Regulation Of Ornithine Decarboxylase Activity Research Articles

Downregulation of MTAP promotes Tumor Growth and Metastasis by regulating ODC Activity in Breast Cancer.

5'-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway and has been reported to suppress tumorigenesis. The MTAP gene is located at 9p21, a chromosome region often deleted in breast cancer (BC). However, the clinical and biological significance of MTAP in BC is still unclear. Here, we reported that MTAP was frequently downregulated in 41% (35/85) of primary BCs and 89% (8/9) of BC cell lines. Low expression of MTAP was significantly correlated with a poor survival of BC patients (P=0.0334). Functional studies showed that MTAP was able to suppress both in vitro and in vivo tumorigenic ability of BC cells, including migration, invasion, angiogenesis, tumor growth and metastasis in nude mice with orthotopic xenograft tumor of BC. Mechanistically, we found that downregulation of MTAP could increase the polyamine levels by activating ornithine decarboxylase (ODC). By treating the MTAP-repressing BC cells with specific ODC inhibitor Difluoromethylornithine (DFMO) or treating the MTAP-overexpressing BC cells with additional putrescine, metastasis-promoting or -suppressing phenotype of these MTAP-manipulated cells was significantly reversed, respectively. Taken together, our data suggested that MTAP has a critical metastasis-suppressive role by tightly regulating ODC activity in BC cells, which may serve as a prominent novel therapeutic target for advanced breast cancer treatment.

Role of ornithine decarboxylase antizyme inhibitor in vivo

Ornithine decarboxylase (ODC) antizyme inhibitor (AZI) has been shown to regulate ODC activity in cell cultures. However, its biological functions in an organism remain unknown. An embryonic stem (ES) cell clone was established, in which the Azin1 gene was disrupted by the gene trap technique. To identify the function of Azin1 gene in vivo, a mutant mouse line was generated using these trapped ES cells. Homozygous mutant mice died at P0 with abnormal liver morphology. Further analysis indicated that the deletion of Azin1 in homozygous mice resulted in the degradation of ODC, and reduced the biosynthesis of putrescine and spermidine. Our results thus show that AZI plays an important role in regulating the levels of ODC, putrescine and spermidine in mice, and is essential for the survival of mice.

Cell-cycle-dependent regulation of ornithine decarboxylase activity in the unicellular green alga Chlamydomonas reinhardtii

Polyamines play an important role in the control of cell growth and cell division. In the unicellular green alga Chlamydomonas reinhardtii as in animal cells, biosynthesis of the 3 commonly occurring polyamines (putrescine, spermidine and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. Therefore, we have investigated the regulation of ODC activity during the cell cycle of Clamydomonas reinhardtii using synchronized cultures. A 2.5-3-fold increase in ODC activity was observed during the transition to the cell division phase. This up-regulation of ODC activity was not due to an increased level of ODC-mRNA as revealed by northern-blot analyses, but correlated with an increased half-life of this particular enzyme (from 1.1 to 3.2 h). Addition of the DNA topoisomerase II inhibitor nalidixic acid during the second half of the growth period caused a transient decrease of ODC activity and a considerable delay of cell divisions. After cell division, a down-regulation of ODC activity was observed which was faster in the dark than in the light and also correlated with changes of the ODC half-life.

In vitro and in vivo effects of the conformationally restricted polyamine analogue CGC-11047 on small cell and non-small cell lung cancer cells

