Ornithine Decarboxylase mRNA Levels Research Articles

Inhibition of ornithine decarboxylase restores hypoxic pulmonary vasoconstriction in endotoxemic mice.

Endotoxemia impairs hypoxic pulmonary vasoconstriction which leads to systemic hypoxemia. This derogation is attributable to increased activity of nitric oxide synthase 2 and arginase metabolism. Gene expression analysis has shown increased expression of ornithine decarboxylase in lungs of endotoxemic mice, a downstream enzyme of arginase metabolism. The aim of this study was to investigate whether inhibition of ornithine decarboxylase increases hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice. Mice received lipopolysaccharides or saline intraperitoneal, and hypoxic pulmonary vasoconstriction was measured using an isolated perfused mouse lung model. Additional mice with and without endotoxemia were pretreated with the ornithine decarboxylase-inhibitor difluoromethylornithine before examination of hypoxic pulmonary vasoconstriction. Hypoxic pulmonary vasoconstriction was defined as the difference of pulmonary arterial pressure between normoxic and hypoxic ventilation. In addition, lung tissue was analyzed using real-time quantitative polymerase chain reaction, Western blot and immunohistochemistry. Lipopolysaccharides caused an up-regulation of ornithine decarboxylase mRNA level (2.73 ± 0.19-fold increase, p < 0.05) as well as ornithine decarboxylase protein level (4.05 ± 0.37-fold increase, p < 0.05). Endotoxemia attenuated hypoxic pulmonary vasoconstriction when compared with untreated control mice (26.3 ± 9.7% vs. 67.0 ± 17.5%). Difluoromethylornithine (20, 100, 500 mg kg−1 body weight intraperitoneal) restored hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice in a dose-dependent way (25.8 ± 9.9%, 57.3 ± 17.2%, 62.3 ± 12.4%) and decreased hypoxic pulmonary vasoconstriction in control mice (53.6 ± 13.6%, 40.0 ± 14.9%, 35.9 ± 12.4%). These results show that endotoxemia induces ornithine decarboxylase expression and suggest that ornithine decarboxylase contributes to the endotoxemia-induced impairment of hypoxic pulmonary vasoconstriction. Inhibition of ornithine decarboxylase might be a target in the therapy of diseases with inflammation impaired hypoxic pulmonary vasoconstriction, like the sepsis-associated acute respiratory distress syndrome (ARDS).

Abstract 4774: Polyamines are rich and induces epigenetic change by inhibition of LSD1 in cancer stem-like cells

Abstract Cancer stem cells (CSC) are highly tumorigenic and resistant to chemotherapy and radiotherapy. We have previously reported that a small fraction of cancer cells with low proteasome activity had CSC-like properties. Ornithine decarboxylase (ODC) is an enzyme which converts ornithine to polyamines including putrescine, spermidine, and spermine. Proteasomal degradation of ODC is known to be ubiquitin-independent. The aim of this study was to explain how low proteasome activity gave CSC-like properties. Western blot showed high ODC protein level in CSC-like cells in cervical cancer and osteosarcoma cell lines, although ODC mRNA level is not high. These findings suggest that ODC protein level is high in CSC-like cells because ODC protein degradation is inactive. To investigate polyamine metabolism in CSC-like cells, we performed GC-MS analysis and found that putrescine, spermidine, and spermine were enriched in CSC-like cells. Furthermore, flux analysis showed conversion from ornithine to polyamines was more rapid in CSC-like cells. We performed a computational structure analysis and found that polyamines bind and inhibit demethylase site of LSD1 which is known to be a demethylase of histone H3K4 and a nuclear homolog of amine oxidases. Spermine was the strongest binding energy. An enzyme assay showed that LSD1 activity was dose-dependently inhibited by polyamines and spermine was the strong inhibitor. Finally, we analyzed performed ChIP sequencing analysis by using HeLa CSC-like cells and found that more global enrichment of H3K4me3 and less global enrichment of H3K4me1 than non-CSC-like cells. In conclusion, polyamines are rich in CSC, and be able to inhibit LSD1 activty and induce epigenetic alterations especially in H3K4 methylation in CSC. Citation Format: Keisuke Tamari, Hideshi Ishii, Masamitsu Konno, Naohiro Nishida, Jun Koseki, Koichi Kawamoto, Fumiaki Isohashi, Yuichiro Doki, Masaki Mori, Kazuhiko Ogawa. Polyamines are rich and induces epigenetic change by inhibition of LSD1 in cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4774. doi:10.1158/1538-7445.AM2017-4774

Apigenin Inhibits Human SW620 Cell Growth by Targeting Polyamine Catabolism.

