Ornithine decarboxylase in difluoromethylornithine-resistant mouse lymphoma cells. Two-dimensional gel analysis of synthesis and turnover

Variant S49 mouse lymphoma cells with increased ornithine decarboxylase activity were obtained by selecting for resistance to alpha-difluoromethylornithine (DFMO), a specific inhibitor of the enzyme. Ornithine decarboxylase was identified as a specifically immunoprecipitable polypeptide that was made at an increased rate in the variant cells. Ornithine decarboxylase was also identified on a two-dimensional gel as a metabolically labeled polypeptide of Mr approximately 55,000 which was synthesized at an increased rate in two independently selected variants. Synthesis of this polypeptide was further augmented by treatment of cells with inhibitors of ornithine decarboxylase activity. The charge of the polypeptide was altered by treatment of either cells or cellular extracts with DFMO, a suicide substrate which binds covalently to the enzyme. This charge alteration and the inactivation of ornithine decarboxylase showed the same dependence on DFMO concentration and both effects were prevented by addition of either ornithine or putrescine. Pulse-chase experiments showed that the half-life of the ornithine decarboxylase polypeptide in these variant cells was 45 min. We conclude that ornithine decarboxylase is made at an increased rate in the resistant variants and that the polypeptide turns over rapidly.     

Identification of ecdysone 25-O-beta-D-glucopyranoside as a new metabolite of ecdysone in the nematode Parascaris equorum.

The mechanism of inactivation of rodent ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) was studied using the inhibitor labelled with 14C in both the 1 and the 5 positions. [1-14C]DFMO was a substrate and was decarboxylated by the enzyme yielding 14CO2. A radioactive metabolite derived from [5-14C]DFMO was bound to the enzyme, and the extent of binding paralleled the irreversible inactivation of ornithine decarboxylase. The partition ratio of decarboxylation to binding was approx. 3.3. These results provide support for the postulated mechanism of action of DFMO [Metcalf, Bey, Danzin, Jung, Casera & Vevert (1978) J. Am. Chem. Soc. 100, 2551-2553], in which enzymic decarboxylation of the inhibitor leads to the generation of a conjugated imine, which then alkylates a nucleophilic residue on the enzyme.     

An essential role for putrescine biosynthesis during meiotic maturation of amphibian oocytes

The objective of this study was to investigate the role of polyamines during meiotic maturation of Xenopus oocytes. The results indicate a rapid and significant increase in the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, during the meiotic maturation induced by either progesterone or human chorionic gonadotropin (HCG). This increase in the enzyme activity was followed by an accumulation of putrescine without any effect on the levels of spermidine or spermine. The inhibition of ODC activity and the accumulation of putrescine levels by α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ODC, also resulted in the inhibition of maturation mediated by progesterone in Xenopus oocytes. DFMO caused an inhibition of both maturation and ovulation induced by HCG in ovarian fragments. This inhibition was readily reversible by exogenous supply of putrescine to the medium. These observations suggest that putrescine plays an important role during the meiotic maturation of amphibian oocytes.     

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.     

Suppression of ribosomal gene expression in oocytes and nurse cells of a polychaete as a result of polyamine synthesis inhibition

The role of the polyamines in ribosomal gene expression was evaluated in the polychaete Ophryotrocha labronica by analyzing the effects of polyamine synthesis inhibition on RNA synthesis during oogenesis, a period characterized by intense nucleolar activity. At various stages of oogenesis adult polychaete females were blocked in their polyamine synthesis by the addition of 10 mM DL-alpha-difluoromethylornithine (DFMO) to the sea water in which they were cultivated. To monitor RNA synthesis during DFMO treatment the animals were pulse-labeled with [5-3H]uridine and processed for autoradiography. Light and electron microscope autoradiographs demonstrate that DFMO treatment suppresses incorporation of label into nucleolar RNA (rRNA) both in the oocytes and their associated nurse cells. The ultrastructural appearance of both cell types reveals interference with nucleolar and ribosomal activity; the endoplasmic reticulum is deprived of ribosomes, and the production of protein granules (vitellogenesis) is reduced. The high specificity of DFMO for polyamine synthesis and the fact that the effects of DFMO were counteracted by addition of a low concentration (10 microM) of putrescine shows that the observed interference with ribosomal gene expression is indeed due to polyamine deficiency.     

