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.     

Quantification and localization of ornithine decarboxylase in the embryonic palate.

Ornithine decarboxylase (ODC; EC4.1.1.17), the key enzyme in polyamine biosynthesis, and intracellular polyamines increase rapidly and markedly in tissues and cells that are actively proliferating as well as differentiating and decrease as these processes cease. ODC activity has also been implicated as playing a role in the proliferation and differentiation of cells derived from the developing palate. Ornithine decarboxylase activity was thus quantified and ODC localized in the developing murine palate in vivo. Levels of ODC activity showed little variation during the ontogeny of the palate, averaging 126 pmol CO2/mg protein/hr. When difluoromethylornithine (DFMO), an irreversible inhibitor of ODC activity, was administered to pregnant mice throughout the period of palate development (days 11-14), palatal tissue ODC activity was reduced by 85%. No craniofacial malformations were observed, however. The lack of a teratogenic effect by DFMO treatment could be due to sufficient remaining ODC activity in craniofacial tissue and/or maintenance of intracellular polyamine levels by the activity of a polyamine transport system. The activity of this system was demonstrated by the ability of palatal tissue in vivo to take up radiolabeled putrescine. The presence of a polyamine transport system was previously suggested by the demonstration of such a system in palate mesenchymal cells in vitro. Dramatic temporal and spatial shifts in tissue patterns of immunolocalization for ODC in developing palatal tissue were also seen. Immunostaining for ODC was evenly distributed in oral, nasal, and medial edge palate epithelial cells on day 12 of gestation. The basal aspects of epithelial cells were, however, more intensely stained. Mesenchymal cells exhibited a peri-nuclear immunostaining pattern. On days 12 and 13 of gestation, the staining patterns for ODC in palate epithelial and mesenchymal cells were comparable. On day 14 of gestation, all regions of the palate epithelium, particularly the medial edge epithelia, were immunostained for ODC, whereas the intensity of staining in the mesenchymal cells was significantly reduced. This study represents essential initial observations toward understanding the role that ODC may play in normal craniofacial development.     

Induction of Brain Ornithine Decarboxylase During Recovery from Metabolic, Mechanical, Thermal, or Chemical Injury

Metabolic, mechanical, thermal, and chemical injury induced ornithine decarboxylase (ODC) activity in rat brain. A two- to sixfold increase in ODC activity was measured at 5-9 h after different modes of injury to the brain. During the early phase of recovery from transient ischemia, when average protein synthesis was less than 50% of control, ODC activity was increased nearly fivefold. The rise in activity could be blocked by anisomycin, or reduced by intracerebral injections of actinomycin D. Drilling burr holes into the skull, injection of the vehicle for actinomycin D, hyperthermia, and freezing lesions all caused increased ODC activity. Neurotoxic chemicals (ammonia, methionine sulfoximine, acrylamide, carbon tetrachloride, and anisomycin) also increased brain ODC activity, whereas other chemicals (mannitol and valine) did not. Treatments known to stimulate the synthesis of heat shock proteins (carotid occlusion, hyperthermia, Cd2+, canavanine, and ethanol) induced ODC activity in the liver, whereas only hyperthermia and ethanol caused significant increases in spleen ODC activity. All increases in ODC activity were blocked by difluoromethylornithine, an irreversible inhibitor of ODC. The cellular response to noxious or stressful stimuli includes the synthesis of a small number of proteins of unknown functions; ODC may be one of these "heat shock" or "trauma" proteins.     

Gastrin and epidermal growth factor induction of ornithine decarboxylase in rat colonic explants

An organ culture system was utilized to examine the effect of gastrin (G-17-I) and epidermal growth factor (EGF) on colonic mucosal ornithine decarboxylase (ODC) activity, and the expression of the ODC gene. Exposure of colonic mucosal explants to either gastrin or EGF (50-500 ng/ml) for only 4 h resulted in a profound stimulation (150-600%) in ODC activity over the basal level. These increases were essentially abolished by difluoromethylornithine (DFMO; 2 nmol/ml) or CaCl2 (2 umol/ml). Gastrin also activated the ODC gene in the colonic mucosa as evidenced by increased steady-state ODC mRNA levels in the colonic mucosal explants after 4 h exposure to the hormone, when compared with the controls. It is concluded that colonic mucosal ODC is responsive to both gastrin and EGF.     

