benzoyladenosine 3',5'-cyclic phosphate with 2-cyanoethyl phosphate in the presence of DCC followed by the removal of protecting groups.     

Thyroid-hormone effects on ornithine decarboxylase.

We examined thyroidectomized, normal and hyperthyroid rats and found that ornithine decarboxylase activity was directly correlated with thyroid functional state in heart and liver and unaffected in brain, testes and spleen, phenomena that correlate with the known effect of thyroid hormone on protein synthesis.     

Fluorescent location of ornithine decarboxylase employing derivatives of the specific inhibitor alpha-difluoromethyl ornithine

Two fluorescent derivatives have been made from alpha-difluoromethyl ornithine by linking the carboxyl group of the ornithine derivatives to fluorescent amines. alpha-difluoromethyl ornithine is a potent inhibitor of ornithine decarboxylase, an enzyme which plays an essential role in cell division. We have used these fluorescent derivatives as probes for ornithine decarboxylase in frozen sections of skin to locate the epithelial cells which are known to contain ornithine decarboxylase. The probes also located squamous cell carcinoma cells in human skin.