Increased Ornithine Decarboxylase Research Articles

A Tear Metabolomic Profile Showing Increased Ornithine Decarboxylase Activity and Spermine Synthesis in Thyroid-Associated Orbitopathy.

About half of patients with Graves’ disease develop an orbitopathy related to an inflammatory expansion of the periorbital adipose tissue and muscles. We used a targeted metabolomic approach measuring 188 metabolites by mass spectrometry to compare the metabolic composition of tears in patients with active (n = 21) versus inactive (n = 24) thyroid-associated orbitopathy. Among the 44 metabolites accurately measured, 8 showed a significant alteration of their concentrations between the two groups. Two short-chain acylcarnitines, propionylcarnitine and butyrylcarnitine, and spermine showed increased concentrations in the tears of patients with active orbitopathy, whereas ornithine, glycine, serine, citrulline and histidine showed decreased concentrations in this group. In addition, the ratio putrescine/ornithine, representing the activity of ornithine decarboxylase, was significantly increased in patients with active compared to inactive orbitopathy (p = 0.0011, fold change 3.75). The specificity of this candidate biomarker was maintained when compared to a control group with unclassified dry eye disease. Our results suggest that the stimulation of ornithine decarboxylase by TSH receptor autoantibodies in orbital fibroblasts could lead to increased synthesis of spermine, through the increased activity of ornithine decarboxylase, that may contribute to periorbital expansion in Graves’ ophthalmopathy.

Impact of nitric oxide on proline and putrescine biosynthesisin Chlamydomonas via transcriptional regulation

Nitric oxide plays an important role in regulating adaption of the model alga Chlamydomonas reinhardtii to various environmental stresses. One response to abiotic stresses is the accumulation of protective molecules such as proline and putrescine. The NO treatment led to a significant accumulation of proline in cells. Quantitative real-time expression analysis of proline metabolic genes in NO-treated cells showed a prolonged upregulation of the gene encoding γ-glutamyl kinase 1 (GGK1) in the glutamate biosynthetic pathway. Furthermore, truncated hemoglobin 2 (THB2)-underexpressing strains with an enhanced endogenous NO demonstrated a higher proline content and GGK1 mRNA abundances than the wild type. In contrast, transcription of the gene encoding ornithine δ-aminotransferase in the ornithine pathway of proline biosynthesis decreased after treatment with NO. This suggests the predominance of the glutamate pathway over the ornithine pathway. We also found that the expression of the proline dehydrogenase gene encoding a key enzyme in proline catabolism was downregulated in NO-treated cells. Chlamydomonas reinhardtii exposed to exogenous NO also showed an increased ornithine decarboxylase 2 mRNA and putrescine content. Our findings indicate a clear link between changes in NO application and proline and putrescine content via transcriptional regulation of respective enzymes.

Ornithine decarboxylase, the rate-limiting enzyme of polyamine synthesis, modifies brain pathology in a mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant neurodevelopmental disorder characterized by variable expressivity. TSC results from inactivating variants within the TSC1 or TSC2 genes, leading to constitutive activation of mechanistic target of rapamycin complex 1 signaling. Using a mouse model of TSC (Tsc2-RG) in which the Tsc2 gene is deleted in radial glial precursors and their neuronal and glial descendants, we observed increased ornithine decarboxylase (ODC) enzymatic activity and concentration of its product, putrescine. To test if increased ODC activity and dysregulated polyamine metabolism contribute to the neurodevelopmental defects of Tsc2-RG mice, we used pharmacologic and genetic approaches to reduce ODC activity in Tsc2-RG mice, followed by histologic assessment of brain development. We observed that decreasing ODC activity and putrescine levels in Tsc2-RG mice worsened many of the neurodevelopmental phenotypes, including brain growth and neuronal migration defects, astrogliosis and oxidative stress. These data suggest a protective effect of increased ODC activity and elevated putrescine that modify the phenotype in this developmental Tsc2-RG model.

