Effects of DL-alpha-difluoromethylornithine on Leishmania donovani promastigotes

Alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been demonstrated to be an effective agent against a variety of parasitic protozoa but not against Leishmania spp. In this report, we show that Leishmania donovani promastigotes in continuous culture are sensitive to the growth inhibitory and cytotoxic effects of DFMO. Incubation of the promastigotes with DFMO obliterates intracellular putrescine pools and depletes spermidine concentrations, which correlates with the onset of growth inhibition. The effects of DFMO on the growth and the intracellular polyamine pools can be reversed completely by the addition of 10 microM putrescine to the culture medium. These results suggest that the treatment of leishmaniasis may be amenable to chemotherapeutic manipulation by DFMO.     

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

Summary The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO) — a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.     

Reversal of the growth inhibitory effect of α-difluoromethylornithine by putrescine but not by other divalent cations

Summary Treatment with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), depletes the putrescine and spermidine content, and reduces the growth rate of Ehrlich ascites tumor cells.The addition of putrescine, which is the immediate precursor of spermidine, promptly replenished the intracellular putrescine and spermidine pools and completely reversed the antiproliferative effect of DFMO. A sequential accumulation of spermine, spermidine and putrescine was observed.1,3-diaminopropane, a lower homolog of putrescine, did not reverse the antiproliferative effect of DFMO, despite its structural similarity and identical positive charge. By inhibiting remaining ODC activity, resistant to 5 mM DFMO, and possibly by inhibiting spermine synthase activity, 1,3-diaminopropane produced a further decrease in total polyamine content by reducing the spermine content.Mg²⁺, which can replace putrescine in many in vitro reactions, completely lacked the capacity to reverse the antiproliferative effect of putrescine and spermidine deficiency.Abbreviations DFMO -difluoromethylornithine - ODC ornithine decarbxylase     

Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.     

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.     

Consequences of aberrant ornithine decarboxylase regulation in rat hepatoma cells

DH23A cells, an α-difluoromethylornithine (DFMO)–resistant variant of rat hepatoma tissue culture cells (HTC), contain high levels of very stable ornithine decarboxylase (ODC). In the absence of DFMO, the high ODC activity results in a large accumulation of endogenous putrescine. Concomitant with the putrescine increase is a period of cytostasis and a subsequent loss of viable cells. In contrast, HTC cells with a moderate polyamine content can be maintained in exponential growth. This suggests that a moderate polyamine concentration is necessary for both optimal cell growth and survival. The cytoxicity observed in the DH23A cells is apparently not due to byproducts of polyamine oxidation or alterations in steady state intracellular pH or free [Ca²⁺]. It is possible to mimic the effects of high levels of stable ODC by treatment of cells with exogenous putrescine in the presence of DFMO. This suggests that overaccumulation of putrescine is the causative agent in the observed cytotoxicity, although the mechanism is unclear. These data support the hypothesis that downregulation of ODC may be necessary to prevent accumulation of cytotoxic concentrations of the polyamines. © 1994 Wiley-Liss, Inc.     

Changing root and shoot architecture with the rolA gene from Agrobacterium rhizogenes: Interactions with gibberellic acid and polyamine metabolism