Apigenin is a nonmutagenic flavonoid that has antitumor properties. Polyamines are ubiquitous cellular polycations, which play an important role in the proliferation and differentiation of cancer cells. Highly regulated pathways control the biosynthesis and degradation of polyamines. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the metabolism, and spermidine/spermine-N1-Acetyl transferase (SSAT) is the rate-limiting enzyme in the catabolism of polyamines. In the current study, the effect of increasing concentrations of apigenin on polyamine levels, ODC and SSAT protein expression, mRNA expression, cell proliferation and apoptosis, and the production of reactive oxygen species (ROS) was investigated in SW620 colon cancer cells. The results showed that apigenin significantly reduced cell proliferation, decreased the levels of spermidine and spermine, and increased previously downregulated putrescine contents. Apigenin also enhanced SSAT protein and mRNA levels and the production of reactive oxygen species in SW620 cells, though it had no significant effect on the levels of ODC protein or mRNA. Apigenin appears to decrease the proliferation rate of human SW620 cells by facilitating SSAT expression to induce polyamine catabolism and increasing ROS levels to induce cell apoptosis.

Chemical hypoxia-induced stimulation of polyamine biosynthesis and ornithine decarboxylase gene transcription in C6 glioma cells

Polyamines, aliphatic polycationic compounds distributed ubiquitously in the living organisms, are considered to be implicated in various cellular processes, and furthermore speculated to play a possible pathophysiological role in physical and mental disorders. In particular, the regulation of polyamine metabolism in damaged glial cells seems interesting and important to understand a possible connection of polyamines with the brain functions. On the other hand, previous studies have shown that ornithine decarboxylase (ODC), the enzyme catalyzing the rate-limiting step of polyamine biosynthesis, can be activated during the period of recovery from various damage in the brain, but little is known about the effects of toxic insults on the expression of ODC gene in neuronal and glial cells. Based on the hypothesis that noxious impacts on the brain might be able to increase polyamine production in glial cells to protect the neuronal cell functions against toxic injuries, the effects of chemical hypoxia on polyamine production and the transcription of ODC gene in C6 glioma cells were investigated using a HPLC assay method and a one-step RT-PCR analysis. In consequence, both cobalt and azide were shown to increase the cellular polyamine contents through elevating ODC mRNA levels under the conditions in which hypoxia-inducible factor-1 (Hif-1) gene was expressed. Thus, chemical hypoxia is speculated to induce ODC gene transcription, thus resulting in the increment of polyamine contents in glial cells, thereby contributing to the recovery of brain function from hypoxic injury.

Abstract 763: Hyperthermia promotes c-myc turnover and reduces ODC expression in colorectal cancer cells.

Abstract Hyperthermia therapy is an FDA-approved procedure that is used to improve the clinical response to radiation and chemotherapy in a variety of solid malignancies. This method involves raising the temperature of tumor-bearing tissues to 41-43°C, which is commonly achieved by using microwave or radiofrequency (RF)-emitting devices. Despite a multitude of treatment centers that are now providing this type of therapy, the cellular and molecular processes are not fully understood. Here, we report that that the oncogenic transcription factor, c-myc has a potentially important role in the anti-cancer mechanism of hyperthermia. By treating colorectal cancer cells (RKO cell line) with heat (incubation at 42°C), we observe a rapid decrease in c-myc levels that is not seen with other transcription factors. We have also observed a similar effect in other cell types, including lung (A549) and neuroblastoma (SH-SY5Y). It is already known that the turnover of c-myc protein is enhanced by phosphorylation at Thr58. Our Western blot data reveal that hyperthermia causes a significant increase in Thr58 phosphorylation, which is accompanied by a reduction in total c-myc levels. c-Myc is frequently deregulated in human cancers, where it promotes the expression of genes that drive tumor cell proliferation. To assess the role of c-myc target genes in the response to hyperthermia, we looked at one such target gene, ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine biosynthesis. We see that the hyperthermia-induced degradation of c-myc is accompanied by a drastic reduction in ODC mRNA and protein levels. Using actinomycin D, we observe that heat has no effect on ODC mRNA stability, suggesting that the decrease in ODC is due to a reduction in gene transcription. In support of this, we find that silencing c-myc expression with siRNA inhibits ODC mRNA and protein levels by a similar amount as hyperthermia. We propose that the down-regulation of ODC caused by c-myc degradation may be an important part of the clinical response to hyperthermia, and future work will evaluate the effect of heat on cellular polyamine levels. Citation Format: Christina T.K. Wales, Aaron T. Jacobs. Hyperthermia promotes c-myc turnover and reduces ODC expression in colorectal cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 763. doi:10.1158/1538-7445.AM2013-763