L-arginine Attenuates Hypobaric Hypoxia-Induced Increase in Ornithine Decarboxylase 1

Chronic hypoxia-induced pulmonaryhypertension and vascular remodeling have been shown to be associated with ornithine decarboxylase 1 (ODC1). However, few animal studies have investigated the role of ODC1 in acute hypoxia. We investigated ODC1 gene expression, morphologic and functional changes, and the effect of L-arginine as an attenuator in lung tissues of rats exposed to acute hypobaric hypoxia at a simulated altitude of 6000 m. Sprague-Dawley rats exposed to simulated hypobaric hypoxia (6000 m) for 24, 48, or 72 hours were treated with L-arginine(L-arginine group, 20 mg/100 g intraperitoneal; n=15) or untreated (non-L-arginine group, n=15). Control rats (n=5) were maintained at 2260 m in a normal environment for the same amount of time but were treated without L-arginine. The mean pulmonary artery pressure was measured by PowerLab system. The morphologic and immunohistochemical changes in lung tissue were observed under a microscope. The mRNA and protein levels of ODC1 were measured by real-time polymerase chain reaction and Western-blot, respectively. Hypobaric hypoxia induced pulmonary interstitial hyperemia and capillary expansion in the lungs of rats exposed to acute hypoxia at 6000 m. The mean pulmonary artery pressure and the mRNA and protein levels of ODC1 were significantly increased, which could be attenuated by treatment with L-arginine. L-arginine attenuates acute hypobaric hypoxia-induced increase in mean pulmonary artery pressure and ODC1 gene expression in lung tissues of rats. ODC1 gene contributes to the development of hypoxic pulmonary hypertension.

Endoplasmic Reticulum Stress is Involved in DFMO Attenuating Isoproterenol-Induced Cardiac Hypertrophy in Rats

Background/Aims: Studies performed in experimental animals have shown that polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with regulation of gene expression. Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, has attracted considerable interest for its antiproliferative role, which it exerts through inhibition of the polyamine pathway and cell turnover. Whether DFMO attenuates cardiac hypertrophy through endoplasmic reticulum stress (ERS) is unclear. Methods: Myocardial hypertrophy was simulated by isoproterenol (ISO). Polyamine depletion was achieved using DFMO. Hypertrophy was estimated using the heart/body index and atrial natriuretic peptide (ANP) gene expression. Cardiac fibrosis and apoptosis were measured by Masson and TUNEL staining. Expression of ODC and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blot analysis. Protein expression of ERS and apoptosis factors were analyzed using Western blot analysis. Results: DFMO treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO down-regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up-regulated the expression of SSAT and Bcl-2. Finally, these changes were partially reversed by the addition of exogenous putrescine. Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

Correlation between endogenous polyamines in human cardiac tissues and clinical parameters in patients with heart failure.

Polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy in experimental animals. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with cyclic adenosine monophosphate (cAMP) increases. The aim of the study was to establish the role of these in the human heart in living patients. For this, polyamines (by high performance liquid chromatography) and the activity of ODC and N(1)-acetylpolyamine oxidases (APAO) were determined in the right atrial appendage of 17 patients undergoing extracorporeal circulation to correlate with clinical parameters. There existed enzymatic activity associated with the homeostasis of polyamines. Left atria size was positively associated with ODC (r = 0.661, P = 0.027) and negatively with APAO-N(1) -acetylspermine (r = -0.769, P = 0.026), suggesting that increased levels of polyamines are associated with left atrial hemodynamic overload. Left ventricular ejection fraction (LVEF) and heart rate were positively associated with spermidine (r = 0.690, P = 0.003; r = 0.590, P = 0.021) and negatively with N(1)-acetylspermidine (r = -0.554, P = 0.032; r = -0.644, P = 0.018). LVEF was negatively correlated with cAMP levels (r = -0.835, P = 0.001) and with cAMP/ODC (r = -0.794, P = 0.011), cAMP/spermidine (r = -0.813, P = 0.001) and cAMP/spermine (r = -0.747, P = 0.003) ratios. Abnormal LVEF patients showed decreased ODC activity and spermidine, and increased N(1) -acetylspermidine, and cAMP. Spermine decreased in congestive heart failure patients. The trace amine isoamylamine negatively correlated with septal wall thickness (r = -0.634, P = 0.008) and was increased in cardiac heart failure. The results indicated that modifications in polyamine homeostasis might be associated with cardiac function and remodelling. Increased cAMP might have a deleterious effect on function. Further studies should confirm these findings and the involvement of polyamines in different stages of heart failure.

