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.     

Inhibition of ornithine decarboxylase by retinoic acid and difluoromethylornithine in relation to their effects on differentiation and proliferation

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.     

Inhibition of placental ornithine decarboxylase by DL-alpha-difluoro-methyl ornithine causes fetal growth restriction in rat

The roles of polyamines in intrauterine growth restriction (IUGR) is studied. The DL-alpha-difluoromethyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) which is a rate limiting enzyme of polyamine synthesis was administrated to pregnant rats so that we obtained rat fetuses with IUGR. The changes of maternal nutrition, damage of the placenta, and the direct effect of DFMO on the fetus were examined in this IUGR model. Administration of DFMO did not induced changes of maternal nutrition except for triglyceride and the fetal metabolic state. But the placental weight, ODC activity, and DNA in the placenta were decreased significantly. The ODC activity in the total placenta decreased to less than 10% of that of the control. Depression of ODC activity in the placenta may be the major cause of IUGR induced by DFMO administration, and polyamines play important roles to carry pregnancy.     

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.     

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)     

A novel mechanism of resistance to alpha-difluoromethylornithine induced by cycloheximide. Growth with abnormally low levels of putrescine and spermidine

Treatment of the chemically transformed fibroblasts BP-A31 and other cell lines with low concentrations of cycloheximide (CHM) for 72 h followed by the removal of the protein synthesis inhibitor leads to the proliferation of alpha-difluoromethylornithine (DFMO)-resistant phenotypes. These drug-resistant cells contain almost no ornithine decarboxylase (ODC) activity and concomitantly very low levels of putrescine and spermidine. Southern blot analysis and measurements of ODC activity and intracellular polyamine levels showed that the described mechanism of inducing resistance to DFMO triggered by CHM does not involve ODC gene amplification, altered transport of the drug or reduced affinity of the enzyme for DFMO.     

Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: Crosstalk between ODC and BCL-2

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-α)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCδ) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKδ pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.     

Differentiation of a mouse neuroblastoma variant cell line whose ornithine decarboxylase gene has been amplified.

Differentiation of mouse neuroblastoma cells has been shown to be accompanied by changes in polyamine metabolism and a decrease in polyamine content. We have previously shown that alpha-difluoromethyl ornithine, a suicide inhibitor of ornithine decarboxylase (ODC, EC 4.1.1.17) and suboptimal concentrations of dibutyryl cAMP (0.1 to 0.2 mM) are effective in inducing the differentiation of mouse Neuro-2a (N2a) neuroblastoma cells. Exogenously added putrescine or spermidine can block the action of DFMO and dibutyryl cAMP, suggesting that polyamines may play a regulatory role in neuroblastoma differentiation. We have now isolated from N2a cells a clonal variant line, DF-40, whose ODC gene has been amplified by 40-fold. The DF-40 cells overproduced the ODC enzyme and contained very high levels of putrescine, spermidine and spermine. Treatment of DF-40 cells with dibutyryl cAMP or DFMO/dibutyryl cAMP led to a more than 80% reduction in polyamine content. Such a decrease did not cause the DF-40 cells to differentiate. Polyamine content in the treated DF-40 cells was still comparable or higher than that in the undifferentiated N2a cells. In contrast, serum-deprivation induced full differentiation of DF-40 cells. Levels of polyamine in the differentiated DF-40 cells, however, were also found to be comparable to that in the undifferentiated N2a cells. Exogenously added polyamines could not block the differentiation of DF-40 cells induced by serum-deprivation, suggesting that the action of polyamines in regulating neuroblastoma differentiation may depend on the presence of serum factors.     

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by alphaMSH and ornithine decarboxylase inhibition

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by alpha-melanocyte-stimulating hormone (alphaMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, alphaMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or alphaMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor alpha-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with alphaMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to alphaMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by alphaMSH.     

Polyamine biosynthesis is necessary for interleukin-2-dependent proliferation but not for interleukin-2 production or high-affinity interleukin-2 receptor expression

DL-alpha-Difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), inhibited concanavalin A-induced proliferation of splenic mononuclear cells (SMNC). The inhibition was not reversed by interleukin-2 (IL-2) addition. Although DFMO did not affect the production of IL-2 or the expression of high-affinity IL-2 receptor, IL-2-dependent proliferation of SMNC was inhibited by DFMO, and the inhibition was reversed by exogenous putrescine. The inhibition of IL-2-dependent DNA synthesis appeared to be related to the decrease in intracellular polyamines. When the proliferation of SMNC was induced by IL-2, ODC activity was also increased. A similar result was obtained in the proliferation of an IL-2-dependent T cell line, CTLL. The time course of ODC induction was similar to that of IL-2 production by concanavalin A-stimulated SMNC. These results indicate that polyamine biosynthesis is necessary for IL-2-dependent proliferation, but not for IL-2 production or IL-2 receptor expression.     

Ehrlich ascites tumour cells become refractory to alpha-difluoromethylornithine at a certain stage of growth

When Ehrlich ascites tumour cells are induced to proliferate by serum stimulation, the ornithine decarboxylase (ODC) activity increases rapidly and reaches two to three peaks during the first 24 h. Inhibition of the first peak in ODC activity (occurring at 4 h) by adding alpha-difluoromethylornithine (DFMO) within 2 h of serum stimulation, results in maximal growth inhibition. Under these conditions, similar degrees of polyamine depletion are achieved. When DFMO is added 3 h after seeding, however, enough polyamines have already accumulated during the initial burst in ODC activity to reduce the antiproliferative effect of the drug. The antiproliferative effect is further reduced when DFMO is added 6 h after seeding. When DFMO is added 23 h after seeding, i.e. after maximal accumulation of polyamines, there is no inhibition of cell proliferation. These findings are important to consider both when designing experimental as well as clinical regimens for this drug.     

