Accumulation Of Ornithine Decarboxylase mRNA Research Articles

Phosphatidylinositol 3-Kinase Is Required for the Induction of Ornithine Decarboxylase in Leukemia Cells Stimulated to Growth

The involvement of phosphatidylinositol 3-kinase (PI3K) in the induction of ornithine decarboxylase (ODC) was investigated by using specific PI3K inhibitors. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, treatment with LY294002 inhibited cell growth and provoked a complete block of the induction of ODC activity (IC50∼2 μM) and ODC protein. Some reduction in the accumulation of ODC mRNA was also observed, whereas ODC turnover was not affected significantly. Wortmannin, another specific inhibitor of PI3K, structurally unrelated to LY294002, also inhibited ODC induction with an IC50of about 10 nM. These results indicate that PI3K activity is required for the induction of ODC, possibly affecting both ODC mRNA level and translation. Since p70 S6 kinase (p70S6K) is considered an important mediator of PI3K action in several experimental systems, the effect of rapamycin, which can lead to selective inhibition of p70S6K, was also investigated. Rapamycin inhibited p70S6Kactivity and produced ODC inhibiting effects similar to those elicited by LY294002. However, LY294002 and wortmannin at concentrations which inhibited almost completely PI3K activity did not decrease p70S6Kactivity, suggesting that p70S6Kdoes not mediate the PI3K effects on ODC, but may lie on a separate pathway in this experimental model.

Inhibition of the expression of ornithine decarboxylase and c-Myc by cell-permeant ceramide in difluoromethylornithine-resistant leukaemia cells.

Ceramide has emerged as a novel lipid mediator in cell growth and apoptosis. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, the cell-permeant analogues of ceramide N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide) inhibited the induction of ornithine decarboxylase (ODC) activity with IC50 of 8.3 and 1.5 microM respectively. This effect was strictly related to the ability to inhibit cell growth and [3H]thymidine incorporation. The suppression of cell growth was also associated with apoptosis. The addition of bacterial sphingomyelinase resulted in a significant, but limited, reduction of ODC induction and [3H]thymidine incorporation. Bacterial lipopolysaccharide, which may act as a ceramide analogue, also inhibited the induction of the enzyme. Moreover, C6-ceramide largely prevented the accumulation of ODC mRNA and its precursor, ODC heterogeneous nuclear RNA, that accompanied the induction of ODC activity. A slight increase in ODC turnover was also observed. The DNA-binding activity of some transcription factors known to bind and transactivate the ODC gene was investigated by gel mobility-shift assay under the same experimental conditions. However, only the binding of Myc/Max was negatively affected by the treatment with C6-ceramide. Furthermore, the amount of immunoreactive c-Myc, which increased after stimulation of the cells to growth, was strongly reduced by C6-ceramide. These results suggest that the inhibition of c-Myc and ODC expression may be early events in the response of leukaemia cells to ceramide.

Premature expression of sucrase-isomaltase triggered by corticoid-dependent changes in polyamine metabolism.

A possible link between the corticoid-elicited premature expression of intestinal sucrase-isomaltase (SI) and endogenous changes in polyamine metabolism was investigated in preweaned rats. Starvation at postnatal day 12 caused a precocious expression of SI mRNA and activity. A rapid upsurge of serum corticosterone was observed during the first hour of isolation, occurring in parallel with a transient enhancement of ornithine decarboxylase (ODC) expression and followed by an increase in mucosal polyamine content. Administration of the antiglucocorticoid RU-38486 completely prevented the starvation-evoked stimulation of ODC. The treatment of the sucklings with RU-38486 or with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC, dramatically reduced the amount of SI mRNA. When exogenous hydrocortisone (HC) was administered to 12-day-old sucklings nourished by their dam, an important accumulation of ODC mRNA was observed in the intestinal mucosa 4 h after treatment. Sucklings receiving HC and treated concomitantly with either RU-38486 or DFMO exhibited a reduced amount of ODC mRNA and a dramatic decline in both SI mRNA and activity. Altogether these data support the view that the premature induction of SI expression is dependent on changes in ODC expression and polyamine metabolism that can be elicited either by endogenous changes or by exogenously administered glucocorticoids.