Effects of Gibberellic Acid And Putrescine on 'Thompson Seedless' Grapes

Effects of Gibberellic Acid And Putrescine on 'Thompson Seedless' Grapes Magdalene Koukourikou, Eleftheria Zioziou, Anastasia Pantazaki, Nikolaos Nikolaou, Demetrios Kyriakidis Abstract Gibberellic acid (GA3 ) or putrescine (Put) were sprayed on ‘Thompson seedless’ grapevine clusters, at three different fruit developmental stages, i.e. before (E-L 18), during (E-L 23) and after bloom (E-L 27, 29, 31). Both substances stimulated cluster and berries growth. Berry weight of vines treated with GA3 or Put was approximately 2 and 2.7 fold greater than control, respectively. Treatments significantly increased berry length and width. The smallest than usual number of GA3 applications reduced berry drop and maintained it at control levels while Putrescine improved further more berry attachment. GA3 increased ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) activity compared to the control. ODC activity in clusters and rachis was much higher than the corresponding ADC activity after the first and last GA3-treatment. Finally, the activity for both enzymes was higher in rachis than in berries. Full Text: PDF DOI: 10.15640/aijb.v3n2a2

Spermine synthase overexpression in vivo does not increase susceptibility to DMBA/TPA skin carcinogenesis or Min-Apc intestinal tumorigenesis

Numerous studies have demonstrated a link between elevated polyamine biosynthesis and neoplastic growth, but the specific contribution of spermine synthase to epithelial tumor development has never been explored in vivo. Mice with widespread overexpression of spermine synthase (CAG-SpmS) exhibit decreased spermidine levels, increased spermine and a significant rise in tissue spermine:spermidine ratio. We characterized the response of CAG-SpmS mice to two-stage skin chemical carcinogenesis as well as spontaneous intestinal carcinogenesis induced by loss of the Apc tumor suppressor in ApcMin/+ (Min) mice. CAG-SpmS mice maintained the canonical increases in ornithine decarboxylase (ODC) activity, polyamine content and epidermal thickness in response to tumor promoter treatment of the skin. The induction of S-adenosylmethionine decarboxylase (AdoMetDC) activity and its product decarboxylated AdoMet were impaired in CAG-SpmS mice, and the spermine:spermidine ratio was increased 3-fold in both untreated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated skin. The susceptibility to 7,12-dimethylbenz[a]anthracene (DMBA)/TPA skin carcinogenesis was not altered in CAG-SpmS mice, and SpmS overexpression did not modify the previously described tumor resistance of mice with targeted antizyme expression or the enhanced tumor response in mice with targeted spermidine/spermine-N1-acetyltransferase expression. CAG-SpmS/Min mice also exhibited elevated spermine:spermidine ratios in the small intestine and colon, yet their tumor multiplicity and size was similar to Min mice. Therefore, studies in two of the most widely used tumorigenesis models demonstrate that increased spermine synthase activity and the resulting elevation of the spermine:spermidine ratio does not alter susceptibility to tumor development initiated by c-Ha-Ras mutation or Apc loss.

