Ornithine Decarboxylase Gene Expression Research Articles

Effect of parathyroid hormone related protein, and dihydrotestosterone on proliferation and ornithine decarboxylase mRNA in human prostate cancer cell lines.

Parathyroid hormone related protein (PTHrP) has been identified as the major hormone responsible for the syndrome of humoral hypercalcemia of malignancy (HHM). Recent studies have shown that a large number of prostate tumors demonstrate the presence of PTHrP despite the fact that prostate cancer is rarely associated with the HHM syndrome. Other studies have indicated that PTHrP behaves as an early response gene, which stimulates ornithine decarboxylase (ODC) enzyme activity, an enzyme, involved in the biosynthesis of polyamines. It is therefore possible that PTHrP regulates prostate tumor cell proliferation via ODC gene expression. In the present study, we evaluated the effects of PTHrP and/or dihydrotestosterone (DHT) treatment on DNA synthesis by thymidine incorporation in androgen-dependent (LnCaP) and androgen-independent (PC3) human prostate adenocarcinoma cell lines. In addition, we utilized Northern blot analysis to investigate the effect of PTHrP [1-34] alone or in combination with DHT on ODC mRNA. PTHrP [1-34] treatment resulted in an increase in thymidine uptake in PC3 cells by 50%, whereas no such increase was seen in LnCaP cells. However, in the LnCaP cells, in the presence of DHT, PTHrP stimulated DNA synthesis to a level greater than that seen with DHT alone. DHT (10 nM) treatment resulted in an induction of PTHrP as well as ODC mRNAs in the androgen-dependent (LnCaP) but not in androgen-independent (PC3) cell line. PTHrP [1-34] treatment resulted in induction of ODC mRNA in the LnCaP cells. Addition of DHT resulted in a further increase in the ODC mRNA expression. These data suggest that PTHrP may play a role in prostate cancer cell proliferation and the increased ODC gene expression may be one possible mechanisms responsible for this phenomenon.

Transformation by H-ras can result in aberrant regulation of ornithine decarboxylase gene expression by transforming growth factor-?1

Inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation and occasionally, tumour cell proliferation is actually stimulated by transforming growth factor beta(1) (TGF-beta(1)). The present study demonstrates a novel link between alterations in TGF-beta(1) regulation during cellular transformation and malignant conversion and the expression of ornithine decarboxylase (ODC) which is a key rate limiting activity in the biosynthesis of polyamines and which is an enzyme that plays an important role in cell growth and differentiation. H-ras transformed mouse 10T(1/2) cell lines of varying degrees of malignant potential were examined for possible TGF-beta(1)-mediated alterations in ODC expression. Selective induction of ODC gene expression occurred. This induction was dependent upon the cellular phenotype expressed and the mechanisms responsible for the regulation of the TGF-beta(1)-mediated alterations in ODC expression varied as a function of malignant potential. The TGF-beta(1)-mediated alterations in ODC gene expression involves de novo protein synthesis, transcriptional, and post-transcriptional events. Evidence is also presented to suggest a possible role for protein kinase C-mediated events, protein phosphatases, and G-protein-coupled events in the TGF-beta(1)-mediated regulation of ODC expression in H-ras transformed cells. Evidence is also presented to suggest a possible role for cellular polyamines in the TGF-beta(1)-mediated alterations in ODC expression in H-ras transformed cells. Additionally, alterations in cellular polyamines were shown to influence TGF-beta(1) gene expression in H-ras transformed cells and that these alterations occurred, in part, through post-transcriptional events. The TGF-beta(1)-mediated regulation of ODC expression in H-ras transformed cells of varying degrees of malignant potential appears to be complex, multifaceted, and interactive. This study illustrates the importance of TGF-beta(1)-mediated regulation of ODC expression as a result of H-ras mediated cellular transformation and malignant progression, and further suggests that this TGF-beta(1)-mediated regulation constitutes an integral part of an altered growth regulatory program.

ODC mRNA as a prognostic factor for predicting recurrence in meningiomas.

