Ornithine Decarboxylase mRNA Research Articles

Minor contribution of an internal ribosome entry site in the 5′-UTR of ornithine decarboxylase mRNA on its translation

The mechanism of synthesis of ornithine decarboxylase (ODC) at the level of translation was studied using cell culture and cell-free systems. Synthesis of firefly luciferase (Fluc) from the second open reading frame (ORF) in a bicistronic construct transfected into FM3A and HeLa cells was enhanced by the presence of the 5′-untranslated region (5′-UTR) of ODC mRNA between the two ORFs. However, cotransfection of the gene encoding 2A protease inhibited the synthesis of Fluc. Synthesis of Fluc from the second cistron in the bicistronic mRNA in a cell-free system was not affected significantly by the 5′-UTR of ODC mRNA. Synthesis of ODC from ODC mRNA in a cell-free system was inhibited by 2A protease and cap analogue (m 7GpppG). Rapamycin inhibited ODC synthesis by 40–50% at both the G 1/S boundary and the G 2/M phase. These results indicate that an IRES in the 5′-UTR of ODC mRNA does not function effectively.

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.

Effect of vasoactive intestinal peptide on gastric adenocarcinoma

Vasoactive intestinal peptide (VIP) is a gastrointestinal hormone in the secretin-VIP family. It has been reported that VIP affects some tumor growth, and there is a VIP autocrine regulation in some cancers. However, the effect of VIP on gastric adenocarcinoma is not clear yet. The aim of the present study was to investigate the effect of VIP on gastric adenocarcinoma, especially autocrine regulation of VIP on gastric adenocarcinoma. VIP mRNA and protein, and its receptor mRNA (VIPR(1) and VIPR(2)) were measured in 15 normal antrum mucosa, 20 gastric adenocarcinoma tissues, and the SGC7901 gastric adenocarcinoma cell line by using reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, or radioimmunoassay methods. The effect of the VIP protein and its antagonist (D-p-Cl-Phe6, Leu17)-VIP on SGC7901 cell growth was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method. The expressions of c-myc mRNA and ornithine decarboxylase (ODC) mRNA in SGC7901 cells before and after the incubated VIP protein and/or its antagonist were also measured by RT-PCR method. The VIP mRNA expression in gastric adenocarcinoma tissues was significantly higher than that in normal antrum mucosa (P < 0.01). The VIP-positive immunoreactivity cells existed in 40% of gastric adenocarcinoma tissues, but not in normal tissues (P < 0.01). The VIP-positive immunoreactivity nerve fibers were observed in normal tissues, but not in adenocarcinoma tissues (P < 0.01). The expression rate of VIPR(1) mRNA in adenocarcinoma tissues was significantly lower than that in normal tissues, but that of VIPR(2) mRNA in the two kinds of tissues were similar (P > 0.05). In addition, the expression quantity of VIPR(1) mRNA and VIPR(2) mRNA in adenocarcinoma tissues was significantly lower than that in normal tissues (P < 0.05). SGC7901 cells expressed not only VIP mRNA and the VIP protein, but also VIPR(1) and VIPR(2) mRNA. 10(6) SGC7901 cells secreted 13.15 +/- 8.54 pg VIP on average. VIP did not affect the proliferation of SGC7901 cells, but the antagonist stimulated the proliferation of SGC7901 cells from 10(-5) to 10(-8) mol/L concentration incubated for 24-96 h. VIP downregulated the expressions of c-myc and ODC mRNA, but its antagonist upregulated their expressions. The expression of VIP mRNA upregulates, but the expressions of VIPR mRNA downregulates in gastric adenocarcinoma tissues. The gastric adenocarcinoma tissues contain endocrine cells to secrete VIP, which show malignant specialities. The VIP autocrine regulation exists in SGC7901 cells, and potentially inhibits the proliferation of the cells by downregulating the expressions of c-myc and ODC mRNA. It suggests that VIP may play an important role in the regulation of the growth of gastric cancer cells.