Role of ornithine decarboxylase in breast cancer

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis that decarboxylates ornithine to putrescine, has become a promising target for cancer research. The aim of this study is to investigate the role of ODC in breast cancer. We detected expression of ODC in breast cancer tissues and four breast cancer cell lines, and transfected breast cancer cells with an adenoviral vector carrying antisense ODC (rAd-ODC/Ex3as) and examined their growth and migration. ODC was overexpressed in breast cancer tissues and cell lines compared with non-tumor tissues and normal breast epithelial cells, and there was a positive correlation between the level of ODC mRNA and the staging of tumors. The expression of ODC correlated with cyclin D1, a cell cycle protein, in synchronized breast cancer MDA-MB-231 cells. Gene transfection of rAd-ODC/Ex3as markedly down-regulated expression of ODC and cyclin D1, resulting in suppression of proliferation and cell cycle arrest at G0-G1 phase, and the inhibition of colony formation, an anchorage-independent growth pattern, and the migratory ability of MDA-MB-231 cells. rAd-ODC/Ex3as also markedly reduced the concentration of putrescine, but not spermidine or spermine, in MDA-MB-231 cells. The results suggested that the ODC gene might act as a prognostic factor for breast cancer and it could be a promising therapeutic target.

Regulatory effects of corticosterone on ornithine decarboxylase activity during liver regeneration in rats

The regulation of ornithine decarboxylase (ODC) gene expression and enzyme activity by corticosterone during rat liver regeneration induced by partial hepatectomy (PH) was evaluated. Bilateral adrenalectomies were performed on ether-anesthetized rats 3 days before PH. Corticosterone in sesame oil was injected subcutaneously to adrenalectomized rats. ODC mRNA, ODC protein and enzyme activity were detected by in situ hybridization, Western blot and high performance liquid chromatography (HPLC), respectively. The ODC mRNA levels, protein accumulation and enzyme activity were lower in the intact liver compared to the regenerating liver. After PH, mRNA levels were remarkably enhanced in all groups and peaked at 5 h post-PH, and presented a persistent increase only in adrenalectomy rats during the regeneration process. Corticosterone treatment resulted in a dose-dependent decrease in ODC mRNA content after 5 h post-PH. ODC protein accumulation in adrenalectomy rats was higher than that in sham-adrenalectomy rats, but it decreased in corticosterone-treated (10 mg/kg) rats until 24 h post-PH, with a strong decline seen in 40 mg/kg corticosterone-treated rats. ODC activity was rapidly promoted, and the highest levels were observed at 6 h after PH in all groups. After corticosterone treatment, the activities declined significantly at 6 h post-PH, with the lowest value found in the 40 mg/kg group. Corticosterone treatment results in dose-dependent decreases in ODC mRNA and enzyme protein both in the intact liver and the regenerating liver. The change in ODC activity is partially related to alterations of ODC mRNA and protein accumulation.

Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism

Iron loading has been observed to have a hyperproliferative effect on hepatocytes in vitro and on tumour cells in vivo; removal of this iron being required to induce antitumour activity. Antiproliferative effects of orally active tridentate iron chelator ICL670 (deferasirox) and bidentate iron chelator CP20 (deferiprone), mediated through the chelation of intracellular iron, were compared in rat hepatoma cell line FAO and human hepatoma cell line HUH7. In FAO cell cultures, we have shown that ICL670 decreased cell viability and DNA replication and induced apoptosis more efficiently than an iron-binding equivalent concentration of CP20. Moreover, ICL670 decreased significantly the number of the cells in G(2)-M phase. In the HUH7 cell cultures, ICL670 and a four-time higher iron-binding equivalent concentration of CP20, decreased cell viability and DNA replication in the same range. CP20 increased the number of the cells in G(2)-M phase. However, ICL670 inhibited polyamine biosynthesis by decreasing ornithine decarboxylase mRNA level; in contrast, CP20 increased polyamine biosynthesis, particularly putrescine level, by stimulating spermidine-spermine N(1)-acetyl transferase activity that could activate the polyamine retro-conversion pathway. By mass spectrometry, we observed that ICL670 cellular uptake was six times higher than CP20. These results suggest that ICL670 has a powerful antitumoural effect and blocks cell proliferation in neoplastic cells by a pathway different from that of CP20 and may constitute a potential adjuvant drug for anticancer therapy.

Sexual dimorphism of ornithine decarboxylase in the mouse adrenal: influence of polyamine deprivation on catecholamine and corticoid levels

Adrenal sexual dimorphism is thought to be important in explaining sex-related differences regarding prevalent diseases and the responses to stress and drugs. We report here that in CD1 mice there is marked sexual dimorphism affecting not only gland size and corticoid hormone secretion but also adrenal ornithine decarboxylase (ODC), polyamine, and catecholamine levels in which testosterone appears to be a major determinant. Our results show that adrenal weight, ODC activity, and corticosterone and aldosterone secretion were higher in female than in male mice and that orchidectomy brought these male parameters closer to the values found in females. mRNA levels of steroidogenic proteins SF-1, Dax-1, steroid 21-hydroxylase, and aldosterone synthase appeared to be slightly higher in female than in male adrenals. Immunocytochemical analysis of adrenal ODC revealed that immunoreactivity was higher in females than in males and was located mainly in the cortical cells, and especially in zona glomerulosa, whereas no sex differences in ODC mRNA levels were observed. These results suggest that sex-associated differences in the expression of ODC in the mouse adrenal gland appear to be related mainly to posttranscriptional mechanisms. Combination treatment of mice with alpha-difluoromethylornithine (a suicide inhibitor of ODC) and a polyamine-deficient diet produced a marked decrease in adrenal polyamine and catecholamine levels and a significant reduction in plasma corticosterone and aldosterone concentrations that were not associated with a decrease in the mRNA levels of steroidogenic proteins. All of these data suggest a relevant role for testosterone, ODC, and polyamines in the mouse adrenal function.

Gene Expression of Ornithine Decarboxylase in Lung Cancers and Its Clinical Significance

Lung cancer is one of the most lethal cancers in China because of its high incidence and high mortality. Ornithine decarboxylase (ODC), an important enzyme in polyamine biosynthesis, is increased in cancer cells. Some chemotherapeutic agents aimed at reducing ODC expression show inhibitory effects on cancer cell growth, so ODC can be useful in gene diagnosis and gene therapy of cancers. In this study, we examined the effect of antisense ODC on lung cancer cells. A-549 cells were infected with rAd-ODC/Ex3as, a recombinant adenovirus containing the cytomegalovirus promoter, green fluorescent protein gene and 120 bp antisense ODC. The cell cycle was evaluated by flow cytometry. A nude mouse xenograft model was used in the tumorigenicity test. Reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry were used to study the expressions of ODC on lung cancers. It was found that the growth of cells infected with rAd-ODC/Ex3as was substantially inhibited and cells were arrested at G1 phase. Cells infected with rAd-ODC/Ex3as can suppress tumor formation in a nude mouse xenograft model. The expression of ODC mRNA and ODC protein levels in lung cancer tissues was significantly higher than that in normal tissues (P<0.05), which correlated significantly with the stage of lung cancer (P<0.05). This study suggested that rAd-ODC/Ex3as has antitumor activity in human lung cancer cells. The ODC gene might play an important role in lung cancer and the overexpression of ODC might be related to the occurrence and development of lung cancer.