Asparagine markedly induces the expression of ornithine decarboxylase gene in transformed mammalian cells but not in their untrasformed counterparts

We have previously shown that asparagine alone induces a 10–15-fold increase in ornithine decarboxylase (ODC) mRNA level in DF-40 mouse neuroblastoma cells. The induction is due to an accumulation of ODC mRNA through a post-transcriptional stabilization mechanism (Chen, Z.P. and Chen, K.Y. (1992) J. Biol. Chem., 267, 6946–6951). In the present study we showed that asparagine induced ODC gene expression in v-Ha- ras-transformed 3T3 ( ras-3T3) cells but not in 3T3 cells. Other growth related genes including c- src, c- ras, and c- fos were not affected by asparagine in ras-3T3 cells. Southern blot analysis indicated that the pronounced asparagine effect was not due to ODC gene amplification in ras-3T3 cells. The effect of asparagine on the induction of ODC mRNA could account for the significant increases in the ODC activity in ras-3T3 cells. We also examined the effect of asparagine on ODC gene expression in human KD cells and their transformed counterparts. Our findings strongly suggest that the induction of ODC mRNA by asparagine may represent a component of an altered growth regulatory program associated most prominently with cell transformation.

UVB radiation induces phosphorylation of the epidermal growth factor receptor, decreases EGF binding and blocks EGF induction of ornithine decarboxylase gene expression in SV40‐transformed human keratinocytes

We previously demonstrated that epidermal growth factor (EGF) induces a several-fold increase in ornithine decarboxylase (ODC) activity and the steady-state level of ODC mRNA in cultured SV40-transformed human keratinocytes (1). Pretreatment of cell cultures with ultraviolet B (UVB) radiation resulted in a reduction of EGF-induced ODC activity. To determine whether UVB inhibits the accumulation of ODC mRNA by EGF, cells were pretreated with 20 mJ/cm2 UVB or sham-irradiated and then incubated with 100 ng/ml EGF. Northern blot analysis revealed that UVB irradiation entirely blocked the EGF induction of ODC mRNA. Since the binding of EGF to its plasma membrane receptor is the first step in initiating a biological response, the effect of UVB on EGF binding was evaluated. UVB treatment of cultured keratinocytes resulted in an immediate and dose-dependent reduction of EGF binding. Scatchard analysis revealed that the reduction of EGF binding was due to a 52% decrease in the number of available receptors, from 6.2 x 10(4)/cell to 3.0 x 10(4)/cell. However, UVB decreased the EGF-binding affinity very little (Kd = 0.60 nM in control and Kd = 0.75 nM in UVB-treated Z114 cells). In addition, UVB did not alter the rate of EGF internalization. These data suggest that UVB blocks the signal transduction pathway of EGF that is involved in regulation of ODC gene expression. Immunoblot analysis of extracts from irradiated cells showed that UVB induced tyrosine phosphorylation of EGFR and that the quantity of EGFR protein was unaffected by UVB treatment. Phosphorylation of EGFR may be responsible for decreased binding of EGF to its receptor.

Inhibition of Ornithine Decarboxylase Activity and Cell Proliferation by Ultraviolet B Radiation in EGF-Stimulated Cultured Human Epidermal Keratinocytes