The Myotonic Dystrophy Type 2 Protein ZNF9 Is Part of an ITAF Complex That Promotes Cap-independent Translation

The 5'-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy.

Polyamines upregulate the mRNA expression of cationic amino acid transporter-1 in human retinal pigment epithelial cells

We previously showed that ornithine was mainly transported via cationic amino acid transporter (CAT)-1 in human retinal pigment epithelial (RPE) cell line, human telomerase RT (hTERT)-RPE, and that CAT-1 was involved in ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient cell produced by a OAT specific inhibitor, 5-fluoromethylornithine (5-FMO). We showed here that CAT-1 mRNA expression was increased by ornithne in OAT-deficient RPE cells, which was reversed by an inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethylornithine (DFMO). Polyamines, especially spermine, one of the metabolites of ODC, also enhanced the expression of CAT-1 mRNA. ODC mRNA expression was also increased by ornithine and polyamines, and gene silencing of ODC by siRNA decreased ornithine transport activity and its cytotoxicity. In addition, the mRNA of nuclear protein c-myc was also increased in 5-FMO- and ornithine-treated hTERT-RPE cells, and gene silencing of c-myc prevented the induction of CAT-1 and ODC. Increases in expression of CAT-1, ODC, and c-myc, and the inhibition of these stimulated expression by DFMO were also observed in primary porcine RPE cells. These results suggest that spermine plays an important role in stimulation of mRNA expression of CAT-1, which is a crucial role in ornithine cytotoxicity in OAT-deficient hTERT-RPE cells.

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.

The methionine salvage pathway compound 4-methylthio-2-oxobutanate causes apoptosis independent of down-regulation of ornithine decarboxylase

4-Methylthio-2-oxobutanoic acid (MTOB) is the final compound of the methionine salvage pathway that converts the polyamine byproduct methylthioadenosine to adenine and methionine. Here we find that MTOB inhibits growth of several human cell lines in a dose-dependent manner. Growth inhibition was specific for MTOB as we did not observe any inhibition with other chemically related compounds. MTOB treatment causes apoptosis and reduction of ornithine decarboxylase (ODC) activity but not ODC mRNA. To determine if MTOB exerts its effects primarily via ODC inhibition, we compared the effects of MTOB with the ODC-specific inhibitor difluoromethylornithine (DFMO). We found that MTOB was a more potent inducer of apoptosis than DFMO, lacked activation of caspase 3/7, and was able to induce apoptosis in cells lacking p53. Our results show that MTOB-induced growth inhibition and apoptosis is not simply secondary due to ODC inhibition and implies that MTOB activates apoptosis via other mechanisms.

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.

Changes in polyamine content and localization of Pinus sylvestris ADC and Suillus variegatus ODC mRNA transcripts during the formation of mycorrhizal interaction in an in vitro cultivation system

The involvement of polyamines (PAs) in the interaction between Pinus sylvestris L. seedlings and an ectomycorrhizal fungus Suillus variegatus (Swatz: Fr.) O. Kunze was studied in an in vitro cultivation system. PA concentrations in seedlings were analysed after 1, 3, and 5 weeks in dual culture with S. variegatus, and changes in PA pools were compared with the growth of the seedlings. Pinus sylvestris arginine decarboxylase (ADC) and S. variegatus ornithine decarboxylase (ODC) mRNA transcripts were localized during the formation of mycorrhizas. During mycorrhiza formation, Suillus variegatus ODC transcripts were found in developing hyphal mantle and Hartig net, and P. sylvestris ADC transcripts in specific root parenchyma cells adjacent to tracheids and in mitotic cells of the root apical meristem. However, no unambiguous difference in ADC transcript localization between inoculated and non-inoculated roots was observed. Regardless of the unchanged distribution of ADC transcripts, inoculation with S. variegatus increased free putrescine, spermidine, and spermine concentrations in roots within the first week in dual culture. The concentration of free and conjugated putrescine and conjugated spermidine also increased in the needles due to the fungus. The fungus-induced lateral root formation and main root elongation were greatest between the first and third week in dual culture, coinciding with retarded accumulation or a decrease of free PAs. These results show that accumulation of PAs in the host plant is one of the first indicators of the establishment of ectomycorrhizal interaction between P. sylvestris and S. variegatus in the in vitro system.