Ornithine decarboxylase induction and polyamine biosynthesis are required for the growth of interleukin-2- and interleukin-3-dependent cell lines

The objective of this study was to evaluate induction of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, and subsequent polyamine accumulation in interleukin-2 (IL-2)- and interleukin-3 (IL-3)-dependent growth. The CTLL-20 and FDC-P1 cell lines, which have been shown to be absolutely dependent on IL-2 and IL-3, respectively, were used in these studies. The CTLL-20 and FDC-P1 cells each had different temporal patterns of ODC induction following lymphokine stimulation. ODC levels increased rapidly in the FDC-P1 cells, peaking 4 hr after stimulation with IL-3. In contrast, peak ODC activity in the CTLL-20 cells occurred 18 hr following stimulation with IL-2 and reached eightfold higher levels than those observed in the FDC-P1 cells. Treatment with D,L-alpha-difluoromethylornithine X HCl X H2O (DFMO), a specific irreversible inhibitor of ODC activity, completely abrogated lymphokine-dependent ODC induction in both the CTLL-20 and FDC-P1 cell lines. Similarly, intracellular levels of the polyamines putrescine and spermidine were reduced in both cell lines following DFMO treatment. DFMO treatment reduced both IL-2- and IL-3-dependent proliferation in a dose-dependent manner. However, this inhibition could be reversed by the addition of exogenous putrescine. DFMO treatment had no effect on cell viability. Polyamine-depleted CTLL-20 and FDC-P1 cells showed decreased absorption of IL-2 and IL-3 activity, respectively. However, the addition of exogenous putrescine restored the ability of the cells to absorb the appropriate lymphokine. These data are the first to demonstrate that ODC induction and polyamine biosynthesis are required in lymphokine dependent growth.     

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.     

Effects of diethyldithiocarbamate and endogenous polyamine content on cellular responses to hydrogen peroxide cytotoxicity.

In exponential-phase Chinese-hamster cells, 0.1 mM-diethyldithiocarbamate (DDC) afforded greater than 1 log survival protection to cultures treated before and during exposure to 1 mM-H2O2. Both DDC and H2O2 treatment stimulated the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, within 4 h of exposure. DDC, and to a lesser degree H2O2, also stimulated the activity of spermidine N1-acetyltransferase (SAT), the rate-limiting enzyme in polyamine catabolism. The increase in SAT activity, after exposure to DDC or another stress (heat shock), was inhibited in cells depleted of putrescine and spermidine by alpha-difluoromethylornithine (DFMO), the enzyme-activated suicide inhibitor of ODC. Pretreatment with DFMO or heat shock also induced resistance to H2O2 cytotoxicity. Since SAT activity is low in resting cells, yet stimulation of enzyme activity depends on endogenous spermidine pools, these results suggest that the expression of SAT activity occurs by a mechanism involving a stress-dependent displacement of spermidine into a new intracellular compartment. The stimulation of ODC and SAT activities does not appear to be a necessary component of the mechanism by which DDC protects cells from H2O2 cytotoxicity, although spermidine displacement may be a common facet of the cellular response to stress.     

Ornithine decarboxylase is a mediator of c-Myc-induced apoptosis.

c-Myc plays a central role in the regulation of cell cycle progression, differentiation, and apoptosis. However, the proteins which mediate c-Myc function(s) remain to be determined. Enforced c-myc expression rapidly induces apoptosis in interleukin-3 (IL-3)-dependent 32D.3 murine myeloid cells following IL-3 withdrawal, and this is associated with the constitutive, growth factor-independent expression of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis. Here we have examined the role of ODC in c-Myc-induced apoptosis. Enforced expression of ODC, like c-myc, is sufficient to induce accelerated death following IL-3 withdrawal. ODC induced cell death in a dose-dependent fashion, and alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzyme activity, effectively blocked ODC-induced cell death. ODC-induced cell death was due to the induction of apoptosis. We also demonstrate that ODC is a mediator of c-Myc-induced apoptosis. 32D.3-derived c-myc clones have augmented levels of ODC enzyme activity, and their rates of death were also a function of their ODC enzyme levels. Importantly, the rates of death of c-myc clones were inhibited by treatment with DFMO. These findings demonstrate that ODC is an important mediator of c-Myc-induced apoptosis and suggest that ODC mediates other c-Myc functions.     

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.     

In vivo effects of DL-alpha-difluoromethylornithine on the polyamine and nucleotide phosphate metabolism in P388/S leukemia cells

In vivo effects of DL-alpha-difluoromethylornithine (DFMO) on the metabolism of polyamines and nucleotide phosphates were monitored in P388/S leukemia cells grown intraperitoneally in BDF1 inbred male mice. Inhibiting the ornithine decarboxylase (ODC) activity DFMO depleted putrescine and spermidine to 30-50 and 50-60%, respectively, and increased spermine to 25-60% compared with the controls, when given as 2% solution in drinking water of the tumor-bearing animals. DFMO treatment caused a parallel 56% elevation of total nucleotide content in tumor cells with distinct and significant increase of some nucleotide phosphates. The most pronounced alterations were shown in the intracellular UTP (202%), CTP (103%), ADP (92%) and ATP (71%) concentrations. Changes in polyamine and nucleotide phosphate metabolisms were dependent on tumor progression. A possible explanation of the metabolic events induced by DFMO is discussed.     

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.