Induction of Ornithine Decarboxylase by N-Methyl-D-Aspartate Receptor Activation Is Unrelated to Potentiation of Glutamate Excitotoxicity by Polyamines in Cerebellar Granule Neurons

Abstract: Polyamines positively modulate the activity of the N -methyl- d -aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)—the key regulatory enzyme in polyamine synthesis—and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.     

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.     

Early alterations of polyamine metabolism induced after acute administration of clenbuterol in mouse heart.

An acute treatment of mice with clenbuterol, a beta-adrenergic agonist, produced a marked increase of polyamines levels in heart, particularly during the early phase of administration of the drug. A single dose of 1.5 mg/kg caused as much as a 10 fold induction in activity of ornithine decarboxylase (ODC) and 3 to 4 fold increase in levels of putrescine, spermidine and spermine in mouse heart. Maximum changes were observed 3 to 4 hours post-administration of clenbuterol. This treatment did not produce any change in S-adenosylmethionine decarboxylase activity. The induction of cardiac ODC by clenbuterol was also dose dependent with a peak at about 5 micromol/kg. Co-administration of difluoromethylornithine, an irreversible inhibitor of ODC, or propranolol, a nonspecific beta-antagonist, with clenbuterol completely prevented the induction of ODC activity as well as the increase in polyamine levels in heart. However, pretreatment with alprenolol or metoprolol, the specific beta1 and beta2-antagonists, respectively, produced only partial prevention. The cardiac ODC from controls as well as clenbuterol treated mice exhibited similar affinity (Km) for its substrate, ornithine, while maximum enzyme activity (Vmax) was about 14 fold higher in clenbuterol treated mouse heart than in the control. Clenbuterol produced no change in the level of specific ODC mRNA or the protein, but the enzyme from the drug-treated mouse heart was considerably more stable than the control. Pretreatment of mice with either cycloheximide or actinomycin D followed by administration of clenbuterol could not prevent the induction in ODC activity suggesting that de novo biosynthesis of the enzyme protein or ODC mRNA was not responsible for induction of ODC activity. Post-translational changes in ODC may be responsible for an early increase of ODC activity due to clenbuterol treatment.     

HSG cells differentiated by culture on extracellular matrix involves induction of S-adenosylmethione decarboxylase and ornithine decarboxylase

The human salivary gland (HSG) epithelial cell line can differentiate when cultured on extracellular matrix preparations. We previously identified >30 genes upregulated by adhesion of HSG cells to extracellular matrix. In the current studies, we examined the role of one of these genes, the polyamine pathway biosynthetic enzyme S-adenosylmethionine decarboxylase (SAM-DC) and the related enzyme, ornithine decarboxylase (ODC), on HSG cell differentiation during culture on extracellular matrix. HSG cells cultured on fibronectin-, collagen I gel-, and Matrigel-coated substrates for 12-24 h upregulated SAM-DC and ODC mRNA expression and enzyme activity compared to cells cultured on non-precoated substrates. After 3-5 days, HSG cells grown on Matrigel- or collagen I gel-coated substrates acquired a differentiated phenotype: the cells showed changes in culture morphology and increased expression of salivary gland differentiation markers (vimentin, SN-cystatin, and alpha-amylase). Further, culturing the cells on substrates precoated with an anti-beta1-integrin-antibody promoted differentiation-like changes. HSG cells cultured on collagen I- or Matrigel-coated substrates rapidly entered the cell cycle but showed decreased cell proliferation at longer times. In contrast, cell proliferation was enhanced on fibronectin-coated substrates compared to cells on non-precoated substrates. Treatment with the polyamine synthesis inhibitors, difluoromethylornithine (DFMO), and methylglyoxal bis-(guanylhydrazone) (MGBG), inhibited cell proliferation and delayed (3)H-thymidine incorporation in HSG cells cultured on all of the substrates. Further, inclusion of DFMO and MGBG inhibited or delayed acquisition of the differentiated phenotype in HSG cells cultured on Matrigel- or collagen I gel-coated substrates. This suggests that the adhesion-dependent expression of SAM-DC and ODC contributes to extracellular matrix-dependent HSG cell differentiation.     