Polyamines are essential for normal growth; however, the requirement for, and the metabolism of, polyamines are frequently dysregulated in cancer. Polyamine analogues have demonstrated promising preclinical results in multiple model systems of cancer, but their clinical utility has been limited by apparent toxicity. A representative compound of a new generation of short chain, conformationally restricted polyamine analogues, CGC-11047 has been synthesized and ongoing phase I clinical trials indicate it to be well tolerated at weekly doses of 610 mg (dose escalation is still in progress). Therefore, studies were designed to gain a better understanding of its effects on cellular polyamine biochemistry and efficacy in the treatment of human lung cancer models in vitro and in vivo. Human lung cancers cell lines representing non-small cell and small cell lung cancers were investigated for their growth and biochemical response to CGC-11047. Effects of in vitro treatment with CGC-11047 on cell growth, the activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC), and the expression and activity of the polyamine catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxides (SMO) were measured. Additionally, the overall effects on intracellular polyamine pools were monitored. Finally, the in vivo efficacy of CGC-11047 in the treatment of a nude mouse model of human non-small cell lung cancer was evaluated. CGC-11047 effectively inhibited the growth of both small cell and non-small cell lung cancer cells in vitro. The greatest biochemical effects were observed in the non-small cell lung cancer cells where in addition to a profound down regulation of ODC activity, there was a significant increase in polyamine catabolism leading to a greater degree of polyamine pool depletion and greater accumulation of CGC-11047 when compared with the changes observed for the small cell lines. Importantly, CGC-11047 was found to be highly significant (P < 0.0001) in delaying the progression of established tumors in an in vivo model of human non-small cell lung cancer. CGC-11047 represents a promising new polyamine analogue that warrants further preclinical and, potentially, clinical evaluation in lung cancer.

Stable siRNA-mediated silencing of antizyme inhibitor: regulation of ornithine decarboxylase activity

Ornithine decarboxylase (ODC) is the rate-limiting enzyme involved in the biosynthesis of polyamines essential for cell growth and differentiation. Aberrant upregulation of ODC, however, is widely believed to be a contributing factor in tumorigenesis. Antizyme is a major regulator of ODC, inhibiting ODC activity through the formation of complexes and facilitating degradation of ODC by the 26S proteasome. Moreover, the antizyme inhibitor (AZI) serves as another factor in regulating ODC, by binding to antizyme and releasing ODC from ODC-antizyme complexes. In our previous report, we observed elevated AZI expression in tumor specimens. Therefore, to evaluate the role of AZI in regulating ODC activity in tumors, we successfully down-regulated AZI expression using RNA interference technology in A549 lung cancer cells expressing high levels of AZI. Two AZI siRNAs, which were capable to generate a hairpin dsRNA loop targeting AZI, could successively decrease the expression of AZI. Using biological assays, antizyme activity increased in AZI-siRNA-transfected cells, and ODC levels and activity were reduced as well. Moreover, silencing AZI expression decreased intracellular polyamine levels, reduced cell proliferation, and prolonged population doubling time. Our results directly demonstrate that downregulation of AZI regulates ODC activity, intracellular polyamine levels, and cell growth through regulating antizyme activity. This study also suggests that highly expressed AZI may be partly responsible for increased ODC activity and cellular transformation.

Balsamodendron mukul suppresses benzoyl peroxide and ultraviolet light induced tumor promotional events in Swiss mice

The current study evaluates the efficacy of Balsamodendron mukul Hook ex. Stocks, an indigenous plant, in the modulation of benzoyl peroxide (BPO) treated and ultraviolet (UV) light irradiated tumor promotional events. BPO is a well-known tumor promoter while UV radiations are capable of acting as complete carcinogens. Treatment of the dorsal portions of the mice skins with BPO (20 mg/0.2 ml/animal) and subsequent UV irradiation (0.420 J/m 2/s) caused a significant depletion of reduced glutathione (GSH), it’s metabolizing and phase II enzymes ( p < 0.01). Also, down regulation of the activities of antioxidant enzymes and up regulation of ornithine decarboxylase activity and enhancement in the synthesis of DNA, malondialdehyde (MDA) and hydrogen peroxide (H 2O 2) was observed. However, topical pretreatment with the extract of B. mukul not only restored the content of GSH, MDA and H 2O 2 but it also recovered the activities of above-mentioned enzymes ( p < 0.05) and prevented synthesis of DNA ( p < 0.05) significantly. Although the lower dose was not very effective, the higher dose showed promising results against oxidative stress and hyperproliferative response of BPO and UVB radiations. From the present data we conclude that B. mukul is potentially strong enough to modulate the events of skin tumor promotion.

Overexpression of a dominant-negative ornithine decarboxylase in mouse skin: effect on enzyme activity and papilloma formation.

