Ornithine Decarboxylase Levels Research Articles

Targeted overexpression of ornithine decarboxylase enhances beta-adrenergic agonist-induced cardiac hypertrophy.

These studies were designed to determine the consequences of constitutive overexpression of ornithine decarboxylase (ODC) in the heart. Induction of ODC is known to occur in response to agents that induce cardiac hypertrophy. However, it is not known whether high ODC levels are sufficient for the development of a hypertrophic phenotype. Transgenic mice were generated with cardiac-specific expression of a stable ODC protein using the alpha-myosin heavy-chain promoter. Founder lines with >1000-fold overexpression of ODC in the heart were established, resulting in a 50-fold overaccumulation of putrescine, 4-fold elevation in spermidine, a slight increase in spermine and accumulation of large amounts of cadaverine compared with littermate controls. Despite these significant alterations in polyamines, myocardial hypertrophy, as measured by ratio of heart to body weight, did not develop, although atrial natriuretic factor RNA was slightly elevated in transgenic ventricles. However, stimulation of beta-adrenergic signalling by isoproterenol resulted in severe hypertrophy and even death in ODC-overexpressing mice without further altering polyamine levels, compared with only a mild hypertrophy in littermates. When beta1-adrenergic stimulation was blocked by simultaneous treatment with isoproterenol and the beta1 antagonist atenolol, a significant, although reduced, hypertrophy was still present in the hearts of transgenic mice, suggesting that both beta1 and beta2 adrenergic receptors contribute to the hypertrophic phenotype. Therefore these mice provide a model to study the in vivo co-operativity between high ODC activity and activation of other pathways leading to hypertrophy in the heart.

Antizyme Regulates the Degradation of Ornithine Decarboxylase in Fission Yeast Schizosaccharomyces pombe: STUDY IN THE spe2 KNOCKOUT STRAINS

The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe. To regulate cellular spermidine experimentally, we cloned and disrupted S-adenosylmethionine decarboxylase gene (spe2) in S. pombe. The null mutant of spe2 was devoid of spermidine and spermine, accumulated putrescine, and contained a high level of ODC. Addition of spermidine to the culture medium resulted in rapid decrease in the ODC activity caused by the acceleration of ODC degradation, which was dependent on de novo protein synthesis. A fraction of ODC forming an inactive complex concomitantly increased. The accelerated ODC degradation was prevented either by knockout of antizyme gene or by selective inhibitors of proteasome. Thus, unlike budding yeast, mammalian type antizyme-mediated ODC degradation by proteasome is operating in S. pombe.

Inhibition by interferon α-2b of rat liver regeneration: effect on ornithine decarboxylase and total protein synthesis

Polyamines are key factors in macromolecule synthesis during liver regeneration. It has been postulated that interferon-α (IFNα) decreases putrescine levels in regenerating liver by inhibiting ornithine decarboxylase (ODC) activity, the main enzyme in polyamine biosynthesis. In the present study, we analysed the effects of a pharmacological dose of IFNα on polyamine and ODC levels during the regenerative process following partial hepatectomy in rats. Synthesis of ODC by isolated hepatocytes from IFN-treated rats with regenerating livers was also assessed. Furthermore, we investigated the effect of IFNα-2b on DNA and total protein synthesis in 24-hr regenerating livers. No effect on DNA synthesis was observed at the dose of IFNα-2b used, but total protein synthesis decreased significantly in IFNα-2b-treated rats undergoing liver regeneration (7.0 ± 2.0 and 12.1 ± 1.7% · min−1 in hepatectomized rats treated with IFNα-2b and saline, respectively). ODC levels were also reduced significantly (by 50%) in hepatectomized rats treated with IFNα-2b versus saline. In parallel with the ODC decrease, the concentrations of putrescine and spermidine (63 ± 25 vs 101 ± 15 nmol/g liver and 1.08 ± 0.35 vs 2.14 ± 0.22 μmol/g liver, respectively, in IFNα-2b- and saline-treated hepatectomized rats) showed similar, significant diminutions. Moreover, the incorporation of [35S]methionine into ODC was decreased dramatically in isolated hepatocytes from IFNα-2b-treated hepatectomized rats 12 hr after surgery. In conclusion, the protein synthesis rate in regenerating liver was impaired by therapeutic doses of IFNα-2b. In addition, the results presented in this study suggest that IFNα-2b negatively regulates ODC synthesis, causing a reduction in polyamine levels during liver regeneration.

