Overexpression Of Ornithine Decarboxylase Research Articles

Ornithine decarboxylase functions in both autophagy and apoptosis in response to ultraviolet B radiation injury.

We present a mechanism for how ornithine decarboxylase (ODC) regulates the crosstalk between autophagy and apoptosis. In cancer cells, low-intensity ultraviolet B (UVBL ) induces autophagy while high-intensity UVB (UVBH ) induces apoptosis. Overexpression of ODC decreases UVBL -induced autophagy by inhibiting Atg5-Atg12 conjugation and suppressing the expression of autophagy markers LC3, Atg7, Atg12, and BECN1 proteins. In contrast, when ODC-overexpressing cells are exposed to UVBH radiation, the levels of LC3-II, Atg5-Atg12 conjugate, BECN1, Atg7, and Atg12 increase, while the apoptosis marker cleaved-PARP proteins decrease, indicating that ODC overexpression induced UVBH -induced autophagy but inhibited UVBH -induced cellular apoptosis. Additionally, when exposed to UVBH radiation, silencing BECN1, Atg5, and Atg12 genes results in a decrease in the level of LC3-II proteins but an increase in the level of cleaved-PARP proteins, and apoptotic bodies were significantly increased while autophagosomes were significantly decreased. These findings imply that ODC inhibits apoptosis in cells via the autophagy pathway. The role of Atg12 in ODC-overexpressing cells exposed to UVBH radiation is investigated using site-directed mutagenesis. Our results indicate that the Atg12-D111S mutant has increased cell survival. The Atg12-ΔG186 mutant impairs autophagy and enhances apoptosis. We demonstrate that when ODC-overexpressing cells are silenced for the Atg12 protein, autophagy and apoptosis are strongly affected, and ODC-induced autophagy protects against UVBH -induced apoptosis via the Atg12 protein.

Effects of ODC on polyamine metabolism, hormone levels, cell proliferation and apoptosis in goose ovarian granulosa cells

Ornithine decarboxylase (ODC) plays an indispensable role in the process of polyamine biosynthesis. Polyamines are a pivotal part of living cells and have diverse roles in the regulation of cell proliferation and apoptosis, aging and reproduction. However, to date, there have been no reports about ODC regulating follicular development in goose ovaries. Here, we constructed ODC siRNA and overexpression plasmids and transfected them into goose primary granulosa cells (GCs) to elucidate the effects of ODC interference and overexpression on the polyamine metabolism, hormone levels, cell apoptosis and proliferation of granulosa cells. After interfering with ODC in GCs, the mRNA and protein levels of ODC and the content of putrescine were greatly decreased (P < 0.05). When ODC was overexpressed, ODC mRNA and protein levels and putrescine content were greatly increased (P < 0.05). The polyamine-metabolizing enzyme genes ornithine decarboxylase antizyme 1 (OAZ1) and spermidine / spermine-N1-acetyltransferase (SSAT) were significantly increased, and spermidine synthase (SPDS) was significantly decreased when ODC was downregulated (P < 0.05). OAZ1, SPDS and SSAT were significantly increased when ODC was upregulated (P < 0.05). In addition, after interference with ODC, progesterone (P4) levels in the culture medium of GCs increased greatly (P < 0.05), while the overexpression of ODC caused the P4 level to decrease significantly (P < 0.05). After ODC downregulation, granulosa cell activity was significantly reduced, the apoptosis rate was significantly increased, and the BCL-2 / BAX ratio was downregulated (P < 0.05). Under ODC overexpression, the activity of GCs was notably increased, the apoptosis rate was significantly reduced, and the BCL-2 / BAX protein ratio was upregulated (P < 0.05). Our study successfully induced ODC interference and overexpression in goose ovarian GCs, and ODC regulated mainly putrescine content in GCs with a slight influence on spermidine and spermine. Moreover, ODC participated in the adjustment of P4 levels in the culture medium of GCs, promoted granulosa cell proliferation and inhibited granulosa cell apoptosis.