Select Nutrients in the Uterine Lumen of Sheep and Pigs Affect Conceptus Development

Interferon tau (IFNT) is the pregnancy recognition signal from ruminant conceptuses. IFNT also acts with P4 to induce expression of genes for transport of nutrients, such as glucose (Gluc) and arginine (Arg) into the uterine lumen to activate mechanistic mammalian target of rapamycin (MTOR) cell signaling that stimulates proliferation, migration, gene transcription and mRNA translation by conceptus trophectoderm (Tr). In ewes, Arg and Gluc increase significantly in the uterine lumen between Days 10 and 15 of pregnancy due to increased expression of transporters for Gluc (SLC2A1 and SLC5A1) and Arg (SLC7A2B) by uterine epithelia. Arg and Gluc stimulate proliferation, migration and mRNA translation by Tr. Arg increases expression of GTP cyclohydrolase 1 (GCH1) and IFNT mRNAs while Arg and Gluc increase ornithine decarboxylase, nitric oxide synthase 2, and GCH1 mRNAs and proteins by Tr cells. GCH1 is required for synthesis of tetrahydrobiopterin, an essential cofactor for all NOS isoforms. Arg is metabolized to nitric oxide and polyamines that increase proliferation and migration of Tr cells. In pigs, Gluc, Arg, leucine (Leu) and glutamine (Gln) increase in the uterine lumen between Days 12 and 15 of pregnancy due to enhanced expression of transporters for Gluc and amino acids. Transporters for Gluc in porcine uterine LE (SLC2A1) and conceptus trophectoderm (SLC2A2) are abundant. Transporters for glutamate and neutral (SLC1A1, SLC1A4) and cationic (SLC7A1, SLC7A2, SLC7A7, SLC7A9) amino acids are expressed in uterine LE and SLC7A3 mRNA is expressed in conceptus Tr. Arg and Leu increase MTOR cell signaling and proliferation of pig Tr, as do Gluc and fructose. Azaserine, an inhibitor of hexosamine biosynthesis, inhibits effects of Gluc and fructose. Thus, select nutrients in the uterine lumen affect gene transcription and mRNA translation to affect conceptus development.

Abstract 821: miRNA profiling and target identification during normal pancreata transformation to PanINs to pancreatic ductal cancer progression in p48Cre/+-LSL-KrasG12D/+ mice: Implication for human pancreatic cancer chemoprevention and treatment

Abstract miRNA expression levels and their target genes during development of pancreatic intraepithelial neoplasia (PanINs), more so, in ductal adenocarcinomas (PDAC) remain largely unknown. Understanding miRNA regulated disease progression from PanINs to PDAC would provide opportunities to develop novel chemopreventive and therapeutic strategies. miRNA and mRNA were isolated from pancreatic tissues (normal, PanINs and PDAC from LSL-KrasG12D/+ mice). Eighty-eight miRNAs were profiled through RT-PCR using miRNA Array. miRNA target genes were identified by bioinformatics (Target Scan, Microcosm) and confirmed by RT-PCR and IHC. During PanIN 1 stage, miR-1, miR-183, Let7g, miR-124, let7-f, miR-10a, and miR-16 were significantly down-regulated (142, 4, 12, 5, 30, 7, 5 folds respectively), in contrast to miR25 (14 fold up-regulation). At PanIN-2 & -3 stages, 22 miRNAs were over-expressed and 11 miRNAs, miR-133b, miR-122, miR-196a, miR-148a, miR-92a, miR-137, miR-212, miR-1, miR363, miR-219, miR-193 (17, 9, 10, 4, 19, 22, 5, 11, 4.5, 4.3, 9.5 folds, respectively) were down-regulated compared to normal pancreas. Whereas, in PDAC, miR-184 (7 folds) and miR-150 (8 folds) were significantly over expressed and miR-133b, miR-222, miR92a, miR-140, miR-148a, miR-7a (15, 31, 116, 4, 4.2, 156 folds, respectively) were down-regulated based on volcano plot analysis. Notably, miR-1 was down-regulated during all PanIN stages (PanIN1 to 3). Also, miR-133b, miR-122, miR-92a, and miR-148a were down-regulated at PanIN 3 & PDAC. Most prominently, at PanIN 1 stage, Ras MAPK pathway (let7-g, let7-f, miR124) was activated through HDAC4 controlled by miR-1. Also, pathfinder results suggest that insulin (let7-g and let7-f) and glutamate receptor (miR-10a, miR16) signaling were disrupted at PanIN 1 stage. miRNA data suggests that disruption of glucose metabolism leads to increased FAS controlled by miR-1. MiR-25 up-regulation suggests an effect on TGF-β signaling through importin-β family proteins at PanIN1 stage. Importantly, miR-92a showed increase of ornithine decarboxylase (ODC) activity at PanIN-3 and PDAC. Similarly, miR-150 and miR-145a, regulators of β-catenin and Gadd45a, respectively, were noted to be deregulated at the PanIN 3 and PDAC. Overall, altered miRNAs target genes from PanIN progression to PDAC confirmed by RT-PCR and IHC include, ODC, HDAC4, IR substrate 1, TGFβ receptor protein 1, FAS, cyclin D1, profilin 2, exportin 4, glutaminase, p38, VEGF, p21, Bcl-2, β-catenin, p2X7, E2F1, AKT, and Gadd45a. Our data supports that miRNAs and their regulatory genes provide pathways involved in PanINs progression to PDAC. This information may help in targeting miRNAs or their regulated genes at different stages of pancreatic tumor progression. (Supported by NIH N01 CN-53300) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 821. doi:10.1158/1538-7445.AM2011-821