Irradiation of EGF-stimulated human keratinocytes in vitro with ultraviolet B (UVB) radiation inhibited both ornithine decarboxylase (ODC) activity and cellular proliferation. A dose-dependent reduction in ODC activity occurred in primary cultures of adult facial keratinocytes and neonatal foreskin keratinocytes, and in an SV40-transformed keratinocyte cell line derived from neonatal foreskin. When SV40-transformed keratinocytes were treated with epidermal growth factor (EGF), ODC activity was induced up to 21 times in the absence of ultraviolet radiation. However, pre-treatment with UVB significantly reduced the EGF induction of ODC. For example, 85% less ODC activity was observed in cultures treated with EGF (10 ng/ml) plus 2.5 mJ/cm2 of UVB than cultures treated with EGF alone. To assess the effect of UVB on cell proliferation, normal human epidermal keratinocytes grown in medium containing EGF were irradiated with 5 and 10 mJ/cm2 UVB. At days 3 and 5 post-irradiation a significant (up to 78%) decrease in proliferation was observed. Nevertheless, the mean proportion of viable to dead cells remained similar in both UVB-treated and non-irradiated cell cultures. Northern blot analysis of total RNA isolated from irradiated and sham-irradiated cultures showed that UVB caused approximately a one third reduction in steady-state ODC mRNA levels in EGF-stimulated keratinocyte cultures. Because ODC is an enzyme required for cell proliferation, we propose that the UVB-induced decrease in cell proliferation may result at least in part from UVB inhibition of ODC mRNA accumulation and reduced enzyme activity.

Comparison of androgen regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase gene expression in rodent kidney and accessory sex organs.

Androgen regulation of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities and accumulation of the mRNAs encoding these two enzymes in rodent kidney and accessory sex organs were studied. The ODC mRNA concentration and enzyme activity were increased by androgens in kidney, prostate, and seminal vesicle of 3-day castrated mice and rats, whereas AdoMetDC mRNA and protein levels were androgen inducible only in rodent accessory sex organs. ODC and AdoMetDC mRNAs were regulated in prostate and seminal vesicles in a coordinate fashion, with the maximal levels reached within 24-48 h of steroid exposure. The extent of induction was similar for the two gene products, and ODC and AdoMetDC mRNA accumulation occurred primarily in epithelial cells of accessory sex organs, as shown by in situ hybridization studies. The murine ODC promoter contains an androgen-responsive element (ARE)-like sequence at about -910 nucleotides from the cap site; this element binds to androgen receptor in vitro, albeit with much lower affinity than some other AREs. In transient expression studies with CV-1 cells, an ODC promoter construct (pODCCAT) conferred androgen responsiveness upon the reporter gene. ODC mRNA accumulation is androgen regulated in epithelial cells of proximal tubules in both murine and rat kidney; however, different subtypes of the proximal tubular cells respond in the two species, as revealed by in situ hybridization studies. Expression of the ODC gene was induced more markedly and for a longer duration in the murine than in the rat kidney, but the initial response occurred faster in the rat kidney. The relatively slow kinetics of ODC mRNA accumulation in mouse kidney were not due to recruitment of new cells to respond; rather, in situ hybridization studies indicated that there was progressive accumulation of the mRNA in the responding cells. Collectively, these data indicate that the genes for two key enzymes in polyamine biosynthesis are not regulated in an identical fashion in different androgen target tissues, such as rodent kidney and accessory sex organs.

Studies on the relationship between cell proliferation and cell death: Opposite patterns of SGP-2 and ornithine decarboxylase mRNA accumulation in pha-stimulated human lymphocytes

To assess the relationship between cell proliferation and cell death, the mRNA accumulation of ornithine decarboxylase (ODC) and sulfated glycoprotein 2 (SGP-2) were measured in human peripheral blood lymphocytes (HPBL) 2-6 hours after stimulation with phytohemagglutinin (PHA). ODC is the rate limiting enzyme of polyamines biosynthesis and its early induction in mitogen-stimulated lymphocytes has been reported. On the other hand, SGP-2, a glycoprotein present in most mammalian tissues, is induced in classical models of apoptosis, such as dexamethasone-treated thymocytes. Indeed, a consistent amount of SGP-2 mRNA in quiescent HPBL, an early and progressive decrease of SGP-2 mRNA and a parallel increase of ODC mRNA accumulation, were observed, in PHA-stimulated HPBL, suggesting that concomitant repression of SGP-2 and induction of ODC genes contribute for the cell entering the cell cycle.