CHOP, a Basic Leucine Zipper Transcriptional Factor, Contributes to the Antiproliferative Effect of IL-1 on A375 Human Melanoma Cells Through Augmenting Transcription of IL-6

Interleukin-1 (IL-1) inhibits the proliferation of A375 human melanoma cells. We have demonstrated previously that p38 mitrogen-activated protein kinase (MAPK) mediated the antiproliferative effect of IL-1 partially through the downregulation of activity and protein level of ornithine decarboxylase (ODC). In this study, we investigated the role of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), one of the p38 MAPK target transcriptional factors. The mRNA level of CHOP was not affected by IL-1 treatment in A375-6 cells. Unexpectedly, CHOP was constitutively phosphorylated, and IL-1 or p38 MAPK inhibitor, SB203580, did not affect the phosphorylation level. However, A375-6 cells exhibited enhanced sensitivity to IL-1 by transfecting CHOP expression plasmid and reduced sensitivity to IL-1 by antisense CHOP mRNA expression plasmid. Furthermore, CHOP appeared to regulate positively IL-6 production at the transcriptional level. The experiments using CHOP muteins revealed that dimerization ability - but not p38 MAPK-dependent phosphorylation or DNA binding activity - is important for the IL-6 inducing activity of CHOP. These results indicate that CHOP contributes to the IL-1 growth-inhibitory signal through augmenting IL-6 production.

Androgen responsiveness and intrarenal localization of transcripts coding for the enzymes of polyamine metabolism in the mouse

Polyamines, spermidine (SPD), and spermine (SPM) are intracellular polycations required for cell growth and differentiation. Their biosynthetic precursor, the diamine putrescine (PUT), is produced by regulatory ornithine decarboxylase (ODC). Spermidine/spermine N1-acetyltransferase (SSAT) is the ODC counterpart in the degradation pathway which retroconverts SPM and SPD into PUT. Castration of male mice for 7 days resulted in a 40% decrease of the renal levels of both SSAT and ODC transcripts. Administration of 5-α-dihydrotestosterone (DHT) to castrated mice for the last 3 days before sacrifice caused the levels of ODC and SSAT mRNAs to increase by 250% and 180%, respectively. Thus activation of the retroconversion pathway of polyamine metabolism appears to contribute towards the increase in PUT production known to be caused by androgens in the mouse kidney. In situ hybridization histochemistry experiments showed that the SSAT transcript is expressed only by the epithelial cells of the straight and convoluted distal tubules of the nephron, while the expression of the ODC transcript is confined to the epithelium of the convoluted and straight portion of the proximal tubules. The separation of the biosynthetic from the degradation pathway along the nephron suggests that PUT is mostly produced in the distal tubule, where it may play a physiological role, independent of androgen action, in protecting tubular cells from the very low osmolarity to which they are exposed in this nephron segment.Key words: Adenosylmethionine decarboxylase, spermidine/spermine N1-acetyltransferase, ornithine decarboxylase, mouse kidney, polyamines.

Effect of immobilization and concurrent exposure to a pulse-modulated microwave field on core body temperature, plasma ACTH and corticosteroid, and brain ornithine decarboxylase, Fos and Jun mRNA.