The response of several murine embryonal carcinoma cell lines to stimulation of differentiation by alpha-difluoromethylornithine

In an attempt to better establish the relationship between polyamine levels and the differentiation of embryonal carcinoma cells, we have examined the ability of alpha-difluoromethylornithine (DFMO), a known inducer of differentiation in one embryonal carcinoma cell line, to stimulate the differentiation of embryonal carcinoma cells from a variety of cell lines. Differentiation was monitored using a variety of criteria including morphological alterations and changes in biochemical and antigenic parameters. Depending on their response to difluoromethylornithine, three classes of cell lines could be identified, those which 1) differentiate extensively, 2) differentiate poorly, and 3) fail to differentiate. Three different classes of embryonal carcinoma cell lines reflect differential changes in polyamine levels resulting from inhibition of ornithine decarboxylase enzyme activity by DFMO. The specific cell lines which exhibit large decreases in both ornithine decarboxylase activity and polyamine levels also show extensive differentiation. The cell lines which show only moderate decreases in enzyme activity and polyamines differentiate poorly while the cell lines which fail to respond to DFMO in that polyamines do not drop below the threshold level necessary to induce differentiation fail to differentiate. These studies suggest that decreases in intracellular polyamines induce EC cell differentiation in vitro.     

Superinduction of ornithine decarboxylase by halogenated ribofuranosylbenzimidazoles.

1. The effect of dichlororibofuranosylbenzimidazole (DiCl-RB), an inhibitor of hnRNA synthesis and casein kinase-2 activity, on ornithine decarboxylase (ODC) was investigated in a difluoromethylornithine (DFMO) resistant, ODC overproducing cell line. 2. In cells growing in the absence of DFMO, DiCl-RB provoked a marked, but transient increase in ODC activity and immunoreactive ODC content. 3. The ODC response to DiCl-RB was prevented by cycloheximide and was not due to stabilization of the enzyme. 4. The dibromo derivative analogue (DiBr-RB) exerted similar effects on ODC, but was effective at lower concentrations. 5. The halogenated ribofuranosylbenzimidazoles were ineffective in cells growing in the presence of DFMO and containing higher levels of ODC protein.     

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.     

Further studies on difluoromethylornithine in African trypanosomes

DL-alpha-Difluoromethylornithine (DFMO), a specific enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC) was previously shown to cure mice infected with Trypanosoma brucei brucei, a parasite of game and cattle in Africa and Trypanosoma brucei rhodesiense, a human African Sleeping Sickness pathogen. Our studies now indicate that DFMO blocks ornithine decarboxylase and lowers trypanosome polyamine levels in vivo. Polyamine uptake in T.b. brucei also resembles that previously described for mammalian cells. The therapeutic potential of DFMO can now also be extended to another human pathogen, Trypanosoma brucei gambiense. Finally, DFMO acts synergistically with another drug, bleomycin, to cure acute trypanosome infections, and furthermore, this same drug combination provides a new approach to the treatment of trypanosomal infections of the central nervous system.     

An evaluation of the involvement of polyamines in modulating MCF-7 human breast cancer cell proliferation and progesterone receptor levels by estrogen and antiestrogen

The present studies were undertaken to determine the importance of the polyamine biosynthetic pathway in cellular proliferation and hormone-regulated progesterone receptor synthesis in estrogen receptor-containing breast cancer cells. Treatment of MCF-7 cells with difluoromethylornithine (DFMO), the irreversible inhibitor of the enzyme ornithine decarboxylase (ODC), prevented estradiol-induced cell proliferation in a dose-dependent fashion. DFMO inhibition of estradiol-induced cell proliferation was completely recoverable by the addition of exogenous putrescine while putrescine alone did not stimulate proliferation of control cells. ODC activity was 4-fold greater in estrogen-treated cells and DFMO (5 mM) fully inhibited ODC activity. DFMO was able to suppress only slightly further the proliferation of antiestrogen (tamoxifen) treated cells and putrescine was able to recover this DFMO inhibition. In contrast to the suppressive effect of DFMO on cell proliferation, DFMO had no effect on the ability of estrogen to stimulate increased (4-fold elevated) levels of progesterone receptor. Hence, while ODC activity appears important for estrogen-induced cell proliferation, inhibition of the activity of this enzyme has no effect on the ability of estradiol to increase cellular progesterone receptor content.     

Action of DL-alpha-difluoromethyl ornithine on Acanthamoeba culbertsoni.