Regulation of ornithine decarboxylase mRNA by phorbol esters and insulin in normal and C‐kinase‐deficient rat hepatoma cells

Tumor-promoting phorbol esters and insulin produce similar effects in Reuber H35 rat hepatoma cell proliferation, including increased ornithine decarboxylase (ODC) enzyme activity, DNA synthesis, and mitogenesis. We investigated ODC mRNA accumulation in cells treated with either insulin or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Both agents caused rapid accumulation of ODC mRNA: for TPA, it was maximal 3 hr after treatment (4-6-fold greater than control cells) and returned quickly to control levels; for insulin, it was significantly longer, continuing to increase for at least 6 hr. Simultaneous treatment with TPA and insulin led to additive effects on ODC mRNA. Induction of ODC by TPA was blocked by down-regulation or inhibition of protein kinase C (PKC), consistent with a PKC-mediated mechanism. In contrast, PKC down-regulation had little effect on ODC induction by insulin. Furthermore, although both agents stimulated ribosomal S6 protein phosphorylation in cells containing normal amounts of PKC, the response to TPA was abolished in PKC-depleted cells; the effect of insulin was only slightly inhibited. TPA caused a rapid redistribution of essentially all of the PKC activity from the cytosolic to the membrane fraction of the cells, whereas insulin had no effect on PKC distribution. These results suggest that although insulin and TPA share some common cytoplasmic signalling pathways, their effects on phosphorylation of nuclear proteins and transcription of ODC may be mediated by distinct factors.

Regional increases in ornithine decarboxylase mRNA levels in the rat brain after partial mesodiencephalic hemitransection as revealed by in situ hybridization histochemistry

Changes in the level of ornithine decarboxylase mRNA after partial mesodiencephalic hemitransection were evaluated in various regions of the rat brain by means of in situ hybridization histochemistry coupled with computer-assisted image analysis. On days 1 and 2 after the lesion, increased accumulation of ornithine decarboxylase mRNA was observed on the lesioned side in various telencephalic regions (e.g. neostriatum and frontoparietal cortex), and in the pars compacta of the substantia nigra. Both in the frontoparietal cortex and substantia nigra a decreasing gradient of ornithine decarboxylase mRNA activation was observed going far from the site of the lesion. Seven days after the operation, ornithine decarboxylase mRNA levels returned to control values on the lesioned side but increased in some regions, such as the frontoparietal cortex, on the intact side. The present results demonstrate that the parent cell body biosynthetic machinery is activated by the mechanical lesion of the axons at the level of ornithine decarboxylase gene expression. The increase of ornithine decarboxylase mRNA is not as large as the enhancement in ornithine decarboxylase activity previously shown, suggesting that the response to the lesion may also involve changes in the rate of translation of ornithine decarboxylase mRNA and/or in the rate of degradation of ornithine decarboxylase protein.

Androgen-regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase genes

Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) are two key enzymes in polyamine biosynthesis. Both the ODC and the AdoMetDC gene is regulated by androgens in accessory sex organs of mice and rats, whereas only the ODC gene is androgen-responsive in rodent kidney. Androgenic responses in murine and rat kidneys are, however, dissimilar in that the induction of ODC activity and ODC mRNA accumulation is transient in the rat but sustained in the murine renal cells. In addition, in situ hybridization experiments with single-stranded cRNA probes revealed that ODC gene expression occurs in different subpopulations of epithelial cells of the proximal tubules in mice and rats. ODC and AdoMetDC genes are androgen-regulated in the same cell types of the accessory sex organs, as judged by hybridization histochemistry. Sequencing of the promotor region of the murine ODC gene has indicated the presence of several DNA elements for binding of transcription factors/regulatory proteins, including a putative androgen-response element at about 900 nucleotides upstream of the transcription start site.