Exposure of humans and rodents to radiofrequency (RF) cell phone fields has been reported to alter a number of stress- related parameters. To study this potential relationship in more detail, tube-restrained immobilized Fischer 344 rats were exposed in the near field in a dose-dependent manner to pulse-modulated (11 packets/s) digital cell phone microwave fields at 1.6 GHz in accordance with the Iridium protocol. Core body temperatures, plasma levels of the stress-induced hormones adrenocorticotrophic hormone (ACTH) and corticosterone, and brain levels of ornithine decarboxylase (Odc), Fos and Jun mRNAs were measured as potential markers of stress responses mediated by RF radiation. We tested the effects of the loose-tube immobilization with and without prior conditioning throughout a 2-h period (required for near-field head exposure to RF fields), on core body temperature, plasma ACTH and corticosteroids. Core body temperature increased transiently (+/-0.3 degrees C) during the initial 30 min of loose-tube restraint in conditioned animals. When conditioned/tube-trained animals were followed as a function of time after immobilization, both the ACTH and corticosterone levels were increased by nearly 10-fold. For example, within 2-3 min, ACTH increased to 83.2 +/- 31.0 pg/dl, compared to 28.1 +/- 7.7 pg/dl for cage controls, reaching a maximum at 15-30 min (254.6 +/- 46.8 pg/dl) before returning to near resting levels by 120 min (31.2 +/- 10.2 pg/dl). However, when non-tube-trained animals were submitted to loose-tube immobilization, these animals demonstrated significantly higher (3-10-fold greater) hormone levels at 120 min than their tube-trained counterparts (313.5 +/- 54.8 compared to 31.2 +/- 10.2 pg/dl; corticosterone, 12.2 +/- 6.2 microg/dl compared to 37.1 +/- 6.4 microg/dl). Hormone levels in exposed animals were also compared to those in swim-stressed animals. Swimming stress also resulted in marked elevation in both ACTH and corticosterone levels, which were 10-20 fold higher (541.8 compared to 27.2-59.1 pg/dl for ACTH) and 2-5 fold higher (45.7 compared to 8.4- 20.0 microg/dl for corticosteroids) than the cage control animals. Three time-averaged brain SAR levels of 0.16, 1.6 and 5 W/ kg were tested in a single 2-h RF-field exposure to the Iridium cell phone field. When RF-exposed and sham-exposed (immobilized) animals were compared, no differences were seen in core body temperature, corticosterone or ACTH that could be attributed to near-field RF radiation. Levels of Odc, Fos and Jun mRNA were also monitored in brains of animals exposed to the RF field for 2 h, and they showed no differences from sham-exposed (loose-tube immobilized) animals that were due to RF-field exposure. These data suggest that a significant stress response, indicated by a transient increase in core body temperature, ACTH and corticosterone, occurred in animals placed in even the mild loose-tube immobilization required for near-field RF exposure employed here and in our other studies. Failure to adequately characterize and control this immobilization response with appropriate cage control animals, as described previously, could significantly mask any potential effects mediated by the RF field on these and other stress-related parameters. We conclude that the pulse-modulated digital Iridium RF field at SARs up to 5 W/kg is incapable of altering these stress-related responses. This conclusion is further supported by our use of an RF-field exposure apparatus that minimized immobilization stress; the use of conditioned/tube-trained animals and the measurement of hormonal and molecular markers after 2 h RF-field exposure when the stress-mediated effects were complete further support our conclusion.

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli.

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.

Tumour promoter mediated altered expression and regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase in H-ras-transformed fibrosarcoma cell lines