The multiplication of A. culbertsoni in the peptone medium was not inhibited by 10-20 mM concentration of alpha-difluoromethyl ornithine (DMFO) while a partial and transient inhibition of cell multiplication was observed by 10-20 mM DFMO in proteose peptone, yeast extract, glucose (PYG) medium. Ornithine decarboxylase (ODC) activity in the cells and cell free extracts was strongly inhibited by DFMO, excluding enzyme refractoriness and impermeability of cells for DFMO as the possible causes of DFMO resistance. The presence of polyamines in the peptone and PYG media as well as uptake of polyamines by the amoebae has been demonstrated. The growth and multiplication of A. culbertsoni in chemically defined medium was not affected by 1-5 mM DFMO while 10-20 mM DMFO yielded partial inhibition. A lowering of diaminopropane levels and enhancement of spermidine levels was observed in DFMO inhibited cells and level of ODC was drastically reduced in the inhibited cultures. Uptake of polyamines from the growth media may partly account for DFMO resistance of A. culbertsoni. Alternative mechanisms for DFMO resistance are indicated.     

Changes in the polyamine and nucleotide metabolism of P388 leukemia cells treated with DL-alpha-difluoromethylornithine in culture

Influence of DL-alpha-difluoromethylornithine (DFMO) treatment on the growth kinetics, labelling index, extra- and intracellular polyamine and nucleotide concentrations was monitored in cultured P388 leukemia cells. A substantial decrease of cell proliferation was observed when the cells were continuously treated with 1-5 mM DFMO. Depletion of cellular polyamines, mostly of putrescine and spermidine, was seen with a concomitant but delayed increase of spermidine and spermine levels in the culture medium. Changes of DNA content and of labelling index of untreated and treated cells seem to indicate that DFMO arrested cells in G1/S transition. The results presented here provide additional in vitro evidence on the characteristic changes in the metabolic imbalance of ornithine in tumor cells induced by DFMO via inhibition of ornithine decarboxylase and ornithine carbamoyl transferase activities.     

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.     

Growth regulatory effects of cyclic AMP and polyamine depletion are dissociable in cultured mouse lymphoma cells

Treatment of mouse lymphoma S49 cells with D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in S49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a S49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases.     

Role of polyamines in the proliferation and steroidogenic activities of bovine adrenocortical cells in culture

Difluoromethylornithine (DFMO), a selective inhibitor of ornithine decarboxylase, was used to probe the possible role of polyamines in the regulation of proliferation and steroidogenic activities of bovine adrenocortical cells in primary culture. The presence of DFMO in the culture medium not only suppressed the polyamine increase observed in proliferating control cells but resulted in a rapid depletion of the putrescine and spermidine cellular content, while spermine remained at a basal level. The proliferation of DFMO-treated cells was rapidly blocked and resumed at a normal rate upon addition of putrescine to the medium. DFMO-treated cells showed an impaired steroidogenic response to ACTH while adenylate cyclase stimulation was not altered. Thus, while ornithine decarboxylase and polyamines may be required for adrenocortical cell replication, deprivation of these compounds did not facilitate the expression of differentiated cell functions, as observed with granulosa cells.     

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 enhances the roscovitine-induced apoptosis through the activation of mitochondria in HCT116 colon carcinoma cells

Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential in various cancer types which are characterized by the accumulation of transformed cells due to impaired apoptotic machinery. Roscovitine, a CDK inhibitor showed to be a potent apoptotic inducer in several cancer cells. Polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. In this study, we explored the potential role of polyamines in roscovitine-induced apoptosis in HCT116 colon cancer cells. Roscovitine induced apoptosis by activating mitochondrial pathway caspases and modulating the expression of Bcl-2 family members. Depletion of polyamines by treatment with difluoromethylornithine (DFMO) increased roscovitine-induced apoptosis. Transient silencing of ornithine decarboxylase, polyamine biosynthesis enzyme and special target of DFMO also increased roscovitine-induced apoptosis in HCT116 cells. Interestingly, additional putrescine treatment was found pro-apoptotic due to the presence of non-functional ornithine decarboxylase (ODC). Finally, roscovitine altered polyamine catabolic pathway and led to decrease in putrescine and spermidine levels. Therefore, the metabolic regulation of polyamines may dictate the power of roscovitine induced apoptotic responses in HCT116 colon cancer cells.     

Structural Insight into DFMO Resistant Ornithine Decarboxylase from Entamoeba histolytica: An Inkling to Adaptive Evolution

Background

Polyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. α-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO.

Methodology/Principal Finding

The basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCΔ15). Structure was determined by molecular replacement method and refined to 2.8 Å resolution. The orthorhombic crystal exhibits P2₁2₁2₁ symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 Å. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure.

Conclusions/Significance

We determined the tetrameric crystal structure of EhODCΔ15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI.     

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.