Myocardial ischemia and hypoxia exert opposite effects on the expression of amiloride binding protein 1 (Abp1)

The polyamine putrescine is required for normal cell growth and division. Putrescine formation is enhanced during myocardial ischemia presumably due to increased ornithine decarboxylase activity. The goal of this study was to assess the role of the diamine oxidase Abp1 in regulating putrescine degradation in the ischemic rat heart. At 24 h, 3 and 6 weeks after myocardial infarction Abp1 mRNA was significantly reduced compared to sham‐operation. Inhibition of Abp1 by amiloride prevented the in vitro differentiation of C2C12 myoblasts. Exposure of mice at 8% O2 (6 h) increased Abp1 mRNA in the hearts 11‐fold (P<0.02, n=5). Abp1 mRNA was also increased in several cell lines at 1% O2 (24 h). Reporter gene assays revealed an approx. 3,5‐fold stimulation of the Abp1 promoter in transfected C2C12 myoblasts at 1% O2. Promoter activity was also significantly enhanced by the Wilms' tumor transcription factor (Wt1), which is activated in hypoxia and controls myocardial blood vessel formation. Abp1 transcripts were reduced 60% in the hearts of Wt1‐deficient vs. wild‐type murine embryos (P<0.05, n=6). Silencing of Wt1 by RNAi reduced the Abp1 mRNA in M15 cells by more than 60%. In conclusion, myocardial ischemia and hypoxia exert opposite effects on Abp1 expression. A cooperative regulation of the Abp1 promoter by Wt1 and hypoxia may contribute to adequate putrescine levels in the developing and ischemic heart.

Effects of exposure to DAMPS and GSM signals on ornithine decarboxylase (ODC) activity: I. L-929 mouse fibroblasts