A transgenic mouse line expressing a truncated form of the ornithine decarboxylase (ODC) dominant-negative mutant K69A/C360A under the control of the keratin 6 promoter has been established (K6/ODCdn mice). These mice were backcrossed onto both the DBA/2J and C57BL/6J backgrounds for subsequent tumorigenesis experiments utilizing an initiation/promotion protocol. In short-term experiments, expression of the ODCdn protein product was induced in the epidermis within 24 h after application of the tumor promoter tetradecanoyl phorbol acetate (TPA) to the skin, and ODC activity in the epidermis of K6/ODCdn mice was reduced by at least 75% compared with littermate controls. However, in tumorigenesis experiments utilizing a variety of initiator (7,12-dimethylbenz[a]anthracene; DMBA) and promoter (TPA) concentrations, K6/ODCdn mice formed at least as many tumors as their littermate controls regardless of background strain. In experiments utilizing chrysarobin, a tumor promoter with a different mechanism of action than TPA, again there was no significant difference in tumor formation between K6/ODCdn mice and littermate controls. Similarly, when K6/ODCdn mice were crossed with K5/ODC mice, a transgenic line described previously which forms tumors without application of a promoting agent, double transgenic mice formed as many tumors as mice expressing the K5/ODC transgene alone. Analysis of epidermis following multiple TPA applications revealed a dramatic spike in ODC activity in both K6/ODCdn mice and non-transgenic mice after six applications, and western blot analysis suggested a stabilization of endogenous wild-type ODC in K6/ODCdn transgenic mice. ODC activity, endogenous protein and polyamines were also elevated in tumors from K6/ODCdn mice. The accumulation of endogenous ODC protein is most probably the result of competition from the transgene-derived ODCdn protein for binding of antizyme, which is known to regulate ODC activity by stimulating degradation of the ODC protein.

Proliferation of intestinal crypt cells by gastrin-induced ornithine decarboxylase.

To determine whether the gastrin stimulated intestinal crypt cell (IEC-6) proliferation by induction of ornithine decarboxylase (ODC). IEC-6 cells were grown in DMEM containing 50 mL x L(-1) dialyzed fetal bovine serum for 24h and then were treated with gastrin. The proliferative capability of the cells was monitored subsequently on d 1, 2, 3, and 4 after treatment with MTT assay at aborbance 570 nm.The cellular ODC mRNA expression, ODC activity, and putrescine content were examined by RT-PCR method, radiometric technique and high-performance liquid chromatography(HPLC) analysis respectively after 12h of treatment. On d1 after exposure of IEC-6 cells to pentagastrin, the proliferation increased initially and reached a peak on d3 at 250 microg x L(-1) concentration. Pentagastrin 500microg x L(-1) increased cell proliferation on day 1 and day 2, and then decreased. Compared with control group, pentagastrin 250 microg x L(-1) increased ODC mRNA level by 1.09-fold (P<0.05), ODC activity by 1.71-fold(P<0.01), and putrescine content 5.30-fold (P<0.01), respectively. Similarly, pentagastrin of 500 microg x L(-1) also increased ODC mRNA level by 1.16-fold (P<0.05), ODC activity 1.63-fold(P<0.05), and putrescine content 4.41-fold (P<0.01), respectively. But there was not significant difference between them. Gastrin is an agent which promotes IEC-6 cell proliferation involved in regulating ODC activity mechanism.

Nitric oxide inhibits ornithine decarboxylase via S-nitrosylation of cysteine 360 in the active site of the enzyme.

Ornithine decarboxylase is the initial and rate-limiting enzyme in the polyamine biosynthetic pathway. Polyamines are found in all mammalian cells and are required for cell growth. We previously demonstrated that N-hydroxyarginine and nitric oxide inhibit tumor cell proliferation by inhibiting arginase and ornithine decarboxylase, respectively, and, therefore, polyamine synthesis. In addition, we showed that nitric oxide inhibits purified ornithine decarboxylase by S-nitrosylation. Herein we provide evidence for the chemical mechanism by which nitric oxide and S-nitrosothiols react with cysteine residues in ornithine decarboxylase to form an S-nitrosothiol(s) on the protein. The diazeniumdiolate nitric oxide donor agent 1-diethyl-2-hydroxy-2-nitroso-hydrazine acts through an oxygen-dependent mechanism leading to formation of the nitrosating agents N(2)O(3) and/or N(2)O(4). S-Nitrosoglutathione inhibits ornithine decarboxylase by an oxygen-independent mechanism likely by S-transnitrosation. In addition, we provide evidence for the S-nitrosylation of 4 cysteine residues per ornithine decarboxylase monomer including cysteine 360, which is critical for enzyme activity. Finally S-nitrosylated ornithine decarboxylase was isolated from intact cells treated with nitric oxide, suggesting that nitric oxide may regulate ornithine decarboxylase activity by S-nitrosylation in vivo.