Peroxisome proliferator-activated receptor gamma and spermidine/spermine N1-acetyltransferase gene expressions are significantly correlated in human colorectal cancer

BackgroundThe peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that regulates adipogenic differentiation and glucose homeostasis. Spermidine/spermine N1-acetyltransferase (SSAT) and ornithine decarboxylase (ODC) are key enzymes involved in the metabolism of polyamines, compounds that play an important role in cell proliferation. While the PPARγ role in tumour growth has not been clearly defined, the involvement of the altered polyamine metabolism in colorectal carcinogenesis has been established. In this direction, we have evaluated the PPARγ expression and its relationship with polyamine metabolism in tissue samples from 40 patients operated because of colorectal carcinoma. Since it is known that the functional role of K-ras mutation in colorectal tumorigenesis is associated with cell growth and differentiation, polyamine metabolism and the PPARγ expression were also investigated in terms of K-ras mutation.MethodsPPARγ, ODC and SSAT mRNA levels were evaluated by reverse transcriptase and real-time PCR. Polyamines were quantified by high performance liquid chromatography (HPLC). ODC and SSAT activity were measured by a radiometric technique.ResultsPPARγ expression, as well as SSAT and ODC mRNA levels were significantly higher in cancer as compared to normal mucosa. Tumour samples also showed significantly higher polyamine levels and ODC and SSAT activities in comparison to normal samples. A significant and positive correlation between PPARγ and the SSAT gene expression was observed in both normal and neoplastic tissue (r = 0.73, p < 0.0001; r = 0.65, p < 0.0001, respectively). Moreover, gene expression, polyamine levels and enzymatic activities were increased in colorectal carcinoma samples expressing K-ras mutation as compared to non mutated K-ras samples.ConclusionIn conclusion, our data demonstrated a close relationship between PPARγ and SSAT in human colorectal cancer and this could represent an attempt to decrease polyamine levels and to reduce cell growth and tumour development. Therefore, pharmacological activation of PPARγ and/or induction of SSAT may represent a therapeutic or preventive strategy for treating colorectal cancer.

Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: Crosstalk between ODC and BCL-2

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-alpha)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCdelta) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKdelta pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.

Ornithine decarboxylase gene is overexpressed in colorectal carcinoma

To investigate the ornithine decarboxylase (ODC) gene expression in colorectal carcinoma, ODC mRNA was assayed by RT-PCR and ODC protein was detected by a monoclonal antibody against fusion of human colon ODC prepared by hybridoma technology. Total RNA was extracted from human colorectal cancer tissues and their normal counterpart tissues. ODC mRNA levels were examined by RT-PCR. ODC genes amplified from RT-PCR were cloned into a prokaryotic vector pQE-30. The expressed proteins were purified by chromatography. Anti-ODC mAb was prepared with classical hybridoma techniques and used to determine the ODC expression in colon cancer tissues by immunohistochemical and Western blotting assay. A cell line, which could steadily secrete anti-ODC mAb, was selected through subcloning four times. Western blotting reconfirmed the mAb and ELISA showed that its subtype was IgG2a. RT-PCR showed that the ODC mRNA level increased greatly in colon cancer tissues (P<0.01). Immunohistochemical staining showed that colorectal carcinoma cells expressed a significantly higher level of ODC than normal colorectal mucosa (98.6+/-1.03% vs 5.26+/-5%, P<0.01). ODC gene overexpression is significantly related to human colorectal carcinoma. ODC gene expression may be a marker for the gene diagnosis and therapy of colorectal carcinoma.

Cisplatin up-regulates the in vivo biosynthesis and degradation of renal polyamines and c-Myc expression.

Time-dependent changes in polyamine metabolism and c-Myc expression are reported in kidney of mice treated with cisplatin, a widely used anticancer drug. We show that cisplatin significantly induces the expression of two enzymes critical to proper homeostasis of cellular polyamines, ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT). We also document the cross-talk between signalling pathway(s) induced by cisplatin injury to renal tubules and the testosterone/androgen receptor pathway. Their interaction results in a decrease in testosterone-induced ODC activity and ODC mRNA level, and in differential modulation of SSAT expression. Moreover, cisplatin and antifolate CB 3717, another nephrotoxic drug examined, severalfold up-regulate expression of c-Myc mRNA, albeit with different kinetics. However, cisplatin, contrary to CB 3717, does not induce renal hepatocyte growth factor (HGF)/c-Met expression being without effect on HGF mRNA level and significantly down-regulating c-Met transmembrane receptor message. In conclusion, these in vivo studies document significant cisplatin-induced modulation of polyamine biosynthesis/degradation and up-regulation of c-Myc expression, and suggest that c-Myc transcription factor is involved in the induction of ODC in kidney injured with antifolate, but not with cisplatin.