Purpose: A temporary increase in ornithine decarboxylase (ODC) activity was reported in lysed L-929 fibroblasts after exposure to the microwaves emitted by Digital Advanced Mobile Phone System (DAMPS-835 MHz, 2.5 W/kg, 8 hours). Confirmation of these data was undertaken, given the suggested potential physiopathological consequences, i.e., tumour promotion.Materials and methods: Murine L-929 fibroblasts were exposed at various Specific Absorption rates (SAR) to (DAMPS) or Global System for Mobile communications (GSM) signals using different set-ups. Cell ODC activities were assayed using 14CO2 generation from 14C-labeled L-ornithine.Results: ODC activity in live L-929 cells showed no significant alteration after exposure at an SAR of 2.5 W/kg, for one hour at the end of exposure to 50 Hz-modulated DAMPS-835 using Transverse Electro-Magnetic (TEM) cells. No significant alteration in ODC activity was observed at 6 W/kg, with active fans to regulate temperature (37°C). Tests using cell lysed after exposure in another temperature-controlled set-up (waveguides) did not confirm the published studies reporting increased ODC activity in Radio-Frequency radiation (RFR)-exposed L-929 cells. In the second part of the study, no alteration of ODC activity was detected when L-929 cells were exposed to different RFR signals: 217 Hz modulated GSM-900 (wire-patch antenna) or GSM-1800 (waveguides), and lysed before ODC measurement.Conclusion: We conclude that under our exposure conditions, DAMPS-835 and GSM signals have no influence on ODC activity in L-929 cells.

Effects of exposure to DAMPS and GSM signals on Ornithine Decarboxylase (ODC) activity: II. SH-SY5Y human neuroblastoma cells

Purpose: An increase in Ornithine Decarboxylase (ODC) activity was reported in L929 murine fibroblast cells after exposure to a digital cellular telephone signal. This result was not confirmed by several other studies, including the one reported in a companion paper. As a partner in the Perform-B programme, we extended this study to human neuroblastoma cells (SH-SY5Y), using well-defined waveguide systems to imitate exposure to radiofrequency radiation (RFR): Digital Advanced Mobile Phone System (DAMPS) or Global System for Mobile communications (GSM) signals emitted by mobile phones.Materials and methods: Human neuroblastoma cells (SH-SY5Y) were exposed at various Specific Absorption Rates (SAR) to DAMPS or GSM signals using different set-ups. Cell ODC activities were assayed using 14CO2 generation from 14C-labeled L-ornithine.Results: SH-SY5Y cells were incubated for 20 hours, and were blindly exposed to 50 Hz-modulated DAMPS-835 or 217 Hz-modulated GSM-1800 for 8 or 24 h using Information Technologies in Society (IT'IS) waveguides equipped with fans. After cell lysis, ODC activity was determined using 14C-labeled L-ornithine. ODC activity was estimated by the 14CO2 generated from 14C-labeled L-ornithine, as generated d.p.m. 14CO2/h/mg protein. The results showed that, irrespective of the signal used (835 MHz/DAMPS, or 1800 MHz/GSM) and exposure conditions (duration and SAR), human SH-SY5Y neuroblastoma cells did not exhibit any alteration in ODC enzyme activity.Conclusion: This work did not show a significant effect of mobile phone RFR exposure on ODC activity in neuroblastoma cells (SH-SY5Y).

Methylation status of CpG islands in the promoter region of genes differentially expressed in colonic mucosa from adenoma patients and controls in response to altered vegetable intake

Vegetables may protect against colorectal cancer (CRC) via changes in gene expression involved in anticarcinogenic mechanisms. There is considerable evidence that aberrant DNA methylation plays an important role in carcinogenesis. Furthermore, DNA methylation can be affected by dietary components. Therefore, in the present study, we investigated the DNA methylation status of CpG dinucleotides within the promoter region of the four genes protein kinase C b 1, ornithine decarboxylase 1, fos proto-oncogene and 5,10-methylenetetrahydrofolate reductase in the colon of female sporadic adenoma patients and healthy controls. These genes were chosen because their expression was modulated in response to altered vegetable intake, they are functionally relevant for CRC; they have CpG islands in their promoter region, and a methylation-specific restriction enzyme is available to permit quantitative assay. No significant differences in extent of methylation in colon DNA were detected for any of the four genes in both adenoma polyp patients and healthy controls after altering vegetable intake. Interestingly, before the intervention, ornithine decarboxylase 1 promoter methylation was lower in the colonic mucosa of the adenoma polyp patients when compared with healthy control subjects, which may explain the increased ornithine decarboxylase 1 activity in CRC reported in the literature. In conclusion, we found no evidence that changes in promoter methylation were responsible for differences in expression of four genes in the human colonic mucosa in response to altered vegetable intake. The mechanism(s) responsible for this altered gene expression and, indeed, potential effects on methylation of other genes remain to be determined.