The effects of rolA on root and shoot architecture have been ascribed to a deficiency in gibberellic acid (GA₃) and to changes in polyamine metabolism. Using tobacco, we examined interactions among GA₃, a polyamine accumulation inhibitor (α-DL-difluoromethylornithine or DFMO) and the rolA gene controlled by the 35S CaMV promoter. We measured the effects of these three agents on architecture and polyamine accumulation in excised roots and whole plants grown in vitro. Previous work showed that DFMO or genetic transformation with the rolA gene from Agrobacterium rhizogenes, controlled by the 35S promoter (P_35S-rolA), caused excised tobacco roots to grow faster with altered root system architecture. We show that gibberellic acid (GA₃) reversed the effects of DFMO on the architecture of excised root systems, but neither reversed the effects of DFMO on growth, nor the changes in growth and architecture associated with P_35S-rolA. GA₃ treatment alone resulted in increased agmatine levels, suggesting that the inhibition of the effects of DFMO on architecture was through a stimulation of the arginine decarboxylase (ADC) pathway, GA₃ alone also inhibited the accumulation of putrescine and tyramine conjugates in excised roots. In tobacco plants growing in vitro DFMO and P_35S-rolA were associated with reduced shoot height, which was partially restored by GA₃ treatment; however, GA₃ also stimulated shoot height in the controls. GA₃ did not lessen the leaf wrinkling associated with P_35S-rolA. P_35S-rolA increased root number in young seedlings in vitro, and increased root system length in seedlings grown in soil. As in excised roots, the developmental changes linked to DFMO and P_35S-rolA were accompanied by reductions in putrescine titers. GA₃ treatment stimulated putrescine accumulation in stems and leaves, and partially reversed the negative effects of DFMO and P_35S-rolA on putrescine accumulation in roots, stems and leaves. Again, the restoration of putrescine pools appeared to be through a stimulation of the ADC pathway, since agmatine accumulated in plants exposed to GA₃. In general, the effects of DFMO and P_35S-rolA on phenotype and polyamine metabolism were coordinated, and in many cases these effects were similarly modulated by GA₃, reinforcing the previous conclusion that the phenotypic effects of rolA in roots and shoots occur through interference with polyamine metabolism and that the putrescine conjugates are particularly important in regulating root system growth and architecture. We were unable, however, to discem consistent evidence for a direct role for GA₃ in establishing the RolA phenotype.     

Tolerance to Putrescine Toxicity in Chinese Hamster Ovary Cells Is Associated with Altered Uptake and Export

When Chinese hamster ovary (CHO) cells were cultured with low concentrations of putrescine (< 5 mM) their cell cycle time increased significantly and a fraction of the cells died. A cell line tolerant to the cytotoxic and growth inhibitory effects of millimolar concentrations of putrescine was developed by growing CHO cells over many months in increasing concentrations of the polyamine. A putrescine-tolerant cell line was obtained which was capable of growing in concentrations up to 25 mMputrescine and displayed growth and cell division rates similar to the original untreated/parental CHO cells. The tolerant cells grown in putrescine displayed relatively high intracellular putrescine yet the cell-associated putrescine concentration was estimated to be 10-fold less than the culture medium level. This high concentration of cellular putrescine diminished within 60 min when the cells were changed to non-putrescine-containing media. The putrescine-tolerant phenotype was further characterized in regards to the mechanisms involved in putrescine uptake, efflux, and biosynthesis. The parental and tolerant cell lines had similar or identical levels of cellular spermidine and spermine and no differences in the acetylated polyamine pools or diamine oxidase activity. The activity of ornithine decarboxylase was also similar in the two cell lines in both the presence and the absence of ornithine. The tolerant cells, however, had a decreased uptake rate for putrescine. The tolerant cell line also showed a greatly enhanced ability to export putrescine, especially when treated with ornithine, suggesting that an upregulated polyamine export system may be present in the tolerant cells which could be responsible for the increased cell survival in high putrescine concentrations. The data are discussed in regard to the potential for identifying the transport protein(s) responsible for the maintenance of nontoxic intracellular concentrations of putrescine in a tolerant cell line grown in putrescine.     

Correlation between transglutaminase activity and polyamine levels in human neuroblastoma cells. Effect of retinoic acid and alpha-difluoromethylornithine

The human neuroblastoma cell line SK-N-BE can be induced to differentiate by retinoic acid (RA) or by alpha-difluoromethylornithine (DFMO). The former inducer produces neurite outgrowth, 60% reduction of growth rate, overexpression of neural antigens, and enhanced gamma-aminobutyric acid (GABA) and acetylcholinesterase levels. In contrast, DFMO causes cell body elongation, complete growth inhibition, and higher binding of antibodies directed against neuroectodermal antigens. Polyamine metabolism is also differently affected by the two agents. In particular a large spermine catabolism is induced by RA, while DFMO treatment leads to a small increase in the level of this compound. The neural differentiation induced by RA is accompanied by a marked increase in transglutaminase activity and its induction is paralleled by a transient increase of putrescine and spermidine. The putrescine and spermidine depletion determined by DFMO is accompanied instead by a large inhibition of transglutaminase activity. The inhibiting effect of DFMO treatment on transglutaminase is reversed by the addition of 1 mM putrescine to the culture medium. In the presence of both RA and DFMO a mixed morphological and biochemical pattern is observed. The possibility that the expression of transglutaminase associated to cellular differentiation may be modulated by the level of its substrates is also discussed.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Downregulation of T cell growth factor production by ornithine decarboxylase and its product putrescine: -α-Difluoromethylornithine suppresses general protein synthesis but augments simultaneously the production of interleukin-2

