In vitro killing of Babesia bovis by the ornithine analog alpha-monofluoromethyldehydroornithine methyl ester

The effects of the 2 ornithine decarboxylase inhibitors alpha-difluoromethylornithine (DFMO) and alpha-monofluoromethyldehydroornithine methyl ester (delta MFMO-ME) on growth of Babesia bovis blood stages in vitro were tested. The DFMO had no apparent effects on in vitro growth of B. bovis nor on the morphology of the parasite at concentrations up to 20 mM. In contrast, delta MFMO-ME had cytotoxic effects on B. bovis at 0.5 mM which were more pronounced at 5 mM. delta MFMO-ME caused both a decrease in percentage parasitized erythrocytes and a degeneration of parasites after 12 h exposure, and the magnitude of both effects was dose-dependent. The effects of delta MFMO-ME were not reversible for B. bovis precultured for 12 h (5 mM) or 24 h (0.5 mM) in drug before culturing the parasite in drug-free medium. Unexpectedly, 300 microM putrescine did not reverse the effects of delta MFMO-ME on B. bovis blood stage, raising the possibility that inhibition of ornithine decarboxylase is not responsible for these effects.     

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.     

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or α-difluoromethylornithine

Abstract We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and α-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 μM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G₁ and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 × 10⁵), medium (5 × 10⁵), or high (2 × 10⁶) cell densities in a 25 cm² flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.     

Effects of cyclosporine and alpha-difluoromethylornithine on the growth of mouse colon cancer in vitro

The growth and survival of mouse (MC-26) colon carcinoma in vitro and in vivo are significantly reduced by inhibitors of polyamine biosynthesis. alpha-Difluoromethylornithine (DFMO), is a specific and irreversible inhibitor of ornithine decarboxylase (ODC); the rate-limiting enzyme in polyamine biosynthesis. DFMO treatment inhibits the growth of MC-26 colon cancer cells and decreases MC-26 cell survival both in vitro and in vivo. In the present study, we examined the effects of cyclosporine (CsA) on growth, survival, and polyamine levels in MC-26 colon cancer in vitro. CsA had inhibitory effects on MC-26 colon cancer growth which were similar to DFMO; these effects were blocked by the addition of the polyamine, putrescine. The combination of CsA (8.3 X 10(-4) mM) and DFMO (0.5 mM or 1.0 mM) inhibited MC-26 cell survival to a greater extent than either agent alone. These results suggest that CsA given in combination with other agents which inhibit polyamine synthesis may be useful for the treatment of colon cancer.     

Inhibition of growth of Giardia lamblia by difluoromethylornithine, a specific inhibitor of polyamine biosynthesis

Difluoromethylornithine (DFMO) is a specific and irreversible inhibitor of ornithine decarboxylase, an enzyme which catalyzes the first step in the biosynthetic pathway of the polyamines. We tested the effect of DFMO on the growth of Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis. Growth of G. lamblia was inhibited by DFMO at concentrations of greater than or equal to 1.25 mM. Culture doubling time increased with increasing DFMO concentration. Growth inhibition was reversed if spermidine was added within 53 h of addition of DFMO; no growth was observed if spermidine was added later, indicating eventual parasite death. The growth of E. histolytica and T. vaginalis, two unrelated mucosal-dwelling parasites of humans, was not inhibited by 20 mM DFMO. These studies indicate that polyamine biosynthesis from ornithine is required for growth of G. lamblia.     

Polyamine depletion induces G<Subscript>1</Subscript> and S phase arrest in human retinoblastoma Y79 cells

Background  

Polyamines and ornithine decarboxylase (ODC) are essential for cell proliferation. DL-α-difluoromethylornithine (DFMO), a synthetic inhibitor of ODC, induces G₁ arrest through dephosphorylation of retinoblastoma protein (pRb). The effect of DFMO on cell growth of pRb deficient cells is not known. We examined the effects of DFMO on pRb deficient human retinoblastoma Y79 cell proliferation and its molecular mechanism.     