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.

Comparative Effect of the Oral Iron Chelators ICL670A and CP20 on Cell Proliferation, Apoptosis and Polyamine Metabolism in Rat and Human Liver Cell Cultures.

Iron overload increases the mitotic index in rat hepatocyte cultures stimulated by EGF. Iron, implicated in tumoral cell proliferation, promotes the development of the hepatocellular carcinoma. Conversely, iron chelation decreases cell proliferation. Polyamines play also an important role in cell proliferation. The purpose of the present work was to compare the effect of the two oral iron chelators ICL670A and CP20 on cell proliferation, apoptosis and polyamine metabolism in rat and human liver cell cultures.We used three experimental models: the rat hepatoma cell line FAO, the rat liver epithelial cell line RLEC and the human hepatoma cell line HUH7. Chelator cell uptake was analyzed by mass spectrometry. DNA replication and cell viability were measured by thymidine incorporation and mitochondrial succinate dehydrogenase activity. Cytotoxicity was evaluated by extracellular LDH activity. Cell cycle analysis was performed by flow cytometry. Apoptosis was quantified by DNA fragmentation and caspase 3 activity. Polyamine metabolism was analyzed by measuring their intracellular concentrations by mass spectrometry as well as the ornithine decarboxylase (ODC) and spermine-spermidine acetyl transferase (SSAT) mRNAs levels. ICL670A entered the cells four times better than CP20. We observed that ICL670A (20 μM) decreased DNA replication (−75%) and cell viability (−40%) while a comparable CP20 stoechiometric concentration (30 μM) had no significant effect. ICL670A exhibited also a higher cytotoxicity than CP20. The decrease in DNA replication induced by ICL670A was correlated to a cell cycle block in S phase; CP20 (30 μM) increased slightly the cell number in G2/M phase while a higher CP20 concentration (150 μM) blocked clearly cell cycle in G2/M phase. By measuring DNA fragmentation and the activity of the executioner caspase 3, we demonstrated a high apoptotic effect of ICL670A (20 μM) compared to CP20 (30 μM). In the HUH7 human cell line, we observed that ICL670A (20 μM) decreased the intracellular levels of polyamines which was correlated to a drop in ODC and SSAT mRNAs levels. Conversely, an increase in SSAT mRNA level was observed in the presence of CP20 (150 μM) leading to an enhancement of putrescine and spermidine levels.In conclusion, ICL670A and CP20 inhibit cell proliferation by two distinct ways. ICL670A compared to CP20:enters better the cells;exerts a higher antiproliferative and apoptotic effect;modulates polyamine metabolism by inhibiting ODC and SSAT enzyme activities.

Helicobacter pylori-induced Macrophage Apoptosis Requires Activation of Ornithine Decarboxylase by c-Myc

Helicobacter pylori infection causes chronic inflammation of the gastric mucosa that results from an ineffective immune response. We have demonstrated that one underlying mechanism is induction of macrophage apoptosis mediated by polyamines. The transcription factor c-Myc has been linked to induction of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. We determined whether H. pylori stimulates transcriptional activation of ODC in macrophages, whether this occurs via c-Myc, and whether these events regulate activation of apoptosis. H. pylori induced a significant increase in ODC promoter activity that peaked at 6 h after stimulation and was closely paralleled by similar increases in ODC mRNA, protein, and enzyme activity. By 2 h after stimulation, c-Myc mRNA and protein expression was induced, protein was translocated to the nucleus, and there was specific binding of a consensus probe for c-Myc to nuclear extracts. Both an antennapedia-linked inhibitor of c-Myc binding (Int-H1-S6A,F8A) and transfection of a c-Myc dominant-negative construct significantly attenuated H. pylori-induced ODC promoter activity, mRNA, enzyme activity, and apoptosis in parallel. Transfection of ODC small interfering RNA inhibited ODC activity and apoptosis to the same degree as inhibition of c-Myc binding. These studies indicate that c-Myc is an important mediator of macrophage activation and may contribute to the mucosal inflammatory response to pathogens such as H. pylori by its effect on ODC.