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells.

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.

Cell-cycle-dependent regulation of ornithine decarboxylase activity in the unicellular green alga Chlamydomonas reinhardtii

Cell-cycle-dependent regulation of ornithine decarboxylase activity in the unicellular green alga Chlamydomonas reinhardtii

NADH: ubiquinone oxidoreductase inhibitors block induction of ornithine decarboxylase activity in MCF-7 human breast cancer cells.

Rotenone is the classical inhibitor of NADH: ubiquinone oxidoreductase and its analogue deguelin is a potent inhibitor of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase mRNA steady state level and enzyme activity in mouse 308 cells (Gerhäuser et al. 1995). In MCF-7 human breast cancer cells, rotenone, deguelin and two structurally-unrelated miticides (pyridaben and fenazaquin) inhibit not only NADH: ubiquinone oxidoreductase but also induced ornithine decarboxylase activity with IC50 values of < 1 to 70 nM. Rotenone inhibits ornithine decarboxylase activity equally well as induced by TPA, insulin-like growth factor I and 17 beta-oestradiol. Pyridaben is the most potent of the four inhibitors not only for NADH: ubiquinone oxidoreductase activity (bovine heart enzyme) and TPA-induced ornithine decarboxylase activity and mRNA steady state level but also for TPA-induced reactive oxygen species. It is therefore proposed that NADH: ubiquinone oxidoreductase inhibitors block multiple and possibly reactive oxygen species-modulated pathways which regulate ornithine decarboxylase activity.

EXPRESSION OF INTESTINAL ORNITHINE DECARBOXYLASE ANTIZYME DURING POSTNATAL DEVELOPMENT IN RATS.

227 Increased ornithine decarboxylase (ODC) activity has been shown to play an essential role in intestinal mucosal maturation and regeneration in the rat. However, the cellular mechanisms regulating the expression of ODC activity during postnatal development are not clear. ODC antizyme (AZ), a noncompetitive inhibitory protein involving in the degradation of ODC bothin vivo and in vitro, has been shown to play a pivotal role in the regulation of ODC activity. Aims: To investigate the expression of intestinal AZ during postnatal development and its relationship with intestinal maturation. Methods: Rat small intestinal mucosa was obtained from postnatal day 5, 10, 15, 17, 21, 24 and 30. Intestinal mucosa was assayed for ODC and sucrase activities. Intestinal AZ and sucrase mRNA were measured by Northern blot analysis. Intestinal AZ protein levels were measured by Western blot analysis. TheXhoI/Bam HI fragment in the vector pTSA-Hyg2 containing rat AZ cDNA clone, was used as probe for AZ Northern analysis.Results: Intestinal sucrase activities were low until postnatal day 19 when its activities increased steadily up to day 30. Intestinal ODC activities remained low from postnatal day 5 to day 17. Sharp increases of ODC activities were noted after day 17, with a peak at day 24 and a decline at day 30. Sucrase mRNA showed same patter as that of sucrase activity with increases after postnatal day 19. Intestinal AZ mRNA and proteins are followings (% of 5 d/o): TableConclusions: 1) Intestinal AZ mRNA and proteins are well expressed before weaning which indicate AZ may play a role in maintaining low and quiescent ODC activities before weaning. 2). Other mechanisms are involved in the induction and degradation of ODC activity around the weaning period since AZ proteins at 30 day are not increased.