Treatment of EL-4 lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin-2 (IL-2) and the expression of IL-2-specific mRNA within 4–8 h. This system is an ideal model for studies on the induction of a differentiated function in a homogeneous lymphoid cell population by a defined signal. TPA induces also an increase of ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4–8 h. A similar increase of intracellular putrescine and polyamine concentrations can be achieved by administration of 2 mM putrescine to the culture medium. However, putrescine cannot induce the production of IL-2 in the absence of TPA and cannot reconstitute the IL-2 production in cultures with PGE₂ or cyclosporine A, i.e., two well-known immunosuppressive substances which inhibit ODC activity. Putrescine has rather a counter-regulatory effect as concluded from the observation that the TPA-induced TCGF production and IL-2-specific mRNA expression are augmented (superinduced) by the ODC inhibitor -α-difluoromethylornithine (DFMO) and again suppressed after the administration of putrescine or polyamines to DFMO-treated cultures. The glycolytic activity, general protein synthesis ([³H]leucine incorporation), and the cell cycle progression from G₂/M to G₁, in contrast, are inhibited by DFMO and reconstituted by putrescine. This demonstrates that the cells are able to sacrifice to a large extent several vital functions including their general protein synthesis and to devote themselves at the same time to a fulminant production of their functionally most relevant protein IL-2. This process is downregulated by ODC and its product putrescine. A correlation between increased IL-2 production and accumulation of cells in the G₂/M phase was also observed in cultures treated with hydroxyurea or with a combination of amethopterin and adenosine.     

Effects of exogenous polyamines and difluoromethylornithine on seed germination and root growth of Arabidopsis thaliana

Effects of exogenous polyamines and difluoromethylornithine (DFMO) on seed germination and seedling root growth of Arabidopsis thaliana were investigated. Root growth was stimulated by low concentrations of putrescine but was increasingly inhibited by high concentrations of putrscine. DFMO inhibited root growth and this inhibition was reversed by applying putrescine. In contrast, both spermidine and spermine had no effect on root growth but inhibited seed germination. The results suggest a possible requirement of endogeneous putrescine for normal root growth of Arabidopsis seedlings.Abbreviations DFMO difluoromethylornithine - DFMA difluoromethylarginine - ODC ornithine decarboxylase - Put Putrescine - Spd Spermidine - Spm Spermine     

Leishmania infantum: polyamine biosynthesis and levels during the growth of promastigotes.

1. Decarboxylation of polyamine precursors: L-ornithine and L-methionine was determined along the growth curve of Leishmania infantum promastigotes in vivo, reaching maximum values on day 2 post-inoculum (mid-logarithmic phase). 2. Maximum values of L-ornithine and L-methionine decarboxylation were: 1.97 +/- 0.28 nmol CO2/hr/10(7) promastigotes and 3.18 +/- 0.34 nmol CO2/hr/10(7) promastigotes, respectively. 3. Total (free + conjugated) polyamine content was closely related with the proliferative stage of Leishmania infantum promastigotes. 4. D,L-alpha-difluoromethylornithine (DFMO) and Berenil depleted putrescine levels in a concentration-dependent manner. 5. Total and free putrescine/spermidine ratio varied significantly with the proliferative stage. Minimum values were found in late logarithmic phase (day 3 post-inoculum). 6. Small but detectable amounts of free spermine were detectable along the growth curve of Leishmania infantum promastigotes.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

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.     

Effects of inhibitors of ornithine and S-adenosylmethionine decarboxylases on L6 myoblast proliferation

The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.     