Inhibition of Growth of Giardia lamblia by Difluoromethylornithine,a Specific Inhibitor of Polyamine Biosynthesis1

Difluoromethylornithine (DFMO) is a specific and irreversible inhibitor of ornithine decarboxylase, an enzyme which catalyzes the first step in the biosynthetic pathway of the polyamines. We tested the effect of DFMO on the growth of Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis. Growth of G. lamblia was inhibited by DFMO at concentrations of ≥ 1.25 mM. Culture doubling time increased with increasing DFMO concentration. Growth inhibition was reversed if spermidine was added within 53 h of addition of DFMO; no growth was observed if spermidine was added later, indicating eventual parasite death. The growth of E. histolytica and T. vaginalis, two unrelated mucosal-dwelling parasites of humans, was not inhibited by 20 mM DFMO. These studies indicate that polyamine biosynthesis from ornithine is required for growth of G. lamblia.     

Ornithine Decarboxylase Activity and Edema Formation in Cerebral Ischemia of Conscious Gerbils

Abstract: General anesthetic agents often affect the biochemical and physiologic changes triggered by cerebral ischemia. This study examined the regional activities of ornithine decarboxylase (ODC) in gerbils subjected to 5 min of bilateral carotid occlusion without anesthesia. At 2, 4, and 6 h of reperfusion, significant ODC activity was observed in both the cortex and the hippocampus. Pretreatment with α-difluoromethylornithine (DFMO) significantly blocked the ODC activity at 2, 4, and 6 h. Significant edema formation was found at 2, 4, and 6 h. At 2 h, edema formation was unaffected by administration of DFMO. However, DFMO treatment reduced later edema formation at 4 and 6 h. These results demonstrate that ODC activity and edema formation are delayed in gerbils after the induction of transient ischemia even with the removal of anesthetic agents and their potentially protective effects. These findings suggest that ODC activity and its induction of delayed cerebral edema are specific to cerebral ischemia and not to an anesthetic effect. DFMO treatment reduced both the ODC activity and edema formation, indicating a role for polyamines in postischemic edema formation.     

Plasmodium berghei: inhibition of the sporogonous cycle by alpha-difluoromethylornithine

alpha-Difluoromethylornithine (DFMO), an enzyme inhibitor of ornithine decarboxylase, inhibits the sporogonous cycle of the malaria parasite Plasmodium berghei in the mosquito vector Anopheles stephensi. DFMO was administered to the mosquitoes dissolved either in the sugar solution at their disposal in the cages or through blood meals taken from treated mice. The mice subsequently bitten by mosquitoes treated with DFMO by both routes of administration did not contract malaria.     

Polyamines in marine macroalgae: Levels of putrescine, spermidine and spermine in the thalli and changes in their concentration during glycerol-induced cell growth in vitro

The levels of putrescine (Put), spermidine (Spd) and spermine (Spm) were analyzed in naturally collected samples of the marine macroalgae Dyctiota dichotoma, Gelidium canariensis and Grateloupia doryphora . Polyamines (PAs) appeared in free (35–134 μg g⁻¹ fresh weight) and bound TCA-insoluble form (1 667–2 624 μg g⁻¹ fresh weight). Axenic in vitro cultures of sporelings from G. doryphora were established in the medium containing glycerol. This medium promoted growth and morphogenesis and also increased the free and bound PA levels in the sporelings. Tracer experiments using 70 kBq [U-¹⁴C]-glycerol showed significant quantities of radioactivity in Put, Spd and Spm after 20 h of incubation. The effects of glycerol on growth were inhibited by the ornithine decarboxylase (EC 4.1.1.17) inhibitor α -difluoromethylornithine (DFMO). The presence of DFMO in the incubation medium with [U-¹⁴C]-glycerol also reduced the radioactivity in PAs.     

Depletion of 9L rat brain tumor cell polyamine content by treatment with d,l-α-difluoromethylornithine inhibits proliferation and the G1 to S transition

d,l-α-Difluoromethylornithine (DFMO), an irreversible inactivator of ornithine decarboxylase, inhibited 9L monolayer culture rat brain tumor cell proliferation at concentrations as low as 1 mM DFMO to about 25% of control growth when cells were seeded at an initial density of 5 × 10⁵/flask. DFMO reduced intracellular putrescine content to <5% of control by 8 h and spermidine content to <5 % of control by 48 h post-treatment. Cytostasis caused by 10 or 25 mM DFMO could both be reversed and blocked by addition of exogenous putrescine. Cells pretreated for 48 h with DFMO and then replated in its absence could not enter exponential growth until polyamine production resumed. Addition of exogenous putrescine at the time of replating allowed pretreated cells to resume exponential growth at the same time as controls. Flow cytometry revealed that the fraction of cells in G1 increased until polyamine accumulation resumed, implying the presence of a G1-S block. Within 6 h of replating, there was a decrease in the fraction of control cells in G1. These observations support the hypothesis that entry of 9L cells into S phase depends on an adequate intracellular pool of polyamines.     