Distribution and regulation of ornithine decarboxylase activity along the villus–crypt axis in the small intestine

Distribution of ornithine decarboxylase activity in rat intestinal villi and crypts was determined by serially sectioning frozen mucosa and measuring enzyme activity in pools of sections composed of villi or crypts. Contents of the pools was determined by histological examination of representative sections, and simultaneous measurement of sucrase as a marker of villus samples demonstrated excellent separation of villi and crypts. In fasted and ad lib fed rats, enzyme activity was highest in the villus–crypt junctional area and in crypts ( P<0.05). Refeeding after a fast increased enzyme activity 15-fold, with greatest activity in villus tips and the villus–crypt junctional area. Luminal 0.4 M glycine stimulated enzyme activity only in villus and villus–crypt junctional samples, while luminal 10 mM putrescine stimulated activity only in crypts. Parenteral epidermal growth factor caused increased enzyme activity in all mucosal areas, but the 18–28-fold increase in the three villus samples (top, middle and bottom) was significantly greater ( P<0.05) than the 7–9-fold increase in crypt and junctional samples. In rats refed after a fast, parenteral putrescine (2 mmol/kg) depressed enzyme activity in all mucosal areas. Ornithine decarboxylase activity is usually greatest in junctional and crypt cells, and villus and crypt cells respond differently to luminal and systemic stimuli.

Multisite Phosphorylation of Ornithine Decarboxylase in Transformed Macrophages Results in Increased Intracellular Enzyme Stability and Catalytic Efficiency

Ornithine decarboxylase (ODC) is the initial inducible enzyme in the polyamine biosynthetic pathway. In the transformed macrophage-derived RAW264 cell line, ODC was overproduced and existed in both unphosphorylated and phosphorylated forms. To date, the only protein kinase known to phosphorylate mammalian ODC is casein kinase II (CKII). ODC was phosphorylated in vitro by CKII and subjected to exhaustive sequential proteolysis with trypsin and V8 protease. Two-dimensional peptide mapping showed only a single phosphopeptide; two-dimensional phosphoamino acid analysis of the phosphopeptide revealed only 32P-labeled serine. ODC was metabolically radiolabeled with 32Pi in RAW264 cells and also subjected to proteolysis, two-dimensional peptide mapping, and phosphoamino acid analysis. Two phosphopeptides were generated from the metabolically radiolabeled ODC, including one that migrated similarly to the peptide phosphorylated by CKII in vitro. Each of the in situ radiolabeled ODC peptides contained both 32P-labeled serine and threonine residues. Thus, in RAW264 cells, ODC is phosphorylated on at least one serine residue in addition to that phosphorylated by CKII and on at least two threonine residues. Phosphorylated ODC had an increased stability to intracellular proteolysis compared with unphosphorylated ODC, their half-lives being 49.2 +/- 3.78 and 23.9 +/- 2.6 min (p = 0.001), respectively. The phosphorylated and unphosphorylated forms of ODC were independently purified to homogeneity. Kinetic analysis revealed that the catalytic efficiency of the phosphorylated form of ODC was 50% greater than that of the unphosphorylated form; the unphosphorylated ODC had a Vmax of 20.54 +/- 1.65 micromol/min/mg, whereas the phosphorylated form had a Vmax of 30.61 +/- 2.6 micromol/min/mg (p = 0.005). Phosphorylation of ODC by CKII has no effect on enzyme activity. Taken together, these findings demonstrate that regulation of ODC activity is governed by as yet unidentified protein kinases.

Relationship between Ornithine Decarboxylase and Cytoskeletal Organization in Cultured Human Keratinocytes: Cellular Responses to Phorbol Esters, Cytochalasins, and α-Difluoromethylornithine

Changes in cell shape occur during the cell cycle and influence cell proliferation and differentiation. In order to study how altered cell proliferation and cell shape are interrelated, we have studied ornithine decarboxylase (ODC) regulation in cultured normal human epidermal keratinocytes (NHEK). Cytoskeletal disruptors have been reported to modulate regulation of ODC; the products of ODC, the polyamines, influence actin polymerization rates in vitro , and polyamine auxotrophs have profoundly disrupted cytoskeletons. Therefore, altered ODC levels could be involved in signaling changes in cell shape and an intact cytoskeleton could transduce signals to regulate ODC levels. We had previously observed that the phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA), which profoundly alters cell shape, markedly suppresses ODC biosynthesis in NHEK solely at posttranscriptional/protein synthesis levels. TPA treatment caused NHEK to rapidly assume a rounded morphology that was accompanied by a change in actin organization, as determined by rhodamine-phalloidin labeling. Immunolocalization of ODC showed a perinuclear/nuclear distribution in untreated NHEK and a more diffuse pattern after TPA treatment that was apparent within 15-30 min. Changes in ODC enzyme activity are not significant until 60 min after TPA treatment. NHEK treated with cytochalasin B or D to inhibit actin polymerization exhibited a diffuse ODC localization that could he reversed by removal of the cytochalasin; inhibition of ODC by α-difluoromethylornithine caused a diffuse ODC localization. All treatments resulted in cytoskeletal remodeling. These data are the first evidence for a distinct subcellular localization for ODC and suggest that changes in ODC localization may be an initial step in regulation of ODC activity. Furthermore, changes in ODC activity cause an altered cytoskeleton, suggesting one means by which growth regulatory signals can be transduced to the cytoskeleton from various signaling pathways.