In proliferating neoplastic cells, activity of the enzyme ornithine decarboxylase (ODC) increases. Among other brain tumors, ODC activation could also be observed in meningiomas. In the present study, we have investigated ODC gene expression in primary and recurrent meningiomas at the transcriptional level. ODC mRNA (messenger ribonucleic acid), ODC activity, number of mitoses, and Ki-67 index as a marker for nuclear proliferation were quantified in three different groups of meningiomas: tumors without recurrence in a 8.4 years median follow-up period, tumors with recurrence within a median follow-up of 3.0 years, and their corresponding recurrent tumors. ODC mRNA level was significantly higher in meningiomas with later recurrence as compared to meningiomas without recurrence (p < or = 0.01), whereas it declined in the recurrences of the second group (p < or = 0.001). In contrast, ODC activity showed no difference between the two groups of primary tumors, but a significant increase of enzyme activity could be observed in the recurrences as compared to the correponding primary tumors (p < or = 0.001). Likewise, an increase of the Ki-67 index could be detected in the recurrent group (p < or = 0.001). These results suggest that ODC mRNA may represent a prognostic factor for predicting recurrence in meningiomas.

Spermine Oxidation Leads to Necrosis with Plasma Membrane Phosphatidylserine Redistribution in Mouse Leukemia Cells

Oxidation by copper/quinone-containing serum amine oxidases (SAO) is a well-known cause of polyamine cytotoxicity. Spermine oxidation exerts potent immunosuppressive effects in animal cells, but the cell death mechanism involved remains unclear. We compared biochemical and morphological parameters of SAO-mediated cell death in L1210 mouse leukemia cells with normal or amplified ornithine decarboxylase gene expression with those observed during apoptosis induced by deregulated polyamine uptake or by okadaic acid. None of the characteristic features of apoptotic cell death (e.g., chromatin condensation, nuclear fragmentation, internucleosomal DNA cleavage, poly(ADP-ribose) polymerase cleavage) were observed during spermine oxidation-mediated cell death, which was clearly necrotic by morphological criteria. Inhibition of a wide spectrum of caspases did not prevent SAO-dependent cell death, whereas N-acetylcysteine completely abolished the cytotoxic effects of spermine oxidation. Catalase only delayed spermine oxidation-induced cell death without affecting its modality or preventing depletion of intracellular glutathione, suggesting that both H2O2 and aminoaldehydes generated by SAO-mediated spermine oxidation contribute to SAO-induced necrosis. Interestingly, redistribution of phosphatidylserine to the outer leaflet of the plasma membrane, usually a diagnostic feature of apoptosis, preceded necrotic cytolysis triggered by spermine oxidation. Thus, L1210 cell death caused by SAO-mediated spermine oxidation has all the attributes of primary necrosis, but is also accompanied by loss of phospholipid asymmetry, indicating that the latter phenomenon may not be unique to apoptosis. Phosphatidylserine exposure, a potent engulfment signal for phagocytes, might contribute to the immunosuppressive effects of plasma polyamines through a controlled and rapid necrotic process involving SAO.

Sequence, expression and functional analysis of the Coccidioides immitis ODC (ornithine decarboxylase) gene

The ornithine decarboxylase ( ODC) gene of the human respiratory fungal pathogen, Coccidioides immitis ( Ci) was cloned, sequenced, chromosome-mapped, and expressed in Escherichia coli ( Ec). The genomic, cDNA and translated sequences are presented. Transformation of an ODC null mutant strain of Ec (EWH 319) with the Ci ODC gene was conducted to confirm function of the protein encoded by the fungal gene. Activity of the enzyme by the bacterial transformant was inhibited by 1, 4-diamino-2-butanone (DAB), a known inhibitor of eukaryotic ODC. Temporal expression of the Ci ODC gene during the parasitic cell cycle is constitutive, based on results of RT–PCR. However, results of enzyme activity assays of cell homogenates obtained at different stages of parasitic cell development in vitro showed that the functional protein is present only during periods of isotropic growth and segmentation, and these morphogenetic events can be arrested by the addition of DAB. The observed absence of a difference in steady-state mRNA transcript amounts, and the developmentally correlated variation in levels of enzyme activity, suggest a translational or post-translational mechanism of ODC regulation. Since no PEST sequence was detected in the Ci ODC, enzyme regulation by programmed protein degradation as reported for many other eukaryotic ODCs may not occur in this case. ODC activity appears to play a key role in the morphogenesis of Ci, and the enzyme could be a rational target for therapy of disseminated coccidioidomycosis.