Nitric oxide mediates either proliferation or cell death in cardiomyocytes. Involvement of polyamines

Summary Nitric oxide (NO) is a molecule involved in several signal transduction pathways leading either to proliferation or to cell death. Induction of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, represents an early event preceding DNA synthesis. In some cell types increased ODC activity seems to be involved in cytotoxic response. We investigated the role of NO and ODC induction on the events linked to cell proliferation or to cell death in cultured chick embryo cardiomyocytes. Exposure of cardiomyocytes to tumor necrosis factor (TNF) and lipopolysaccharide (LPS) caused NO synthase (NOS) and ODC induction as well as increased incorporation of [³H]-thymidine. This last effect was blocked by a NOS inhibitor and was strongly reduced by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Sodium nitroprusside (SNP), an exogenous NO donor, inhibited the increases of NOS and ODC activities and abolished the mitogenic effect of TNF and LPS. Moreover, SNP alone caused cell death in a dose dependent manner. The cytotoxicity of SNP was not affected by DFMO while it was prevented by antioxidants. The results suggest that different pathways would mediate the response of cardiomyocytes to NO: they can lead either to ODC induction and DNA synthesis when NO is formed through NOS induction or to growth inhibition and cell death, when NO is supplied as NO donor. Increased polyamine biosynthesis would mediate the proliferative response of NO, while the cytotoxicity of exogenous NO seems to involve some oxidative reactions and to depend on the balance between NO availability and cellular redox mechanisms.     

Synergistic induction of rat hepatic ornithine decarboxylase by multiple doses of cobalt chloride

The level of rat hepatic ornithine decarboxylase (ODC) induced by repetitive administration of Co2+ was determined by affinity labeling with [3H]difluoromethylornithine. Such a treatment with Co2+ ion induced ODC level to a 10-fold greater extent than single dose of the metal ion or well-known inducers of the enzyme, such as thioacetamide or carbon tetrachloride. The half life of ODC activity induced by repetitive treatment with Co2+ (95 min) was substantially increased to about 10-fold over the value obtained from the enzyme induced by single treatment with the metal ion (10 min). ODC activity induced by repetitive treatment with Co2+ was separated into two peaks by DEAE-Sepharose column chromatography. The two independently collected fractions of ODC peaks exhibited different affinity for pyridoxal 5'-phosphate in vitro and sensitivity to cycloheximide in vivo.     

Ornithine decarboxylase activity associated with a particulate fraction of brain

The enzymic decarboxylation of ornithine by adult rat brain largely occurs in the particulate fraction. The activity is primarily due to ornithine decarboxylase (ODC) as evidenced by several criteria: i) the concurrent production of equimolar amounts of CO₂ and putrescine, ii) the sensitivity of the reaction to difluoromethylornithine (DFMO), a specific inhibitor of ODC, iii) the lack of major effect of two inhibitors of ornithine-2-oxo-acid transaminase, upon the DFMO-sensitive component of decarboxylation, iv) the failure to profoundly reduce decarboxylation activity in the presence of a large excess of many aminoacids which could compete for non-specific decarboxylases. The insoluble ODC activity appears largely within synaptosomal and mitochondrial-enriched morphological fractions, yet cannot be attributed to trapped soluble ODC. Particulate ODC has a pH optimum and kinetic parameters that differ from those of soluble cerebral ODC.     

Effect of elevated levels of ornithine decarboxylase on cell cycle progression in skin.