Signaling Pathways Regulating Ornithine Decarboxylase Activity in the Embryonic Chicken

The pathways regulating ornithine decarboxylase (ODC) activity in the chick embryo were studied to determine which kinase-signaling pathways regulate ODC activity levels during development. Insulin-dependent tyrosine kinase, protein kinase C and cAMP-dependent protein kinase were activated by the addition of insulin, tetradecanoylphorbol-12,13-acetate, and forskolin, respectively. All three drugs increased ODC activity and forskolin combined with insulin increased ODC activity above the increase caused by either drug alone. These results suggest that all three signaling pathways regulate ODC activity during development and that common intermediates exist among the pathways downstream of the kinases.

Secretin inhibits induction of ornithine decarboxylase activity by gastrin in duodenal mucosa and IEC-6 cells

Ornithine decarboxylase (ODC) catalyzes the first rate-limiting step in polyamine biosynthesis, and increased ODC activity is one of the earliest biochemical events associated with the induction of cellular proliferation. The current study examines the regulation of ODC activity in rat duodenal mucosa and IEC-6 cells (a line of normal rat intestinal crypt cells) in response to the trophic hormone, gastrin, and its inhibitor, secretin. Rats were fasted 22 h before the various treatments, and ODC activity was measured in scraped duodenal mucosa. Gastrin significantly increased ODC activity within 3 h to 4.3 times control levels. The effect of gastrin was totally inhibited by 5 micrograms/kg secretin. In doses of 5 or 10 micrograms/kg, secretin had no effect on basal ODC. Epidermal growth factor (EGF) and refeeding fasted rats also significantly increased ODC activity in duodenal mucosa, but the effects of EGF and refeeding were not prevented by secretin. In cultured IEC-6 cells, ODC activity was significantly increased after exposure to gastrin, 5% dialyzed fetal bovine serum (FBS), EGF, and asparagine. Secretin in doses ranging from 10(-10) to 10(-6) M caused a linear and significant inhibition of the stimulation of ODC activity by gastrin. No dose of secretin affected basal ODC activity or enzyme activity stimulated by 5% dialyzed FBS, EGF, or asparagine in IEC-6 cells. The ODC mRNA levels in IEC-6 cells were also increased after exposure to gastrin. Administration of secretin significantly prevented the stimulated expression of the ODC gene in cells treated with gastrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlations between developmental ornithine decarboxylase gene expression and enzyme activity in the rat brain

The rise and decline in cerebral ODC activity during specific stages of development has been attributed to cytoplasmic intermediates which regulate ornithine decarboxylase activity. Here we examine whether transcriptional regulation contributes to the production of the developmental profiles of ODC activity. Postnatal cerebellar and neocortical tissue were obtained from Long-Evans hooded rats at postnatal days (PND) 5, 10, 15, 20, 25, 30, 90 and probed for ODC and actin gene expression, by Northern analysis. Our results indicate that ODC gene expression in the cerebellum was elevated at PND 5 and 10 followed by a gradual drop to the adult low levels by PND 20. By contrast, high levels of ODC gene expression in the neocortex were seen at PND 5 with an abrupt decrease at day 10 to low adult levels. The expression of the ODC gene in the neocortex follows closely the pattern for the ODC enzyme activity; however, it tends to remain elevated longer in the cerebellum. The levels of actin gene expression exhibited a distinct developmental profile in the postnatally developing cerebellum. However, actin mRNA levels remained unchanged in the neocortex, consistent with the prenatal development of this region. Our findings suggest that ODC gene expression may play an important role in the production of the ontogenetic patterns of ODC activity.

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.