Induction of F9 embryonal carcinoma cell differentiation by inhibition of polyamine synthesis

alpha-Difluoromethylornithine (DFMO), a highly selective inhibitor of ornithine decarboxylase (ODC), induced terminal differentiation of F9 mouse embryonal carcinoma cells in culture. Differentiation was assessed using morphological criteria and the level of plasminogen activator activity. The observed phenotypic changes and the fact that the cells did not synthesize alpha-fetoprotein, indicate that they were parietal endoderm cells. The putrescine, spermidine and spermine content of untreated control cells increased during exponential growth and then decreased gradually with continued time in culture. The increases in putrescine and spermidine contents were prevented by DFMO treatment. In fact, the putrescine and spermidine content decreased below the limits of detection after only one day of treatment. The addition of putrescine to the culture medium at any time within 4 days of DFMO treatment, prevented the DFMO-induced differentiation, suggesting that the effects observed were indeed caused by polyamine depletion. The phenotypic changes induced by DFMO were similar to those induced by retinoic acid, a very potent inducer of embryonal carcinoma differentiation. Although retinoic acid can inhibit ODC activity and putrescine accumulation, it is unlikely that this mechanism of action is responsible for retinoic acid-induced F9 cell differentiation, inasmuch as putrescine addition did not prevent the expression of the differentiated phenotype. Undifferentiated F9 embryonal carcinoma cells exhibited a very short G1 phase, and in this respect they are similar to the cells of the preimplantation mouse embryo. In control (exponentially growing) cultures a majority of the F9 cells were in the S phase, but in DFMO-treated cultures they accumulated in the G1 phase and showed no further proliferative potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel lysine-spermine conjugate inhibits polyamine transport and inhibits cell growth when given with DFMO

Polyamines are ubiquitous molecules with multiple intracellular functions. Cells tightly regulate their levels through feedback mechanisms affecting synthesis, intracellular conversion, and transport. Because polyamines have an important role in regulating cell growth, they are a target for cancer therapeutic development. However, to effectively inhibit cell growth through polyamine depletion one needs to inhibit both polyamine synthesis and import. Although the mammalian polyamine transporter has not been cloned, we have identified ORI 1202, an N(1)-spermine-L-lysinyl amide, as an effective polyamine transport inhibitor. ORI 1202 prevents the cellular accumulation of [(3)H]spermidine over a 20-h test period. ORI 1202 (30-100 microM) effectively inhibits cell growth when used in conjunction with the polyamine synthesis inhibitor alpha-difluoromethylornithine (DFMO; > or =230 microM). Human breast, prostate, and bladder carcinoma cell lines and melanoma cell lines show ORI 1202 EC(50) values in the low micromolar range when tested in conjunction with DFMO. This cytostatic effect correlates with a reduction in the intracellular levels of putrescine and spermidine. When ORI 1202 (45 mg/kg, i.p., tidx5) and DFMO (1% in drinking water) were delivered over 14 days, MDA-MB-231 breast tumor xenografts in nude mice showed 50% growth inhibition. Polyamine depletion therapy provides a cytostatic therapy that could be useful against cancer and other diseases resulting from uncontrolled cell growth.     

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.     

Putrescine does not support the migration and growth of IEC-6 cells

The migration of IEC-6 cells is inhibited when the cells are depleted of polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine (DFMO). Exogenous putrescine, spermidine, and spermine completely restore cell migration inhibited by DFMO. Because polyamines are interconverted during their synthesis and catabolism, the specific role of individual polyamines in intestinal cell migration, as well as growth, remains unclear. In this study, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone)(DEGBG), to block the synthesis of spermidine and spermine from putrescine. We found that exogenous putrescine does not restore migration and growth of IEC-6 cells treated with DFMO plus DEGBG, whereas exogenous spermine does. In addition, the normal distribution of actin filaments required for migration, which is disrupted in polyamine-deficient cells, could be achieved by adding spermine but not putrescine along with DFMO and DEGBG. These results indicate that putrescine, by itself, is not essential for migration and growth, but that it is effective because it is converted into spermidine and/or spermine.