Accumulation of ornithine decarboxylase mRNA accompanies activation of human and mouse monocytes/macrophages

Accumulation of ornithine decarboxylase (ODC) mRNA was investigated in human monocytes and mouse peritoneal macrophages. Treatment of both populations of mononuclear phagocytes with bacterial lipopolysaccharide induced a marked and rapid increase in the accumulation of the ODC gene transcript. A similar phenomenon, albeit less pronounced, was also observed following treatment of human monocytes with human recombinant interferon-γ. These results suggest a role for ODC, and therefore polyamines, in the regulation of mononuclear phagocyte functions.

Myeloid growth factor(s) regulation of ornithine decarboxylase: effects of antiproliferative signals interferon-gamma and cAMP

Colony-stimulating factors (CSFs) stimulate the activation and steady- state mRNA accumulation of an important regulatory enzyme for macromolecular synthesis, ornithine decarboxylase (ODC). Cloned murine CSF-dependent cell lines exhibited a rapid activation of ODC enzyme activity, detectable within ten minutes of stimulations with either interleukin-3 (IL-3), GM-CSF, or G-CSF. This early phase of enzyme activation did not require early protein or mRNA synthesis. The subsequent protracted rise in ODC activity occurring four to six hours after CSF treatment was dependent on increases in steady-state ODC mRNA accumulation and de novo protein synthesis. CSF, therefore, modulates both posttranslational activation of preexisting ODC and stabilization and accumulation of ODC mRNA. Antiproliferative signals, such as cAMP or interferon-gamma (IFN-gamma), effectively inhibited the CSF-directed increase in steady-state ODC mRNA. Cotreatment of the murine NSF 60.8 cell line with IFN-gamma and GM-CSF decreased steady-state ODC mRNA greater than 80% as compared with GM-CSF-treated cells alone. IFN treatment did not cause any appreciable destabilization of mature ODC mRNA, suggesting that its major effect may be at the level of ODC mRNA transcription or posttranscriptional processing. These data indicate that the ODC gene-protein system is an important molecular locus of the effects of myeloid proliferative and antiproliferation signals.

Myeloid Growth Factor(s) Regulation of Ornithine Decarboxylase: Effects of Antiproliferative Signals Interferon-γ and cAMP

Colony-stimulating factors (CSFs) stimulate the activation and steady-state mRNA accumulation of an important regulatory enzyme for macromolecular synthesis, ornithine decarboxylase (ODC). Cloned murine CSF-dependent cell lines exhibited a rapid activation of ODC enzyme activity, detectable within ten minutes of stimulations with either interleukin-3 (IL-3), GM-CSF, or G-CSF. This early phase of enzyme activation did not require early protein or mRNA synthesis. The subsequent protracted rise in ODC activity occurring four to six hours after CSF treatment was dependent on increases in steady-state ODC mRNA accumulation and de novo protein synthesis. CSF, therefore, modulates both posttranslational activation of preexisting ODC and stabilization and accumulation of ODC mRNA. Antiproliferative signals, such as cAMP or interferon-gamma (IFN-gamma), effectively inhibited the CSF-directed increase in steady-state ODC mRNA. Cotreatment of the murine NSF 60.8 cell line with IFN-gamma and GM-CSF decreased steady-state ODC mRNA greater than 80% as compared with GM-CSF-treated cells alone. IFN treatment did not cause any appreciable destabilization of mature ODC mRNA, suggesting that its major effect may be at the level of ODC mRNA transcription or posttranscriptional processing. These data indicate that the ODC gene-protein system is an important molecular locus of the effects of myeloid proliferative and antiproliferation signals.

Regulation of ornithine decarboxylase gene expression in normal and transformed hamster embryo fibroblasts following stimulation by 12-O-tetradecanoylphorbol-13-acetate.