Antizyme Inhibitor 2 (AZIN2/ODCp) Stimulates Polyamine Uptake in Mammalian Cells

One of the processes that regulate intracellular levels of polyamines in mammalian cells is polyamine uptake. We have measured polyamine uptake in COS7 cells for putrescine, spermidine, and spermine, obtaining K(m) values of 4.5, 1.0, and 0.8 mum, respectively. Treatment of nonconfluent cells with cycloheximide stimulated polyamine uptake and prevented the inhibitory effect found in cells preloaded with polyamines, suggesting the existence of a feedback repression mechanism mediated by antizymes. Transient transfected cells with mutated antizyme forms of AZ1, AZ2, and AZ3, which do not require frameshifting, showed a total blockade of polyamine uptake. Transfection of COS7 cells with mouse or human AZIN2, a novel member of the antizyme inhibitor family, recently characterized by our group, markedly stimulated polyamine uptake and counteracted the action of any of the three antizymes in co-transfected cells. The stimulatory effect of AZIN2 on polyamine uptake was abrogated when the putative antizyme binding sequence, formed by residues 117-140 in AZIN2, was deleted. Real time reverse transcription-PCR analysis of antizyme inhibitor transcripts revealed that in brain and testes AZIN2 is more expressed than AZIN1, especially in the testes where the relative expression was about 25-fold higher. Collectively, our results clearly indicate that AZIN2 affects polyamine homeostasis not only by increasing ornithine decarboxylase activity but also by stimulating polyamine uptake, through negating the inhibitory effect of the antizymes. This finding may have physiological relevance, mostly in testes where AZ3 and AZIN2 are mainly expressed.

Mechanism and Tissue Specificity of Nicotine-mediated Lung S-Adenosylmethionine Reduction

We previously reported that chronic nicotine infusion blocks development of Pneumocystis pneumonia. This discovery developed from our work demonstrating the inability of this fungal pathogen to synthesize the critical metabolic intermediate S-adenosylmethionine and work by others showing nicotine to cause lung-specific reduction of S-adenosylmethionine in guinea pigs. We had found nicotine infusion to cause increased lung ornithine decarboxylase activity (rate-controlling enzyme of polyamine synthesis) and hypothesized that S-adenosylmethionine reduction is driven by up-regulated polyamine biosynthesis. Here we report a critical test of our hypothesis; inhibition of ornithine decarboxylase blocks the effect of nicotine on lung S-adenosylmethionine. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of S-adenosylmethionine toward polyamine synthesis and away from methylation reactions; these shifts are reversed by inhibition of ornithine decarboxylase. Because the nicotine effect on Pneumocystis is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection, we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ornithine decarboxylase protein in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on Pneumocystis pneumonia. Earlier we reported that the full effect of nicotine requires 3 weeks of treatment, and here we show recovery is symmetrical, also requiring 3 weeks after treatment cessation. Because this time frame is similar to pneumocyte turnover time, the shift in polyamine metabolism may occur as new pneumocytes are produced.

Mouse skin chemical carcinogenesis is inhibited by antizyme in promotion‐sensitive and promotion‐resistant genetic backgrounds