Alterations in cellular growth are important in the progression of malignant disease. Cell growth regulation by tumour promoters can be complex. The present study demonstrates a novel link between alterations in phorbol ester tumour promoter mediated regulation during malignant conversion and the expression of ornithine decarboxylase and S-adenosylmethionine decarboxylase, key rate-limiting and regulatory activities in the biosynthesis of polyamines. H-ras-transformed mouse 10 T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible phorbol ester tumour promoter mediated changes in ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) gene expression. Selective induction of ODC and SAMDC gene expression was observed, since in contrast to nontransformed parental 10 T1/2 cells, ras-transformed cells capable of benign tumour formation (NR3 cells) and ras-transformed cells capable of metastasis formation (C2 cells) exhibited marked alterations in the levels of ODC and SAMDC gene expression. Increased ODC gene and SAMDC gene expression in response to phorbol-12-myristate-13-acetate (PMA) treatment was found to involve transcriptional events in both NR3 cells and in C2 cells. Post-transcriptional events also played a role in the regulation of ODC gene expression in NR3 cells and in C2 cells, and in the regulation of SAMDC gene expression in C2 cells but not in NR3 cells. In NR3 cells, alterations in ODC and in SAMDC gene expression was an event requiring de novo protein synthesis, whereas in highly malignant C2 cells, protein synthesis inhibition following cycloheximide treatment in cooperation with PMA resulted in an augmentation of both ODC and SAMDC gene expression. Evidence is presented to suggest that the PMA-mediated alterations in ODC and in SAMDC gene expression in NR3 cells and in C2 cells involved protein kinase C - mediated events. The status of the cellular polyamine levels was also an important determinant of the PMA-mediated alterations that occurred in ODC and in SAMDC expression in these H-ras transformed cells. Collectively, these results suggest that PMA can modulate ODC and SAMDC expression in H-ras transformed cells and that the mechanisms involved in the PMA- mediated regulation of ODC and SAMDC gene expression changes as a function of H-ras mediated cellular transformation and malignant progression. This study further suggests a mechanism of PMA stimulation of transformed cells wherein early alterations in the regulatory control of ODC and SAMDC gene expression are important and critical.

Androgen responsiveness and intrarenal localization of transcripts coding for the enzymes of polyamine metabolism in the mouse

Polyamines, spermidine (SPD), and spermine (SPM) are intracellular polycations required for cell growth and differentiation. Their biosynthetic precursor, the diamine putrescine (PUT), is produced by regulatory ornithine decarboxylase (ODC). Spermidine/spermine N1-acetyltransferase (SSAT) is the ODC counterpart in the degradation pathway which retroconverts SPM and SPD into PUT. Castration of male mice for 7 days resulted in a 40% decrease of the renal levels of both SSAT and ODC transcripts. Administration of 5-alpha-dihydrotestosterone (DHT) to castrated mice for the last 3 days before sacrifice caused the levels of ODC and SSAT mRNAs to increase by 250% and 180%, respectively. Thus activation of the retroconversion pathway of polyamine metabolism appears to contribute towards the increase in PUT production known to be caused by androgens in the mouse kidney. In situ hybridization histochemistry experiments showed that the SSAT transcript is expressed only by the epithelial cells of the straight and convoluted distal tubules of the nephron, while the expression of the ODC transcript is confined to the epithelium of the convoluted and straight portion of the proximal tubules. The separation of the biosynthetic from the degradation pathway along the nephron suggests that PUT is mostly produced in the distal tubule, where it may play a physiological role, independent of androgen action, in protecting tubular cells from the very low osmolarity to which they are exposed in this nephron segment.

A retro-inverso peptide homologous to helix 1 of c-Myc is a potent and specific inhibitor of proliferation in different cellular systems.