Ornithine decarboxylase gene expression in Castleman's disease.

Castleman's disease (CD) is a rare atypical lymphoproliferative disorder that is clinically and histologically heterogeneous and is associated with the risk of developing malignant lymphoma. Based on pathological findings CD is divided into two types: a localized form and a multicentric form. The clinical course differs in these two forms. We examined the molecular mechanisms that lie between benign and malignant disease, evaluating a possible implication of oncogenes in the pathogenesis. Since deregulated expression of the gene for ornithine decarboxylase (ODC) has been observed in a variety of human malignancies, we compared ODC expression between the localized and multicentric forms. Using northern blot analysis we found that ODC gene expression clearly differs between the localized and multicentric forms. The findings in this report indicate that the variable pattern of ODC gene expression in the different types of CD could be useful for examining the evolution of this disease.

Tumor promoter-induced ornithine decarboxylase gene expression occurs independently of AP-1 activation

Activator protein 1 (AP-1) transactivation and ornithine decarboxylase (ODC) activity have been established as essential downstream effectors of mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Previous studies have shown that inhibition of either AP-1 transactivation or ODC activity suppressed tumor promoter-induced transformation. By utilizing the JB6 mouse epidermal cell system, the present study determined whether TPA-induced ODC gene expression and activity is independent of AP-1 transactivation. In three independent JB6 (P+) clones, stably expressing dominant negative c-jun, TPA-induced ODC gene expression and activity were similar compared to JB6 P+ cells expressing vector-control alone, while AP-1-dependent transcription was inhibited. Transformation-insensitive JB6 (P-) cells, which lack TPA-inducible c-jun expression, also exhibited similar induction of ODC activity by TPA. alpha-Difluoromethylornithine, an irreversible inhibitor of ODC, attenuated, at an equivalent IC50, both TPA-induced ODC activity and anchorage-independent growth of JB6 P+ cells, despite no inhibition of AP-1 transactivation. Taken together, the results presented indicate that TPA-induced ODC gene expression and activity are independent of AP-1 transactivation. Because inhibition of either AP-1 or ODC precludes TPA-induced transformation, and because ODC is independent of AP-1, we propose that there are at least two pathways to transformation. Each pathway is required but not sufficient for transformation.

ZBP-99 Defines a Conserved Family of Transcription Factors and Regulates Ornithine Decarboxylase Gene Expression

Among transcription factors that regulate ornithine decarboxylase (ODC) gene expression are those that interact with GC-rich promoters, including Sp1 and ZBP-89. Sp1 functions as a transactivator and ZBP-89 as a transrepressor of both the ODC and gastrin promoters. This study reports the cloning and characterization of a second member of the ZBP family that also binds GC boxes. ZBP-99 contains four Krüppel-type zinc fingers that collectively share 91% amino acid sequence similarity and 79% sequence identity with those found in ZBP-89. In addition, there are highly conserved amino acid sequences in the carboxy-terminal segments of the two genes. In spite of their structural similarities, the two proteins are encoded at distinct loci, ZBP-89 on chromosome 3q21 and ZBP-99 on 1q32.1. The predicted open reading frame of ZBP-99 cDNA encodes a 99-kDa protein. Electrophoretic mobility shift assays showed that ZBP-99 protein specifically binds to the GC-rich promoter elements of gastrin and ODC genes. Northern blot analysis showed that a major ZBP-99 transcript of 5.6 kb is expressed ubiquitously at low levels, with elevated expression levels in placenta and in adult kidney, liver, and lymphocytes. Cotransfection of AGS gastric adenocarcinoma and HT-29 colon adenocarcinoma cells with a ZBP-99 expression construct and with an ODC reporter construct show that ZBP-99 repressed basal expression in the two cell lines by 80 and 60%, respectively. Collectively, the data suggest that ZBP-99 binds GC-rich promoters and may complement the activities mediated by ZBP-89.