Elevated polyamine content and increased ornithine decarboxylase (ODC) activity have been associated with neoplastic growth in numerous animal models and human tissues. Antizyme (AZ) is a negative regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. Preliminary evidence, obtained from transgenic mice with tissue specific overexpression of AZ indicates that tumor development can be suppressed by AZ. To extend these studies, we have examined the effect of keratin 5 (K5)- or K6-driven AZ transgenes on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical carcinogenesis of the skin, in promotion-resistant C57BL/6 and promotion-sensitive DBA/2 mice. On both genetic backgrounds, K6-AZ mice showed a reduction in tumor multiplicity, with 85% fewer tumors than wild-type controls on the C57BL/6 background and 50% fewer tumors on the DBA/2 background. K5-AZ mice developed 50% fewer tumors than controls on both backgrounds. The percent of mice with tumors and tumor size were also reduced in the K5-AZ and K6-AZ groups. Tumor and TPA-treated skin sections from K6-AZ mice exhibited the strongest AZ expression, with localization mainly in suprabasal keratinocytes. K6-AZ mice also had slightly reduced cell proliferation rates in tumors and TPA-treated skin. The lack of a more pronounced effect on cell proliferation is probably explained by the observation that AZ staining did not colocalize with proliferating cell nuclear antigen (PCNA), a marker for the proliferative compartment. These studies demonstrate a tumor-suppressive effect of AZ in C57BL/6 and DBA/2 mice, and confirm the importance of ODC and polyamines in tumor development.

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.

Chemopreventive effects of various concentrations of α-santalol on skin cancer development in CD-1 mice

Previous studies from this laboratory have indicated that alpha-santalol (5%) provides chemopreventive effects in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin cancer in CD-1 and SENCAR mice. Skin cancer development is associated with increased ornithine decarboxylase (ODC) activity, DNA synthesis and rapid proliferation of epidermal cells. The purpose of this investigation was to determine the effects of various concentrations (1.25% and 2.5%) of alpha-santalol on DMBA-initiated and TPA-promoted skin cancer development, TPA-induced ODC activity, and DNA synthesis in CD-1 mice. alpha-Santalol treatment at both concentrations (1.25% and 2.5%) prevented the skin cancer development. alpha-Santalol treatment (1.25% and 2.5%) resulted in a significant decrease in the TPA-induced ODC activity and incorporation of [3H]thymidine in DNA in the epidermis of CD-1 mice. There was no significant difference in the effects of 1.25% and 2.5% alpha-santalol on tumour incidence, multiplicity, epidermal TPA-induced ODC activity, or DNA synthesis in CD-1 mice.

Decreased apoptosis and increased ornithine decarboxylase activity in the intestinal mucosa of rats with bilateral ventromedial hypothalamus lesions

It has not been clearly demonstrated whether the ventromedial hypothalamus regulates intestinal cell growth. Ornithine decarboxylase is a key enzyme in polyamine synthesis, which plays an important role in intestinal mucosal growth. The aim of this study was to investigate whether bilateral ventromedial hypothalamus lesions affect mucosal cell growth. This was done by evaluating ornithine decarboxylase activity and apoptosis in rat small intestines. Bilateral ventromedial hypothalamus lesions were produced by thermocoagulation, done with rats under halothane anesthesia 7 days before the experiments. Rats with lesions were pair-fed with sham-operated rats. Total (truncal) vagotomy was performed before the development of ventromedial hypothalamus lesions. Ornithine decarboxylase activity and apoptosis were evaluated in the jejunal mucosa. Ornithine decarboxylase activity in the jejunal mucosa increased significantly 1 week after the development of the bilateral ventromedial lesions, and was attenuated by truncal vagotomy. Apoptosis in the jejunal mucosa was suppressed in rats with ventromedial hypothalamus lesions. In contrast to the effect on ornithine decarboxylase activity, the truncal vagotomies had no effect on apoptosis in rats with lesions. Apoptosis increased in the sham-operated rats after 24-h and 48-h fasting. Apoptosis in the jejunal mucosa of rats with ventromedial hypothalamus lesions did not increase after 24-h fasting. After 48-h fasting, jejunal apoptosis increased in rats with lesions, but not markedly. The ventromedial hypothalamus may regulate cell growth in the intestinal mucosa partly through the vagal nerve; however, the vagal nerve was not related to intestinal apoptosis controlled by the ventromedial hypothalamus.