In 1998 we reported that an L-peptide derived from H1 of c-Myc (Int-H1-S6A,F8A), linked to an internalization sequence from the third a-helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro-inverso (RI-) peptides can assume a 3D conformation very close to their corresponding L-forms and can be recognized by the same monoclonal antibody. We synthesized a RI-peptide form of our original L-peptide: It was much more resistant to serum peptidases than the original molecule (a half life of days rather than hours); in addition, the RI-form of the original Antennapedia internalization sequence was perfectly capable of carrying a D-peptide into human cells. We have studied three different potentially active peptides. L-peptides: Int-H1wt, Int-H1-S6A,F8A. D-peptides: RI-Int -H1-S6A,F8A. We have also studied three presumed control peptides: Int and RI-Int (no H1 motif), H1-S6A,F8A (no internalization sequence). Both 'active' and 'control' peptides have essentially confirmed our expectations, however, in cells treated with the higher concentration (10 mM) of the control peptide RI-Int, non-Myc related side effects could be detected. In order to investigate whether the antiproliferative activities displayed by some of our molecules were indeed related to an interference with the role of c-Myc (and molecules of the family), we chose an iso-amphipathic modified peptide of the H1 motif, with a proximity coefficient >50% and where the major change was at position 7 (F-->A). From a family of 73 H1 motifs belonging to (H1-Loop-H2) hu man sequences, the smallest evolutionary distance from our reference peptide was observed for the H1 of N-Myc, L-Myc, c-Myc, H1-S6A,F8A of c-Myc, and Max, in that order. Our reference peptide was therefore appropriate as a check of whether we were indeed observing activities related to Myc functions. Both Int-H1isoamph and the corresponding RI-Int-H1isoamph peptide were synthesized and studied. In terms of biological targets, we added to the human mammary cancer line of our previous work (MCF-7 cells) a colon cancer line (HCT-116 cells) and also a system of normal cells: human peripheral blood lymphocytes (PBLs) stimulated with phytohemoagglutinin (PHA). Peptides carrying an iso-amphipathic-modified H1 sequence were always very clearly (3-10 times) less active than the corresponding peptides carrying a conserved "H1 of Myc" motif. This finding was noted in five independent situations (all the cellular models considered at the present time): MCF-7 cells treated with L-peptides; MCF-7 cells treated with RI-peptides; HCT-116 cells treated with L-peptides; PBLs treated with L-peptides; PBLs treated with RI-peptides. Modulation of transcription levels of ornithine decarboxylase (ODC), p53, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in PBLs treated with our different molecules, was well compatible with an interference by our active peptides at the level of Myc transcriptional activity. We had already reported a similar observation in MCF-7 cells. On a molar basis, RI-peptides were about 5-10 times more potent and 30-35 times more stable in complete culture medium, than their corresponding L-forms. RI-Int can probably internalize longer peptido-mimetic molecules (for instance molecules mimetic of (H1-Loop-H2), or even more. These possibilities open the way to rodent studies and to more potent/selective Myc inhibitors-two steps closer to a potential drug.

Inhibitory effects of S-methylcysteine and cysteine on the promoting potential of sodium phenobarbital on rat liver carcinogenesis.

The effects of S‐methylcysteine (SMC) and eysteine on the promotion stages of rodent hepatocar‐cinogenesis in a medium‐term bioassay previously developed by Ito were examined. Initiation was induced by a single dose of diethylnitrosamine (DEN), followed by dietary administration of the promoter sodium phenobarbital (NaPB) 2 weeks later, for 6 weeks. Partial hepatectomy was conducted on all the animals at week 3. Inhibitory potential was evaluated by analyzing two markers of carcinogenesis, namely numbers of glutathione S‐transferase placental form (GST‐P)‐positive foci, and proliferating cell nuclear antigen (PCNA). In addition, the level of ornithine decarboxylase (ODC), one of the rate‐limiting enzymes of polyamine metabolism induced by promoters, was analyzed. SMC and cysteine induced significant reduction in the areas of GST‐P‐positive foci. A significant reduction in the PCNA index was observed in the entire liver as well as in GST‐P‐positive areas. SMC also induced down‐regulation of the ODC enzyme activity. Thus, SMC and cysteine were found to inhibit the promotion stage of DEN‐induced hepatocarcinogenesis. No cocarcinogenic effects were evident on administration of either of these chemicals with NaPB.