Alternative splicing facilitates internal ribosome entry on the ornithine decarboxylase mRNA.

Ornithine decarboxylase (ODC) is the ratelimiting enzyme in the biosynthesis of polyamines, which are required for optimal cell growth and proliferation. ODC is overexpressed in many tumors and, conversely, its overexpression induces transformation. We have previously reported that ODC mRNA alternative splicing relieves the translation repression normally imposed by a long and structured 5' untranslated region (UTR), and that the ODC 5' UTR contains an internal ribosome entry site (IRES). Here we show that ODC IRES activity is enhanced following inclusion of alternative sequences generated by splicing at cryptic acceptor sites. Furthermore, the alternative ODC IRES is more sensitive to cell-cycledependent changes in the rate of translation. These findings uncover a new biological property of differentially spliced transcripts. This is the first example of alternative splicing that modulates mRNA translation through the cell cycle in a cap-independent manner.

Albumin stimulates cell growth, L-arginine transport, and metabolism to polyamines in human proximal tubular cells

Pure albumin stimulates proximal tubular epithelial cell (PTEC) proliferation, and may have a role in homeostasis in health, as well as in disrupted PTEC turnover in proteinuric nephropathies. We investigated a role for arginine and its metabolites, the polyamines, in this process, given the ability of polyamines to trigger proliferation in other mammalian cells. [(3)H]-L-arginine uptake was examined after incubation with 20 mg/mL recombinant human serum albumin (rHSA) in HK-2 PTEC monolayers. Nitric oxide synthase (NOS) and arginase activity was measured; NOS, arginase, and ornithine decarboxylase (ODC) expression was identified by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Polyamine synthesis and intracellular amino acid concentrations were compared using high-performance liquid chromatography, and cell growth measured by [(3)H]-thymidine incorporation. In HK-2 PTEC exposed to 20 mg/mL rHSA for 24 hours, cell proliferation as determined by [(3)H]-thymidine incorporation was increased. In parallel, L-arginine transport capacity was increased in a dose- and time-dependent manner. This effect was specific to rHSA, and was not seen with transferrin or immunoglobulin G. The intracellular concentration of L-arginine remained unchanged, although L-ornithine was increased with rHSA incubation. rHSA up-regulated type II arginase mRNA, and increased arginase activity, although no difference in nitric oxide synthase expression or activity was seen. ODC mRNA was increased, as were intracellular polyamine concentrations. alpha-Difluoromethylornithine (DFMO), an ODC inhibitor, reduced intracellular polyamine concentrations and rHSA-induced cell proliferation to control levels. The arginine-ornithine-polyamine pathway appears enhanced in PTEC incubated with rHSA and is involved in cellular proliferation; this may offer novel approaches to understanding progressive proteinuric nephropathies.

Eukaryotic Translation Initiation Factor 4E Activity Is Modulated by HOXA9 at Multiple Levels

The eukaryotic translation initiation factor 4E (eIF4E) alters gene expression on multiple levels. In the cytoplasm, eIF4E acts in the rate-limiting step of translation initiation. In the nucleus, eIF4E facilitates nuclear export of a subset of mRNAs. Both of these functions contribute to eIF4E's ability to oncogenically transform cells. We report here that the homeodomain protein, HOXA9, is a positive regulator of eIF4E. HOXA9 stimulates eIF4E-dependent export of cyclin D1 and ornithine decarboxylase (ODC) mRNAs in the nucleus, as well as increases the translation efficiency of ODC mRNA in the cytoplasm. These activities depend on direct interactions of HOXA9 with eIF4E and are independent of the role of HOXA9 in transcription. At the biochemical level, HOXA9 mediates these effects by competing with factors that repress eIF4E function, in particular the proline-rich homeodomain PRH/Hex. This competitive mechanism of eIF4E regulation is disrupted in a subset of leukemias, where HOXA9 displaces PRH from eIF4E, thereby contributing to eIF4E's dysregulation. In regard to these results and our previous finding that approximately 200 homeodomain proteins contain eIF4E binding sites, we propose that homeodomain modulation of eIF4E activity is a novel means through which this family of proteins implements their effects on growth and development.