Catecholamines participate in the induction of ornithine decarboxylase gene expression in normal and hyperplastic mouse kidney

In the quinazoline antifolate (CB 3717)-induced hyperplastic kidney model, a remarkable increase of ornithine decarboxylase (ODC) activity was paralleled by a smaller, but highly significant augmentation of the ODC transcript level. Catecholamine depletion, evoked by reserpine, strongly impaired antifolate-induced ODC expression; the enzyme activity was almost completely abolished while the mRNA level decreased by 60%. Moreover, under conditions of a depleted catecholamine pool, kidney enlargement was significantly reduced confirming our earlier reports on the indispensability of ODC induction for renal hyperplasia (M. Manteuffel-Cymborowska et al., Biochim. Biophys. Acta, 1182 (1993) 133–141 [1]). In normal mouse kidney catecholamines appeared to be inducers of ODC expression. Use of selective agonists of catecholamine receptors demonstrated the importance of dopamine D2 receptors, and to a lower extent β adrenoreceptors, in the catecholamine mediation of induction of ODC activity and of ODC mRNA levels.These increases were not abolished by an antiandrogen, casodex, suggesting that catecholamine control of ODC expression is an androgen receptor-independent process. The results obtained point to the critical role of renal catecholamines; these biogenic amines are not only involved in the regulation of ODC expression in normal kidney but are also required for the induction of ODC in hyperplastic kidney evoked by antifolate and, as shown recently (M. Manteuffel-Cymborowska et al., Biochim. Biophys. Acta, 1356 (1997) 292–298 [2]), in testosterone-induced hypertrophic kidney.

Interaction of asparagine and EGF in the regulation of ornithine decarboxylase in IEC-6 cells.

Our laboratory has shown that asparagine (ASN) stimulates both ornithine decarboxylase (ODC) activity and gene expression in an intestinal epithelial cell line (IEC-6). The effect of ASN is specific, and other A- and N-system amino acids are almost as effective as ASN when added alone. In the present study, epidermal growth factor (EGF) was unable to increase ODC activity in cells maintained in a salt-glucose solution (Earle's balanced salt solution). However, the addition of ASN (10 mM) in the presence of EGF (30 ng/ml) increased the activity of ODC 0.5- to 4-fold over that stimulated by ASN alone. EGF also showed induction of ODC with glutamine and alpha-aminoisobutyric acid, but ODC induction was maximum with ASN and EGF. Thus the mechanism of the interaction between ASN and EGF is important for understanding the regulation of ODC under physiological conditions. Therefore, we examined the expression of the ODC gene and those for several protooncogenes under the same conditions. Increased expression of the genes for c-Jun and c-Fos but not for ODC occurred with EGF alone. The addition of ASN did not further increase the expression of the protooncogenes, but the combination of EGF and ASN further increased the expression of ODC over that of ASN alone. Western analysis showed no significant difference in the level of ODC protein in Earle's balanced salt solution, ASN, EGF, or EGF plus ASN. Addition of cycloheximide during ASN and ASN plus EGF treatment completely inhibited ODC activity without affecting the level of ODC protein. These results indicated that 1) the increased expression of protooncogenes in response to EGF is independent of increases in ODC activity and 2) potentiation between EGF and ASN on ODC activity may not be due to increased gene transcription but to posttranslational regulation and the requirement of ongoing protein synthesis involving a specific factor dependent on ASN.