High levels of intracellular polyamines promote histone acetyltransferase activity resulting in chromatin hyperacetylation

Polyamines stimulate expression of a variety of genes, including many implicated in cell proliferation. Indeed, aberrant expression of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis, plays a causal role in tumorigenesis. Gene activity is influenced by dynamic changes in acetylation of nucleosomal histones. Although polyamines influence the histone acetyltransferase and deacetylase activities in cell-free systems, their ability to modulate these enzymes in live cells has never been established. To examine the effects of elevated intracellular levels of ODC and polyamines on gene transcription and histone acetylation, cells were infected with a retrovirus containing a cDNA for ODC. ODC overexpression potentiated the stimulatory effects of histone deacetylase inhibitors on reporter gene expression beyond that promoted by ODC or inhibitor treatment alone. Indeed, elevated intracellular levels of ODC promoted hyperacetylation of histones in several epidermal and fibroblast cell types. The ODC-mediated increase in acetylated histones was abrogated when cells were treated with α-difluoromethylornithine, a specific inhibitor of ODC activity, implying a distinct role for polyamines. Specifically, polyamines were found to enhance the action of histone acetyltransferases either directly or indirectly. Our studies document effects of elevated intracellular polyamine levels on histone acetylation in proliferating cells, suggesting a mechanism by which altered polyamine biosynthesis contributes to aberrant expression of genes, facilitating tumor growth. In addition, these studies may have implications for the development of drugs designed to regulate enzymes that modify the acetylation status of histones. J. Cell.Biochem. 77:345–360, 2000. © 2000 Wiley-Liss, Inc.

Translational Trickery and Polyamines

MOLECULAR BIOLOGY Polyamines are essential for the proliferation of eukaryotic cells. Their intracellular concentrations are regulated through the cell cycle and largely determined by the level of ornithine decarboxylase (ODC), the rate-limiting synthetic enzyme. Pyronnet et al. show that mammalian ODC messenger RNA contains a cap-independent internal ribosome entry site that functions exclusively at the G2/M phase of the cell cycle, a time when most protein synthesis is inhibited, thereby ensuring production of the polyamines needed for mitosis. Ivanov et al. investigate a polyamine-enhanced ribosomal frameshifting event that occurs during translation of the mRNA for antizyme 1, a protein that targets ODC for degradation. They show that this programmed frameshifting is conserved from fission yeast to mammals, suggesting that it plays a fundamental role in cell physiology. In a separate study in mice, Ivanov et al. find a new antizyme paralog that is selectively expressed in testis germ cells, suggesting an involvement of this regulatory system in spermatogenesis.— PAK Mol. Cell 5 , 607 (2000); EMBO J. 19 , 1907 (2000); Proc. Natl. Acad. Sci. U.S.A. 97 , 4808 (2000).

High levels of intracellular polyamines promote histone acetyltransferase activity resulting in chromatin hyperacetylation

Polyamines stimulate expression of a variety of genes, including many implicated in cell proliferation. Indeed, aberrant expression of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis, plays a causal role in tumorigenesis. Gene activity is influenced by dynamic changes in acetylation of nucleosomal histones. Although polyamines influence the histone acetyltransferase and deacetylase activities in cell-free systems, their ability to modulate these enzymes in live cells has never been established. To examine the effects of elevated intracellular levels of ODC and polyamines on gene transcription and histone acetylation, cells were infected with a retrovirus containing a cDNA for ODC. ODC overexpression potentiated the stimulatory effects of histone deacetylase inhibitors on reporter gene expression beyond that promoted by ODC or inhibitor treatment alone. Indeed, elevated intracellular levels of ODC promoted hyperacetylation of histones in several epidermal and fibroblast cell types. The ODC-mediated increase in acetylated histones was abrogated when cells were treated with alpha-difluoromethylornithine, a specific inhibitor of ODC activity, implying a distinct role for polyamines. Specifically, polyamines were found to enhance the action of histone acetyltransferases either directly or indirectly. Our studies document effects of elevated intracellular polyamine levels on histone acetylation in proliferating cells, suggesting a mechanism by which altered polyamine biosynthesis contributes to aberrant expression of genes, facilitating tumor growth. In addition, these studies may have implications for the development of drugs designed to regulate enzymes that modify the acetylation status of histones.