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or alpha-difluoromethylornithine

We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and alpha-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 microM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G1 and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 x 10(5)), medium (5 x 10(5)), or high (2 x 10(6)) cell densities in a 25 cm2 flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.     

Inhibition of the differentiation of 3T3-L1 cells by interferon-beta and difluoromethyl ornithine

Both mouse interferon-beta (MuIFN-beta) and the inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethyl ornithine (DFMO), inhibited the differentiation of mouse 3T3-L1 fibroblasts into adipocytes in a dose-dependent manner. DFMO and MuIFN-beta added together to cultures that were induced to differentiate produced an additive anti-differentiation effect. In contrast to this additive cellular effect, DFMO reduced the antiviral activity of MuIFN-beta in both undifferentiated and differentiated cells; DFMO alone had no detectable effect on replication of encephalomyocarditis virus. Putrescine, the product of ornithine decarboxylation, when added to 3T3-L1 cultures (i) enhanced differentiation, (ii) reversed completely the inhibition of differentiation by DFMO, but (iii) had little effect on the antidifferentiation effect of MuIFN-beta. Polyamine content changed four-fold or less in cultures treated with 0.5 mM DFMO and less than two-fold in cultures treated with 100 IU/ml MuIFN-beta for seven days. Thus, it appears not only that MuIFN-beta and DFMO inhibit differentiation of 3T3-L1 cells by different mechanisms but also that the antiviral action of IFN does not involve the regulation of polyamine metabolism by ornithine decarboxylase.     

Targeting the polyamine biosynthetic enzymes: a promising approach to therapy of African sleeping sickness, Chagas’ disease, and leishmaniasis

Summary. Trypanosomatids depend on spermidine for growth and survival. Consequently, enzymes involved in spermidine synthesis and utilization, i.e. arginase, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase, trypanothione synthetase (TryS), and trypanothione reductase (TryR), are promising targets for drug development. The ODC inhibitor α-difluoromethylornithine (DFMO) is about to become a first-line drug against human late-stage gambiense sleeping sickness. Another ODC inhibitor, 3-aminooxy-1-aminopropane (APA), is considerably more effective than DFMO against Leishmania promastigotes and amastigotes multiplying in macrophages. AdoMetDC inhibitors can cure animals infected with isolates from patients with rhodesiense sleeping sickness and leishmaniasis, but have not been tested on humans. The antiparasitic effects of inhibitors of polyamine and trypanothione formation, reviewed here, emphasize the relevance of these enzymes as drug targets. By taking advantage of the differences in enzyme structure between parasite and host, it should be possible to design new drugs that can selectively kill the parasites.     

The effects of some polyamine biosynthetic inhibitors on growth and morphology of phytopathogenic fungi

We have studied the effects of two polyamine biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO) and alpha-difluoromethylarginine (DFMA), and of polyamines (PAs), alone and in combination, on mycelial growth and morphology of four phytopathogenic fungi: Botrytis sp, B. cinerea, Rhizoctonia solani and Monilinia fructicola. The inhibitors were added to a Czapek agar medium to get final concentrations of 0.1, 0.5 and 1.0 mM. DFMO and DFMA, suicide inhibitors of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) respectively, inhibited mycelial growth strongly; the effect was generally more pronounced with DFMA than with DFMO, but each fungus had its own response pattern. The addition of the PAs putrescine (Put) and spermidine (Spd) to the culture medium resulted in a promotion of growth. In Botrytis sp and Monilinia fructicola exposed to inhibitors plus PAs, mycelial growth was actually increased above control values. Mycelial morphology was altered and cell size dramatically reduced in plates containing inhibitors alone, whereas with PAs alone, or in combination with inhibitors, morphology was normal, but cell length and diameters increased considerably. These results suggest that PAs are essential for growth in fungal mycelia. The inhibition caused by DFMA may be due to its arginase-mediated conversion to DFMO.     