We have compared the regulation of ornithine decarboxylase (ODC) gene expression in primary cultures of hamster embryo fibroblasts and in two independently transformed hamster embryo cell lines. Previous studies have demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) can greatly potentiate the serum growth factor induction of ODC enzyme activity in transformed cells, but not in normal hamster embryo fibroblasts. Treatment of either normal or transformed cells with both TPA and serum yielded greater accumulations of ODC mRNA than with either treatment alone, which is consistent with changes at the protein level. However, treatment of the transformed cells with TPA and serum resulted in a greater increase in steady state levels of ODC mRNA than that observed using normal fibroblasts. The time course for the induction of ODC mRNA was similar for both normal and transformed cells with maximal accumulations 4-8 h after treatment. Studies with actinomycin D further suggests that ODC mRNA is comparatively long-lived in both normal and transformed cells. The accumulation of ODC mRNA after stimulation with TPA and serum is blocked by cycloheximide in normal hamster fibroblasts suggesting that this induction is dependent upon protein synthesis. In contrast, cycloheximide did not affect the accumulation of ODC mRNA under similar treatment conditions in transformed cells. This altered regulation of ODC gene expression in transformed hamster embryo fibroblasts cannot be explained by either gene rearrangement or the amplification of an ODC gene. These data suggest that transformation of hamster embryo cells results in a loss of cellular control over ODC gene regulation which includes an alteration in the requirement for protein synthesis for ODC mRNA accumulation.

The mechanisms of ornithine decarboxylase deregulation in c-Ha-ras oncogene-transformed NIH 3T3 cells.

NIH 3T3 cells transformed with the human c-Ha-rasVal-12 oncogene showed markedly enhanced activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, as compared with their nontransformed counterparts. While in normal and in c-Ha-ras proto-oncogene-transfected cells stimulation with serum caused a transient induction of ODC, in cells transfected with the mutant c-Ha-ras oncogene the activity of ODC persisted at high levels for greatly extended periods of time. The amounts of immunoreactive ODC protein roughly paralleled the changes in the enzyme activity. The augmentation of ODC content by transformation could be largely, but not solely, accounted for by an enhanced accumulation of ODC mRNA. Nuclear run-off transcription assays demonstrated that in transformed cells the rate of transcription of the ODC gene was increased but to a much lower extent than the increase in the level of ODC mRNA. The turnover of ODC mRNA, as measured after actinomycin D treatment, was negligible in transformed cells for up to 8 h, whereas in normal cells the messenger content was initially decreased, by 40% within 4 h, and then remained constant. In normal cells, however, actinomycin D depressed the expression of ODC by more than 80%, while in transformed cells the activity of ODC was slightly superinduced, corresponding to the changes of ODC mRNA. These findings suggest that labile proteins may be involved in the regulation of both the stability and translatability of the ODC mRNA. Transformation led also to about 3-fold stabilization of ODC as determined by an exposure of the cells to cycloheximide. The results thus suggest ODC deregulation at multiple levels in the ras-oncogene-transformed cells.

Induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol 13-acetate in hamster fibroblasts. Relationship between levels of enzyme activity, immunoreactive protein, and RNA during the induction process.

Phorbol ester tumor promoters and growth factors rapidly stimulate ornithine decarboxylase activity in the transformed hamster fibroblast line HE68BP. We report here a close correspondence between the time courses and magnitudes of induction of ornithine decarboxylase activity and immunoreactive ornithine decarboxylase protein following treatment of HE68BP cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or refeeding with fresh medium. Cycloheximide addition to induced cells caused a rapid fall in the levels of both ornithine decarboxylase activity and ornithine decarboxylase protein. Northern blot analysis of RNA isolated from HE68BP cells indicated that treatment with TPA and fresh medium increased the amount of two species of mRNA of lengths 2.4 and 2.1 kilobase. This increased accumulation of ornithine decarboxylase mRNA corresponded temporally to that observed at the protein level, with a 15-fold maximal induction 7 h after treatment followed by a rapid decline in hybridizable RNA. These data indicate that stimulation of ornithine decarboxylase activity by TPA or refeeding involves changes in levels of ornithine decarboxylase mRNA as well as changes in the rate of synthesis of ornithine decarboxylase protein.