By crossing TG.AC v-Ha-ras and K6/ODC transgenic mice, we found previously that an activated ras and follicular ornithine decarboxylase (ODC) overexpression cooperate to generate spontaneous tumors in the skin. Cellular proliferation was dramatically increased in the K6/ODC transgenic skin, as evidenced by elevated proliferating cell nuclear antigen and Ki67 expression compared with nontransgenic littermates. Keratinocytes isolated from transgenic skin also displayed increased clonal growth. Paradoxically, expression of the growth inhibition-associated proteins p53, p21Waf1, p27Klp1, and Bax was increased with ODC overexpression in the skin. ODC overexpression did not affect cyclin D/cyclin-dependent kinase 4 (Cdk4)-dependent phosphorylation of retinoblastoma protein but stimulated cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity, with minimal effect on the levels of these proteins. Thus, ODC/polyamine-induced activation of cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity may cooperate with the ras induction of cyclin D/Cdk4/6-associated retinoblastoma protein phosphorylation to not only stimulate proliferation but ultimately contribute to tumor development.     

Expression of a novel human ornithine decarboxylase-like protein in the central nervous system and testes

Ornithine decarboxylase (ODC) is the key enzyme of polyamine synthesis. The physiological activity of ODC is associated with cell proliferation, and high ODC activities are encountered in rapidly growing cancer cells. We have cloned a cDNA for a novel human protein that is 54% identical to ODC and 45% identical to antizyme inhibitor (AZI). mRNA for ODC-paralogue (ODC-p) was found only in the central nervous system and testes, suggesting a role in terminal differentiation rather than cell proliferation. ODC-p occurs at least in eight alternatively spliced forms. In vitro translated ODC-p did not decarboxylate ornithine, whereas, in vivo, one splice variant exerted modest ODC-like activity upon expression in COS-7 cells. ODC-p has a unique mutation in cysteine 360, where this ornithine decarboxylase reaction-directing residue is substituted by a valine. This substitution might lead to an enzymatic reaction that differs from typical ODC activity. ODC-p might also function as a brain- and testis-specific AZI.     

Cellular origin of prolonged induction of ornithine decarboxylase in the rat ovary

The temporal changes of ornithine decarboxylase (ODC) activity were investigated in the immature rat ovary following a single subcutaneous injection of pregnant mare serum gonadotropin (PMSG). A dose-response relationship was established. Maximal ODC activity was obtained at a dose of 25 IU of PMSG. This increase in ODC activity was accompanied by an increase of ovarian weight before reaching a maximum. A 250-fold increase of ODC activity was observed 1 day following a single dose of PMSG (50 IU). The enzyme specific activity only returned to the control level 4-5 days after hormone treatment. Immunoreactive ODC in immature, PMSG-primed rat ovaries and in heavily luteinized rat ovaries was localized utilizing the immunoperoxidase method and an antibody to ODC. Immunoreactive enzyme was confined to the cytoplasm of the granulosa cells but was not present in luteal cells. Thecal cells showed only weak immunostaining. This study provides clear evidence that the granulosa cell is the unique source of ODC activity in response to PMSG treatment. Furthermore, these data support the concept that polyamines play a role in granulosa cell proliferation and hence follicular development.     

Induction of Ornithine Decarboxylase in Cerebral Cortex by Excitotoxin Lesion of Nucleus Basalis: Association with Postsynaptic Responsiveness and N-Methyl-d-Aspartate Receptor Activation

The major cholinergic innervation of the rat cerebral cortex arises from the nucleus basalis in the basal forebrain. Introduction of the excitotoxins kainate or ibotenate into the nucleus basalis by stereotaxic injection results in degeneration of the cholinergic cells. We have investigated the effect of this excitotoxic action on ornithine decarboxylase (ODC) activity and cholinergic responsiveness in the cerebral cortex. A massive and rapid induction of ODC activity was seen in ipsilateral cortex after injection of excitotoxin. A maximal increase in ODC activity of 268 times the control value was seen in ipsilateral cerebral cortex 8 h after lesioning. Thereafter, ODC activity declined but remained significantly greater than control levels for 32 h. Pretreatment of animals with the irreversible ODC inhibitor difluoromethylornithine prevented the induction of ODC by kainate. Tissue content of the ODC product putrescine showed a marked increase in cerebral cortex ipsilateral to the lesion, increasing sevenfold at 24 h, the maximal concentration reached. After 24 h, the level of putrescine decreased but remained significantly elevated above control values for 5 days. Levels of the polyamines spermidine and spermine were unaffected by lesioning. Increases on ODC activity of much smaller magnitude were also seen in brain regions not directly innervated from the ipsilateral nucleus basalis. However, the response in ipsilateral cortex was found to be dependent on an intact projection from nucleus basalis to cortex. The induction of ODC was shown to be prevented by treatment of rats with MK-801, a result indicating the involvement of N-methyl-D-aspartate (NMDA) receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of ornithine decarboxylase in the regulation of cell growth by IL-1 and tumor necrosis factor