Glucocorticoid regulation of ornithine decarboxylase in the postnatal rat lung

Ornithine decarboxylase (ODC) is thought to play a critical role in pulmonary development. The purpose of this study was to characterize the effects of dexamethasone on ODC gene expression and enzyme activity in the lung of rat pups. Subcutaneous administration of dexamethasone (10 mg kg ) was shown to suppress ODC activity in 2-, 6- and 10-day-old rats for as long as 24 h after injection. In contrast, dexamethasone treatment stimulated liver ODC activity indicating that the inhibition of lung ODC is tissue specific. Contrary to expectation, the glucocorticoid enhanced lung ODC expression as indicated by an increased accumulation of ODC mRNA transcripts. The latter effect was associated with an heightened expression of c-myc and max mRNAs, the encoded proteins of which act as transactivators of the ODC gene. Dexamethasone did not alter lung levels of “antizyme” (AZ), an inducible protein that specifically promotes the degradation of the ODC protein enzyme. However, the lack of AZ induction does not necessarily mean that ODC degradation is not the mechanism for the decrease in lung ODC activity of dexamethasone-treated animals. The results obtained indicate that glucocorticoids can downregulate lung ODC activity, and that the effect is mediated by post-transcriptional rather than transcriptional mechanisms. These findings are consistent with the idea that endogenous glucocorticoids play an important role in the modulation of ODC activity and early pulmonary development.

Thrombin stimulates vascular smooth muscle cell polyamine synthesis by inducing cationic amino acid transporter and ornithine decarboxylase gene expression.

Thrombin, a serine protease, is a potent mitogen for vascular smooth muscle cells (SMCs), but its mechanism of action is not known. Since L-ornithine is metabolized to growth-stimulatory polyamines, we examined whether thrombin regulates the transcellular transport and metabolism of L-ornithine by vascular SMCs. Treatment of SMCs with thrombin initially (0 to 2 hours) decreased L-ornithine uptake, whereas longer exposures (6 to 24 hours) progressively increased transport. Kinetic studies indicated that thrombin-induced inhibition was associated with a decrease in affinity for L-ornithine, whereas stimulation was mediated by an increase in transport capacity. Thrombin induced the expression of both cationic amino acid transporter (CAT)-1 and CAT-2 mRNA. Furthermore, thrombin stimulated L-ornithine metabolism by inducing ornithine decarboxylase (ODC) mRNA expression and activity. The stimulatory effect of thrombin on both L-ornithine transport and ODC activity was reversed by hirudin, a thrombin inhibitor, and was mimicked by a 14-amino acid thrombin receptor-activating peptide. Thrombin also markedly increased the capacity of SMCs to generate putrescine, a polyamine, from extracellular L-ornithine. The thrombin-mediated increase in putrescine production was reversed by N(G)-methyl-L-arginine, a competitive inhibitor of cationic amino acid transport, or by alpha-difluoromethylornithine (DFMO), an ODC inhibitor. DFMO also inhibited thrombin-induced SMC proliferation. These results demonstrate that thrombin stimulates polyamine synthesis by inducing CAT and ODC gene expression and that thrombin-stimulated SMC proliferation is dependent on polyamine formation. The ability of thrombin to upregulate L-ornithine transport and direct its metabolism to growth-stimulatory polyamines may contribute to postangioplasty restenosis and atherosclerotic lesion formation.

Heparin inhibits phorbol ester-induced ornithine decarboxylase gene expression in endothelial cells

Glycosaminoglycans regulate angiogenesis by affecting the availability of different growth factors for the endothelial cell (EC). However, little is known about the molecular and functional consequences resulting from direct interaction of these polyelectrolytes with the EC. Here we show that heparin markedly inhibited serum-stimulated DNA synthesis and ornithine decarboxylase (ODC) mRNA expression in human endothelial cells (HEC). About 50% of the serum effect on DNA synthesis and ODC gene expression was prevented by the selective protein kinase C (PKC) inhibitor chelerythrine or by PKC down-regulation. Heparin was ineffective in counteracting that part of the effect of serum that was resistant to PKC inhibition or down-regulation. In serum-free cultured HEC, heparin completely abolished the increase in DNA synthesis and ODC mRNA expression elicited by a number of PKC activators. Cell exposure to difluoromethylornithine, an irreversible inhibitor of ODC enzyme, dramatically antagonised both serum- and phorbol 12-myristate 13-acetate (PMA)-stimulated DNA synthesis. These results suggest that inhibition of PKC-mediated ODC gene expression by glycosaminoglycans may represent an important mechanism in the regulation of HEC proliferation.