Influence of testosterone on regulation of ODC, antizyme, and N1-SSAT gene expression in mouse kidney.

Polyamines are involved in the control of the cell cycle and cell growth. In murine kidney, testosterone enhances gene expression of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. In this study, we document the time course effect of testosterone on 1) gene expression of ODC, antizyme 1 (AZ1), and spermidine/spermine-N1-acetyltransferase (N1-SSAT); 2) ODC activity in proximal convoluted tubules (PCT) and cortical proximal straight tubules (CPST); and 3) renal polyamine levels. Female mice were treated with testosterone for a period of 1, 2, 3, and 5 consecutive days. ODC gene expression was extremely low in kidneys of untreated female mice compared with that of males. Consequently, the renal putrescine level was sevenfold lower in females than in males, whereas spermidine and spermine levels did not differ between sexes. In female kidneys, testosterone treatment sharply increased ODC mRNA and protein levels as well as ODC activity. Testosterone increased the expression of ODC in PCT and CPST over different time courses, which suggests that ODC activity is differentially regulated in distinct tubules. The expression of AZ1 and N1-SSAT mRNA was similar in male and female mouse kidneys. Testosterone treatment enhanced AZ1 and N1-SSAT mRNA levels in a time-dependent manner by unknown molecular mechanisms. Putrescine and spermidine levels increased after testosterone treatment in female kidneys. Surprisingly, although ODC protein and activity were undetectable in female kidneys, the levels of AZ1 mRNA and protein were similar to those in males. Therefore, one may propose that ODC protein could be continuously degraded by AZ1 in female kidneys.

Resveratrol-induced modification of polyamine metabolism is accompanied by induction of c-Fos.

The objective of the current study was to investigate the effect of resveratrol, a naturally occurring polyphenol with cancer chemopreventive properties, on polyamine metabolism in the human colonic adenocarcinoma cell line Caco-2. We demonstrated that inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was due to attenuated ODC protein and mRNA levels (50-200 microM). The naturally occurring resveratrol analog piceatannol (100 microM) also diminished ODC activity, protein and mRNA levels, whereas the green tea polyphenol (-)-epigallocatechin gallate (EGCG; 100 microM) exerted only weak effects on ODC. The transcription factor c-Myc, a positive regulator of the odc gene was attenuated by resveratrol treatment and to a lesser extent by piceatannol and EGCG. S-Adenosylmethionine decarboxylase, an enzyme that synthesizes higher polyamines, was concomitantly inhibited by resveratrol and piceatannol treatment, whereas EGCG did not affect its activity. In addition resveratrol, piceatannol and EGCG enhanced spermidine/spermine N(1)-acetyltransferase activity, an enzyme that degrades polyamines in cooperation with polyamine oxidase. Intracellular levels of spermine and spermidine were not affected, whereas putrescine and N(8)-acetylspermidine concentrations increased after incubation with resveratrol. These events were paralleled by an increase of the activator protein-1 constituents c-Fos and c-Jun. Whereas DNA-binding activity of c-Jun remained unchanged, DNA-binding activity of c-Fos was significantly enhanced by resveratrol and piceatannol, but inhibited by EGCG. The data suggest that growth arrest by resveratrol is accompanied by inhibition of polyamine synthesis and increased polyamine catabolism. C-Fos seems to play a role in this context. Effects of piceatannol on polyamine synthesis were similar, but not as potent as those exerted by resveratrol.