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine synthesis, and polyamines are required for cell growth. As an approach to clarifying the mechanism of action IL-1, the effects of IL-1 on ODC activity were examined in various cell lines whose proliferation was either suppressed or enhanced by IL-1. The proliferation of all cell types used in these experiments was markedly suppressed by a specific ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), substantiating the crucial role of ODC activity for cell proliferation. ODC activity also was considerably suppressed by IL-1 in those cells on which IL-1 exerts an antiproliferative effect, such as a human melanoma cell line (A375) and malignant human mammary cell lines (MCF-7 and T-47D). On the other hand, ODC activity was stimulated in cells that are stimulated to proliferate in response to IL-1, such as a mouse helper T cell line (D10.G4.1), a NK cell-like cell line (YT), and a human glioblastoma cell line (U373 MG). The effect of IL-1 on ODC activity preceded and directly correlated in a dose-dependent manner with its effect on DNA synthesis. Furthermore, putrescine, a product of the ODC reaction and a precursor of polyamines, was able to overcome most, but not all, the antiproliferative action of IL-1 in A375 melanoma cells, which were the most sensitive to suppression by IL-1. However, putrescine did not reverse the cytostatic effect of IL-1 on MCF-7 and T-47D cell lines. In contrast, putrescine, like IL-1, exhibited some co-mitogenic activity on D10.G4.1 cells. Because the biological activities of TNF and IL-1 show considerable overlap, the effect of TNF on ODC activity also was examined. TNF had an antiproliferative effect on A375 cells and stimulated the proliferation of U373 MG cells. The ODC activity in A375 cells was suppressed by TNF, and the ODC activity in U373 MG cells was stimulated by TNF. Putrescine also partially overcame the inhibitory effect of TNF. These results suggest that the regulation of ODC activity may be a key component in the antiproliferative and proliferative action of IL-1 and TNF in some tumor cell types.     

Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

The acquired mutation (V617F) of Janus kinase 2 (JAK2) is observed in the majority of patients with myeloproliferative neoplasms (MPNs). In the screening of genes whose expression was induced by JAK2 (V617F), we found the significant induction of c-Myc mRNA expression mediated by STAT5 activation. Interestingly, GSK-3β was inactivated in transformed Ba/F3 cells by JAK2 (V617F), and this enhanced the protein expression of c-Myc. The enforced expression of c-Myc accelerated cell proliferation but failed to inhibit apoptotic cell death caused by growth factor deprivation; however, the inhibition of GSK-3β completely inhibited the apoptosis of cells expressing c-Myc. Strikingly, c-Myc T58A mutant exhibited higher proliferative activity in a growth-factor-independent manner; however, this mutant failed to induce apoptosis. In addition, knockdown of c-Myc significantly inhibited the proliferation of transformed cells by JAK2 (V617F), suggesting that c-Myc plays an important role in oncogenic activity of JAK2 (V617F). Furthermore, JAK2 (V617F) induced the expression of a target gene of c-Myc, ornithine decarboxylase (ODC), known as the rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO), prevented the proliferation of transformed cells by JAK2 (V617F). Importantly, administration of DFMO effectively delayed tumor formation in nude mice inoculated with transformed cells by JAK2 (V617F), resulting in prolonged survival; therefore, ODC expression through c-Myc is a critical step for JAK2 (V617F)-induced transformation and DFMO could be used as effective therapy for MPNs.