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.

The two ways to produce putrescine in tomato are cell-specific during normal development

To synthesize putrescine, organisms use either ornithine decarboxylase (ODC), arginine decarboxylase (ADC) or both, as is the case for plants. In situ localization of ODC and ADC mRNAs in shoots and roots of tomato indicates that both pathways show a cell-type specialization during normal development. ODC mRNA is localized to mitotically active cells in the shoot and root apical meristem and procambium which accumulated proliferating cell nuclear antigen (PCNA), a cell marker of cell division. However, ADC mRNA was found in PCNA-negative cells no longer dividing of both roots and shoot, providing a localization pattern that fairly complements the one obtained for ODC mRNA.

HSG cells differentiated by culture on extracellular matrix involves induction of S‐adenosylmethione decarboxylase and ornithine decarboxylase

The human salivary gland (HSG) epithelial cell line can differentiate when cultured on extracellular matrix preparations. We previously identified >30 genes upregulated by adhesion of HSG cells to extracellular matrix. In the current studies, we examined the role of one of these genes, the polyamine pathway biosynthetic enzyme S-adenosylmethionine decarboxylase (SAM-DC) and the related enzyme, ornithine decarboxylase (ODC), on HSG cell differentiation during culture on extracellular matrix. HSG cells cultured on fibronectin-, collagen I gel-, and Matrigel-coated substrates for 12-24 h upregulated SAM-DC and ODC mRNA expression and enzyme activity compared to cells cultured on non-precoated substrates. After 3-5 days, HSG cells grown on Matrigel- or collagen I gel-coated substrates acquired a differentiated phenotype: the cells showed changes in culture morphology and increased expression of salivary gland differentiation markers (vimentin, SN-cystatin, and alpha-amylase). Further, culturing the cells on substrates precoated with an anti-beta1-integrin-antibody promoted differentiation-like changes. HSG cells cultured on collagen I- or Matrigel-coated substrates rapidly entered the cell cycle but showed decreased cell proliferation at longer times. In contrast, cell proliferation was enhanced on fibronectin-coated substrates compared to cells on non-precoated substrates. Treatment with the polyamine synthesis inhibitors, difluoromethylornithine (DFMO), and methylglyoxal bis-(guanylhydrazone) (MGBG), inhibited cell proliferation and delayed (3)H-thymidine incorporation in HSG cells cultured on all of the substrates. Further, inclusion of DFMO and MGBG inhibited or delayed acquisition of the differentiated phenotype in HSG cells cultured on Matrigel- or collagen I gel-coated substrates. This suggests that the adhesion-dependent expression of SAM-DC and ODC contributes to extracellular matrix-dependent HSG cell differentiation.

Isolation and Characterization of Ornithine Decarboxylase Gene from Flounder (Paralichthys olivaceus)

Ornithine decarboxylase (ODC) is a homodimeric enzyme dependent on pyridoxal 5'-phosphate. We identified a complementary DNA clone corresponding to ODC from the brain of adult flounder (Paralichthys olivaceus). The flounder ODC cDNA consisted of 2939 bp encoding 272 amino acid residues. The flounder ODC showed 80.3% sequence identity to zebrafish and 70.8% to rat at the amino acid level. Comparison of the structure and nucleotide sequence of the ODC genes revealed that the gene is highly conserved in the flounder, zebrafish, and rat. The presence of ODC mRNA species in brain, kidney, liver, and embryo was confirmed using the reverse transcriptase polymerase chain reaction. The recombinant protein of flounder ODC containing a short histidine tag at the carboxyl terminus was overexpressed in Escherichia coli BL21 (DE3) codon plus using an inducible T7 expression system, and was purified by Ni-NTA affinity chromatography.