Gut-enriched Krüppel-like Factor Represses Ornithine Decarboxylase Gene Expression and Functions as Checkpoint Regulator in Colonic Cancer Cells

Gut-enriched Krüppel-like factor (GKLF, KLF4) is an epithelial-specific transcription factor that expresses in the gastrointestinal tract and mediates growth arrest of colonic epithelium. The molecular mechanisms governing its growth inhibitory effect have not been fully elucidated. In the present study, we showed that induction of GKLF mRNA and protein expression by interferon-gamma treatment was associated with reduction of ornithine decarboxylase (ODC) gene expression and enzyme activity in colon cancer HT-29 cells. Overexpression of GKLF in HT-29 cells significantly reduced ODC mRNA and protein levels as well as enzyme activity and resulted in growth arrest, indicating that ODC might be a downstream target of GKLF. This conclusion was further supported by data showing that GKLF mRNA and protein concentrations were the highest at the G(1)/S boundary of the cell cycle, where ODC mRNA and protein levels were the lowest and that overexpression of GKLF resulted in cell arrested at the G(1) phase. Reporter gene transfection studies and electrophoretic mobility gel shift assays demonstrated that GKLF repressed ODC promoter activity and that these effects appeared to be mediated through interaction with a GC box in the proximal portion of the promoter. Transfection studies using reporter constructs and chromatin immunoprecipitation assays also demonstrated that GKLF inhibited transactivation of the ODC gene by interfering with the binding of Sp1 to the ODC promoter. These results indicate that GKLF may function as a G(1)/S checkpoint regulator and exert its growth arrest effect through down-regulation of ODC gene expression. Furthermore, GKLF is a transcriptional repressor of the ODC gene, and these effects are mediated by interaction with the GC-rich region on the promoter.

Expression of intestinal ornithine decarboxylase during postnatal development in neonatal rats

Ornithine decarboxylase (ODC) has been shown to play an essential role in intestinal growth and maturation in rats. However, the regulatory mechanisms have not been fully elucidated. We studied the mechanisms of expression of intestinal ODC during postnatal development. Rat small intestinal mucosa was obtained from postnatal days 10, 15, 17, 19, 21, 24 and 30. Intestinal mucosa was assayed for ODC and sucrase activities. In addition, intestinal ODC mRNA, and ODC protein levels were also measured. The results showed that the intestinal sucrase activity was low before postnatal day 19. The sucrase activity then increased steadily from day 19 up to day 30. Intestinal ODC activities remained low from postnatal day 10 to day 17. A sharp increase in ODC activity was noted on day 19, which peaked on day 24 (a 20-fold increase from its low basal level) and declined on day 30. Intestinal ODC proteins followed the same pattern of postnatal expression as that of ODC activity. In contrast, ODC mRNA did not show significant change throughout the study period. The possible mechanisms by which intestinal ODC mRNA levels remain practically unchanged during postnatal development are discussed. We conclude that the ontogenic increase in sucrase activity, a marker for intestinal maturation, occurs at the same time to that of the induction of ODC activity. We also suggest that the induction of intestinal ODC activity during postnatal development is the result of post-transcriptional events or other cellular mechanisms. A better understanding of the regulation of polyamine biosynthesis during postnatal development of the small intestine will provide insights contributing to the maturation of the small intestine.

A gene coding for ornithine decarboxylase ( odcA) is differentially expressed during the Mucor circinelloides yeast-to-hypha transition

The differential display technique was used to identify genes from Mucor circinelloides involved in the yeast-to-hypha transition. Using a limited set of primer combinations, cDNA fragments corresponding to mRNAs differentially expressed during the dimorphic transition were isolated. Northern analyses showed that the accumulation of the transcript detected by hybridisation with one of the cDNA fragments increased during the transition and was undetectable at the mycelial stage. Sequence analysis and database searches of this fragment revealed high similarity to ornithine decarboxylase (ODC) encoding genes. The odcA gene of M. circinelloides was isolated from genomic and cDNA libraries and characterised. Electrophoretic karyotyping and hybridisations showed that the odcA gene is single-copy and linked to the leuA and rDNA genes. The single transcript detected (2.1 kb), was considerably longer than the deduced ORF. Through non-radioactive primer extension analysis four transcription initiation sites were mapped to positions −61, −167, −239 and −436 from the start codon. The ODC mRNA levels increased during the yeast-to-hypha transition, reaching a maximum at 120 min, which was accompanied by a rise in ODC enzymatic activity. The expression pattern of the odcA gene showed that in M. circinelloides the ODC levels are transcriptionally regulated, in contrast with other dimorphic fungi in which a post-transcriptional regulation has been proposed.

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.