Polyamine depletion up-regulates c-Myc expression, yet induces G(1) arrest and terminal differentiation of F9 teratocarcinoma stem cells.

The ornithine decarboxylase (ODC) gene is a transcriptional target of c-Myc. Exponentially growing cells usually exhibit high c-Myc levels and high ODC levels, whereas stationary phase cells and terminally differentiated cells have low levels of both proteins. Therefore, we were surprised to find that when F9 teratocarcinoma stem cells were blocked in the G(1) phase of their cell cycle and induced to differentiate by irreversible inhibition of the ODC activity, the expression of c-Myc was up-regulated instead of being down-regulated. During the course of differentiation, the c-myc gene was constitutively expressed, and c-Myc protein accumulated. In transfection experiments, using ODC promoter-reporter gene fusion constructs, the accumulation of c-Myc protein, resulting from polyamine depletion, led to increased reporter gene expression. This finding is consistent with the view that depletion of polyamines relieves the suppression that they exert on c-myc mRNA translation, causing an accumulation of c-Myc protein, which in turn transactivates its target gene, the bona fide ODC gene. Thus, the accumulation of an active c-Myc protein does not preclude differentiative events, nor does it override the growth arrest caused by polyamine depletion. These results suggest a new role for polyamines-as negative regulators of c-Myc expression.

Changes in ornithine decarboxylase activity and putrescine concentrations after spinal cord compression injury in the rat

Traumatic spinal cord injury results in direct physical damage to structures and the generation of local factors contributing to secondary pathogenesis. In the present study, we investigated changes in polyamine metabolism after spinal cord compression injury in the rat. This is a stress induced metabolic pathway, of which an activation may indicate both, secondary pathogenesis or induction of neuroprotective response. Ornithine decarboxylase (ODC) activity, the rate limiting step of polyamine synthesis, and levels of the diamine putrescine, the product of ornithine decarboxylase reaction, were analyzed in control (non-laminectomized) animals and at 2 and 4 h after laminectomy or compression injury at the L4 segmental level. ODC activity was significantly increased 4 h after laminectomy in L4 and in adjacent L3 and L5 segments and compression to L4 produced a further increase 4 h after injury as compared with the intact control group. Putrescine levels were likewise significantly elevated to the same extend in the laminectomized and injured cord as compared with the intact control group. These findings demonstrate increased ODC and putrescine levels in the laminectomized and traumatized spinal cord and suggest that laminectomy may be an important `priming event' that contributes to secondary injury after spinal cord compression injury.

Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation.

Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation Cell proliferation and transformation induced by growth factor stimulation or by carcinogens, viruses, or oncogenes are characterized by an associated increase in polyamine levels, which is mediated by increased polyamine biosynthesis and enhanced uptake of polyamines. Polyamine biosynthesis is catalyzed particularly, in the level of ornithine decarboxylase (ODC). The elevation of cellular polyamine levels on the other hand accelerates the induction of ornithine decarboxylase antizyme (antizyme), which is involved not only in ODC-degradation, but in the negative regulation of polyamine transport. Taking advantage of these characteristics of antizyme, the potential of antizyme as a factor having anti-cell growth and anti-tumor activity was investigated. We show that antizyme can induce cell death associated with a rapid decline of intracellular polyamine contents. The possible anti-tumor activities of ectopically expressed antizyme were tested in p21H-ras (Val 12)-transformed NIH3T3 cells and several human malignant cell lines including a line with loss of p53 expression, and they were shown to be as sensitive as nontransformed NIH3T3 cells in vitro. The in vivo anti-tumor activity was also tested using nude mice inoculated with H-ras transformed NIH3T3 cells that had been transfected with inducible antizyme expression vector and the results showed that antizyme expression in vivo blocks tumor formation in these mice. These results suggest that ectopic antizyme expression is of possible therapeutic benefit in the treatment of cancer, which is mediated by ODC inactivation and intracellular polyamine depletion.