Dietary lipid modulation of Na +/glucose co-transporter (SGLT1), [formula omitted] ATPase, and ornithine decarboxylase gene expression in the rat small intestine in diabetes mellitus

Nutrient transport is increased in streptozotocin-induced diabetes mellitus (DM) in rats. Variations in dietary lipid composition have been shown to modulate nutrient transport during intestinal adaptation in DM. Here, we examined Na +/glucose cotransporter (SGLT1), Na +,K +-ATPase and ornithine decarboxylase (ODC) gene expression in the small intestine in DM. One week after the animals had been given streptozotocin or injected with vehicle, they were randomly allocated to receive either standard chow or semisynthetic isocaloric diets with triglycerides enriched with polyunsaturated fatty acid (PUFA) or saturated fatty acid (SFA). Two weeks later they were killed and brush border membrane (BBM) and basolateral membrane (BLM) fractions and RNA were isolated from the mucosa of the proximal and distal small intestine. Western and Northern blotting were performed with antibodies and cDNA probes specific to SGLT1, the Na +,K +-ATPase α 1 and β 1 subunit isoforms, and ODC. Increased levels of immunodetectable SGLT1, Na +,K +-ATPase α 1 and β 1 subunit, and ODC expression and their corresponding mRNAs were observed in DM versus control rats fed chow. All of these parameters were increased in diabetic and in nondiabetic rats fed SFA as compared with PUFA. These findings suggest that the increased glucose uptake in diabetes, and in response to feeding a saturated as compared with a polyunsaturated diet, is achieved by transcriptional events that modulate transport function and cell proliferation.

180 Elevated expression of ornithine decarboxylase gene expression in scleroderma epidermis

180 Elevated expression of ornithine decarboxylase gene expression in scleroderma epidermis

Catecholamines are required for androgen-induced ODC expression but not for hypertrophy of mouse kidney

Catecholamine depletion, evoked by reserpine, dramatically impaired (5-fold) the testosterone-induced increase of ornithine decarboxylase (ODC) activity in female mouse kidney. However, reserpine did not prevent kidney hypertrophy evoked by testosterone. This is evidenced by the activity of sensitive, biochemical markers of renal hypertrophy, namely arginase and ornithine aminotransferase (OAT), that responded with the increase and decrease of activities to testosterone treatment, respectively [1]. Arginine and ornithine, substrates and/or products of marker enzymes, showed a striking homeostasis as their level was not affected by testosterone and reserpine, and only slightly by DFMO. Northern blot analysis revealed that the ODC mRNA level, that was increased 10-fold by testosterone, was decreased 2-fold in catecholamine-depleted hypertrophic kidney. Thus, ODC transcript level, lowered by reserpine, correlated partially with an attenuated response of ODC activity to testosterone. This was in contrast to DFMO, which inhibited ODC activity, but significantly increased its mRNA content. It is concluded that catecholamines could be involved together with testosterone in regulation of the ODC gene expression in mouse kidney. © 1997 Elsevier Science B.V. All rights reserved.

Effect of thyroxine on pancreatic digestive enzymes and ornithine decarboxylase gene expression in neonatal rats.

Thyroxine has been shown to play a role in the development of exocrine pancreatic enzymes in neonatal rats. To further evaluate the regulatory mechanisms for thyroxine in pancreatic development, we examined the changes in the expression of pancreatic enzymes (amylase and trypsinogen) and ornithine decarboxylase (ODC) genes following daily injection of thyroxine for 5 and 10 days to neonatal rats (5 days old). Total pancreatic proteins and DNA contents as well as the activity of ODC and exocrine enzymes were significantly increased after 5 and 10 days of thyroxine treatment. These increases were associated with parallel alterations (to three to fourfold rise) in steady-state mRNA levels of both amylase and trypsinogen. In contrast, thyroxine only produced a 57-68% increase in steady-state ODC mRNA levels. These data suggest that thyroxine stimulated the express of amylase and trypsinogen genes partly due to increased transcriptional rate and/ or decreased mRNA turnover. Thyroxine also stimulated ODC gene expression. However, the stimulatory mechanisms may involve transnational or posttranslational regulation of ODC and are independent of thyroxine effects.