Opposite effects of fusicoccin and IAA on putrescine synthesis of rice coleop tiles

Changes in polyamine biosynthesis and elongation of etiolated rice coleoptiles ( Oryza sativa L. cv. Taichung Native 1) in response to fusicoccin (FC) and indoleacetic acid (IAA) were investigated. FC stimulated coleoptile elongation at concentrations higher than 1 μ M but caused a decrease in the levels of free putrescine, spermidine and sper-mine, as well as in the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and S -adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50). The extent to which FC caused these effects was dependent on its concentration. Treatment with 100 μ M IAA also induced coleoptile elongation and resulted in a decrease in free spermidine/sper-mine and SAMDC activity. However, treatment with IAA resulted in an increase in free putrescine levels and ADC activity. The extent of coleoptile elongation and putrescine accumulation also depended on IAA concentration. α-Difluoromethylarginine (DFMA), an irreversible inhibitor of ADC. but not α-difluoromethylornithine (DFMO). an irreversible inhibitor of ODC (EC 4.1.1.17), inhibited the LAA-stimulated coleoptile elongation and putrescine accumulation. Addition of putrescine could not reverse the effect of DFMA.     

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.     

Effects of putrescine and inhibitors of putrescine biosynthesis on organogenesis inEuphorbia esula L.

Summary Exogenous putrescine (&#x2264;5 mM) had little effect on root or shoot formation in aseptically isolated hypocotyl segments of leafy spurge (Euphorbia esula L.) grown on full-strength B5 medium. Unexpectedly, putrescine inhibited root and shoot formation in hypocotyl segments grown on B5 medium diluted 10-fold. In the full-strength medium, root and shoot formation were inhibited by 0.5 mM concentrations ofdl-&#x03B1;-difluoromethylornithine (DFMO) anddl-&#x03B1;-difluoromethylarginine (DFMA). DFMO and DFMA are inhibitors of the ornithine decarboxylase and arginine decarboxylase pathways, respectively, of putrescine biosynthesis in plants. Exogenous putrescine (0.5 to 5 mM) did not reverse either the DFMO-or DFMA-induced inhibition of shoot formation. However, the DFMA-induced inhibition of root formation was partially reversed by exogenous putrescine. The auxin, indole-3-acetic acid (IAA), reduced the inhibitory effects of DFMO+DFMA (applied together) on both roots and shoots. In the first few days of culture, the endogenous levels of putrescine and spermidine, but not of spermine, increased in the presence of IAA. The levels of putrescine and spermidine in the tissues did not correlate well with either root or shoot production in the later stages of organ formation; especially in tissues treated with IAA. These results show that there were no obvious correlations between polyamine levels and organogenesis in leafy spurge hypocotyl segments, although residual putrescine or spermidine or both in the tissues at the time of excision may be indirectly involved in the early stages of root formation.     

Trypanosoma brucei ornithine decarboxylase: enzyme purification, characterization, and expression in Escherichia coli

Ornithine decarboxylase from the African trypanosome is an important target for antitrypanosomal chemotherapy. Despite this, the enzyme had not been previously purified or extensively characterized as it is a very low level protein. In this paper we describe the purification of Trypanosoma brucei brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 10(6) nmol CO2/h/mg of protein in the parasite. T. brucei ornithine decarboxylase had a native molecular weight of 90,000 and a subunit molecular weight of 45,000. The isoelectric point of the protein was 5.0. The Km for ornithine was 280 microM and the Ki for the irreversible inhibitor alpha-difluoromethylornithine (DFMO) was 220 microM with a half-time of inactivation at saturating DFMO concentration of 2.7 min. T. brucei ornithine decarboxylase appears similar to mouse ornithine decarboxylase, further supporting our previous suggestion that the selective toxicity of DFMO to the parasite is not due to catalytic differences between the two proteins. Although a small quantity of T. brucei ornithine decarboxylase was purified from T. brucei, extensive structural and kinetic studies will require a more ample source of the enzyme. We therefore expressed our previously cloned T. brucei ornithine decarboxylase gene in Escherichia coli using a vector that contains an inducible lambda promoter. T. brucei ornithine decarboxylase activity was induced in E. coli to levels that were 50 to 200 fold of that present in the long-slender bloodstream form of T. brucei. Ornithine decarboxylase activity in the crude E. coli lysate was 1500-6000 nmol of CO2/h/mg of protein and represented 0.05-0.2% of the total cell protein. The recombinant T. brucei ornithine decarboxylase was purified to apparent homogeneity from the transformed E. coli. The purified recombinant enzyme had kinetic and physical properties essentially identical to those of the native enzyme.