Differential effect on polyamine metabolism in mitogen- and superantigen-activated human T-cells

Polyamines are important for regulation of lymphocyte differentiation and proliferation. Mitogens induce synthesis of ornithine decarboxylase (ODC), the rate limiting enzyme in polyamine biosynthesis. Since mitogens stimulate T-cells by non-physiological routes, the role of polyamine metabolism in T-cell receptor (TCR)-mediated T-cell activation has not been adequately evaluated. The effect of phytohemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) on T-cell ODC and polyamine synthesis was compared. ODC activity was 6–11-fold higher in PHA compared to SEB stimulated T-cells. These differences were not attributed to differences in the magnitude of T-cell proliferation. Kinetics of ODC and polyamine synthesis were also different in PHA- and SEB-stimulated T-cells. In PHA-stimulated cells ODC levels and the induction of putrescine and spermidine synthesis peaked 6 h prior to peak IL-2 production, while in SEB-stimulated cells, ODC levels and polyamine synthesis peaked 6–12 h after IL-2 production. Differences in the temporal relationship between IL-2 production and polyamine induction in mitogen- versus superantigen-stimulated cells may account for the significant inhibition of the proliferative response by α-difluoromethylornithine following PHA but not SEB stimulation. Polyamine metabolism is regulated differently in T-cells stimulated via TCR engagement than with polyclonal mitogens.

Co-operation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin.

Ornithine decarboxylase (ODC) is aberrantly regulated in tumor cells and results in high basal levels of ODC and polyamines in many epithelial tumors. To determine if elevated ODC/polyamine levels can co-operate with a mutant Ha-ras gene in mouse skin tumorigenesis, double transgenic mice were generated by breeding K6/ODC transgenic mice with TG.AC v-Ha-ras transgenic mice. A K6 keratin promoter drives the ODC transgene in K6/ ODC transgenic mice, which results in elevated ODC/ polyamine levels directed to the outer root sheath cells of hair follicles. TG.AC transgenic mice carry a v-Ha-ras transgene while still retaining two normal c-Ha-ras alleles. Transgenic mice that possess only the K6/ODC or the v-Ha-ras transgene did not develop tumors unless treated with either a carcinogen or a tumor promoter, respectively. However, a high percentage of double transgenic mice possessing both the K6/ODC and v-Ha-ras transgenes developed spontaneous tumors. All tumors were well-differentiated keratoacanthomas, some of which progressed to carcinomas within 2 months. The development and the maintenance of these ODC/ras tumors was ODC-dependent since alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the formation and caused the regression of these tumors. These findings indicate that ODC overexpression and an activated Ha-ras are sufficient to produce a high rate of malignant transformation in an animal model. The ODC/ras double transgenic mouse provides a simple in vivo model without the use of chemical carcinogens or tumor promoters in which to test downstream effectors that play a key role in mediating the development of epithelial tumors resulting from the cooperation between ODC and v-Ha-ras.

Effect of Inhibitors of Enzymes Involved in Polyamine Biosynthesis Pathway on Pregnancy in Mouse and Hamster

The syntheses of polyamines, which play a definitive role in cell proliferation and tissue growth during early embryogenesis, are believed to be principally regulated by two enzymes: ornithine decarboxylase (ODC) and polyamine oxidase (PAO). However, when the level of enzyme ODC goes down, another enzyme known as S-adenosyl methyl decarboxylase (SAMDC) is believed to up-regulate the biosynthetic pathway. In the present study, the effects of inhibitors of enzymes SAMDC and PAO were determined in mated hamster and mouse in peri-and immediate postimplantation stages of pregnancy. It was noticed that, whereas the SAMDC inhibitor (MDL-73815) failed to arrest the pregnancy in the hamster in spite of raising the dose to 100 mg/kg, the PAO inhibitor (MDL-72527DA) was found to be 100% effective at 75 mg/kg in both species. The results confirmed the role of enzyme PAO in promoting early development of the embryo.