Expression of the ornithine decarboxylase gene in response to asparagine in intestinal epithelial cells.

Refeeding fasted rats significantly stimulates mucosal growth and ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, but the exact mechanism responsible for induction of ODC at the molecular level is unknown. Of normal dietary constituents, the amino acid asparagine markedly increases ODC activity and mucosal growth when administered intragastrically. The current study examined the expression of the ODC gene in IEC-6 cells (a line of normal rat small intestinal crypt cells) after exposure to asparagine. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum. They were deprived of serum for 24 h before experiments. Exposure to asparagine at the dose of 10 mM resulted in the rapid increase in ODC mRNA levels. The increased expression of the ODC gene began 1 h after and peaked between 3 and 5 h after treatment with asparagine. Maximum increases in ODC mRNA levels were approximately fivefold the normal value. Increased levels of ODC mRNA in cells exposed to asparagine were paralleled by increases in ODC protein and enzyme activity and cellular polyamine levels. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to > 2 h in cells exposed to 10 mM asparagine. The half-life of ODC activity also was increased in asparagine-treated cells. When cellular protein synthesis was inhibited by cycloheximide, asparagine superinduced ODC mRNA levels. Furthermore, asparagine also significantly stimulated DNA synthesis in IEC-6 cells. These results indicate that 1) asparagine stimulates ODC in IEC-6 cells through multiple pathways and 2) increased ODC mRNA levels result partly from a delay in the rate of degradation. These findings suggest that luminal amino acids stimulate gut mucosal growth in association with their ability to regulate ODC gene expression.

Induction of immediate-early, ornithine decarboxylase and antizyme gene expression in the rat small intestine after transient ischaemia

The expression of the immediate early genes (IEG)s c- fos, c- jum and zif/7268, and the genes coding for ornithine decarboxylase (ODC) and its regulatory protein antizyme (AZ), was studied in rat small intestine following transient ischemia. The ischemic stimulus for 10 min alone did not alter the expression of these genes. A rapid and transitory induction of all IEG mRNAs occurred in a coordinated manner peaking at 30 min following recirculation and returned to basal levels 3 hr after recirculation. Protein products of the IEGs accumulated in the smooth muscle layer of the intestine by 2–3 hr after recirculation. Expression of both ODC and AZ mRNAs initially decreased to 70% of control levels 1 hr after recirculation but markedly increased at 2 to 4 hr after recirculation. The functional significance of these changes in gene expression in relation to tissue integrity and function after the ischaemia/reperfusion is discussed.

Androgen induces ornithine decarboxylase gene expression in colonic cell line HT-29.

Carcinoma of the colon is the second most common cancer among men and women combined in the United States. Ornithine decarboxylase (ODC) is the first and key regulatory enzyme in the polyamine biosynthesis pathway and is regulated by various factors. Polyamines are believed to participate in cellular proliferation and differentiation. High levels of polyamines and ODC activity are associated with rapid cell growth, particularly in tumor tissues. Regulation of this enzyme in vivo has important clinical implications. In the present study, we used Northern analysis and mobility shift assay to investigate whether ODC gene expression is regulated by androgens in the three human colonic cell lines, SW620, HT-29, and Caco-2. Cell lines were maintained in Dulbecco's Modified Eagle's medium/F12 supplemented with 5 percent fetal bovine serum. At 60 percent confluency, medium was replaced with steroid-depleted medium, and incubation continued for 24 hours. Following this period, medium was replaced with fresh steroid-free medium containing 1 nM dihydrotestosterone. Dihydrotestosterone stimulated ODC messenger ribonucleic acid expression only in HT-29 colonic cell line. Studies using electrophoretic mobility shift assays of nuclear extracts also showed a binding pattern with SP1 and NF-kappaB regulatory sequences only in testosterone-treated HT-29 cells. These results suggest that androgens may play an important role in the growth of HT-29 colonic cell lines.