Ornithine Decarboxylase Is Sufficient for Prostate Tumorigenesis via Androgen Receptor Signaling

Increased polyamine synthesis is known to play an important role in prostate cancer. We aimed to explore its functional significance in prostate tumor initiation and its link to androgen receptor (AR) signaling. For this purpose, we generated a new cell line derived from normal epithelial prostate cells (RWPE-1) with overexpression of ornithine decarboxylase (ODC) and used it for invitro and invivo experiments. We then comprehensively analyzed the expression of the main metabolic enzymes of the polyamine pathway and spermine abundance in 120 well-characterized cases of human prostate cancer and high-grade prostate intraepithelial neoplasia (HGPIN). Herein, we show that the ODC-overexpressing prostate cells underwent malignant transformation, revealing that ODC is sufficient for de novo tumor initiation in 94% of injected mice. This oncogenic capacity was acquired through alteration of critical signaling networks, including AR, EIF2, and mTOR/MAPK. RNA silencing experiments revealed the link between AR signaling and polyamine metabolism. Human prostate cancers consistently demonstrated up-regulation of the main polyamine enzymes analyzed (ODC, polyamine oxidase, and spermine synthase) and reduction of spermine. This phenotype was also dominant in HGPIN, rendering it a new biomarker of malignant transformation. In summary, we report that ODC plays a key role in prostate tumorigenesis and that the polyamine pathway is altered as early as HGPIN.

Abstract 2135: Chemoprevention of tobacco carcinogen-induced lung cancer by DFMO and Licofelone administered individually or in combination in female A/J mice

Abstract Lung cancer is the leading cause of cancer death among both men and women in the US. Inflammatory eicosanoids and leukotrienes derived from cyclooxygenase (COX)-2 and 5-lipoxygenase (LOX) pathway positively influences lung tumor growth. Overexpression of ornithine decarboxylase (ODC) enzyme activity and polyamines has been associated with proliferation and progression of lung cancer. Thus, targeting both tumor inflammatory molecules and polyamines synthesis may provide additive/synergistic antitumor effects. To overcome clinical toxicity of high dose difluoromethyl ornithine (DFMO), an inhibitor of ODC; and to target both COX-2/5LOX, we tested low dose DFMO combined with Licofelone, a novel dual inhibitor COX-2/5-LOX, in lung cancer model. At 7 weeks of age, mice (25 mice/group) were treated with 10 μmol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) /mouse to induce the lung tumors. Three weeks after the NNK treatment, mice were fed either control or experimental diets containing 200, 400 ppm licofelone or 1500 and 3000 ppm DFMO or low-dose combinations of both agents. Mice were killed at 20 weeks (10 mice/group) and/or 36 weeks (15 mice/group) after NNK-treatment for lung tumor evaluation. Administration of 200 and 400 ppm licofelone significantly suppressed lung tumor (adenoma + adenocarcinoma) formation by &amp;gt;32 to 35% (p43 to 47% (p25 to 30% (p44% (p53% (p60% (p&amp;lt;0.0001) respectively at 17 and 34 weeks exposure. These results suggest that low-dose combinations of licofelone and DFMO inhibit the NNK-induced lung adenocarcinoma formation synergistic/additive manner. Immunohistochemistry analysis revealed that lung tumors from mice exposed to licofelone plus DFMO showed significantly reduced proliferating cell nuclear antigen (PCNA)-positive index, increased accumulation of TUNEL positive cells as compared to lung tumors from NNK-treated mice fed with control diet. These results demonstrate the targeting lung tumor COX-LOX with ODC may provide better efficacy when compared to individual targets on tobacco carcinogen-induced lung adenocarcinomas formation. {Supported by NIH, NCI grants NO1-CN-53300 and Kerley-Cade Chair}. Citation Format: Jagan Mohan Reddy Patlolla, Levy Kopelovich, Li Qian, Laura Biddick, Yuting Zhang, Michael K. Sadeghi, Dhimant Desai, Shantu Amin, Stan Lightfoot, Vernon E. Steele, Chinthalapally V. Rao. Chemoprevention of tobacco carcinogen-induced lung cancer by DFMO and Licofelone administered individually or in combination in female A/J mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2135. doi:10.1158/1538-7445.AM2014-2135

Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15±2.5tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8±1.5tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in tumors induced in these two murine models suggesting the specificity of Notch pathway in this regard. These data provide a novel role of ODC in augmenting tumorigenesis via negatively regulated Notch-mediated expansion of stem cell compartment.

Downregulation of tumstatin expression by overexpression of ornithine decarboxylase

Tumor angiogenesis, a pivotal process for cancer growth and metastasis, requires both upregulation of pro-angiogenic molecules and downregulation of anti-angiogenic molecules. Anti-angiogenesis therapy represents a promising way for cancer treatment. Tumstatin, a novel endogenous angiogenesis inhibitor, inhibits endothelial cell proliferation, pathological angiogenesis and tumor growth. Ornithine decarboxylase (ODC), overexpressed in various cancers, is associated with cell transformation, tumor invasion and angiogenesis. We found that the expression of tumstatin was suppressed in ODC-overexpressing human cancer cells and renal carcinoma tissues. We presumed that ODC overexpression may downregulate the expression of tumstatin. To be able to test this hypothesis, we generated HEK293 cells that overexpress ODC (ODC transfectants) and characterized the following experimental groups: PBS-treated group, mock transfectants, ODC transfectants, ODC transfectants transfected with pcDNA-ODCr (an antisense ODC-expressing plasmid) group and putrescine-treated group. The effect of ODC overexpression on tumstatin expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis and dual luciferase reporter assay. ODC-overexpressing cells and putrescine-treated cells showed suppressed tumstatin mRNA and protein expression, and decreased tumstatin gene promoter activity. Thus, ODC overexpression suppresses the expression of tumstatin, which may provide fundamental evidence for the combination of anti-angiogenic therapy and conventional therapy for cancer treatment.

Polyamine and methionine adenosyltransferase 2A crosstalk in human colon and liver cancer

Methionine adenosyltransferase (MAT) is an essential enzyme that is responsible for the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and precursor of polyamines. MAT1A is expressed in normal liver and MAT2A is expressed in all extrahepatic tissues. MAT2A expression is increased in human colon cancer and in colon cancer cells treated with mitogens, whereas silencing MAT2A resulted in apoptosis. The aim of the current work was to examine the mechanism responsible for MAT2A-dependent growth and apoptosis. We found that in RKO (human adenocarcinoma cell line) cells, MAT2A siRNA treatment lowered cellular SAMe and putrescine levels by 70–75%, increased apoptosis and inhibited growth. Putrescine supplementation blunted significantly MAT2A siRNA-induced apoptosis and growth suppression. Putrescine treatment (100pmol/L) raised MAT2A mRNA level to 4.3-fold of control, increased the expression of c-Jun and c-Fos and binding to an AP-1 site in the human MAT2A promoter and the promoter activity. In human colon cancer specimens, the expression levels of MAT2A, ornithine decarboxylase (ODC), c-Jun and c-Fos are all elevated as compared to adjacent non-tumorous tissues. Overexpression of ODC in RKO cells also raised MAT2A mRNA level and MAT2A promoter activity. ODC and MAT2A are also overexpressed in liver cancer and consistently, similar MAT2A-ODC-putrescine interactions and effects on growth and apoptosis were observed in HepG2 cells. In conclusion, there is a crosstalk between polyamines and MAT2A. Increased MAT2A expression provides more SAMe for polyamines biosynthesis; increased polyamine (putrescine in this case) can activate MAT2A at the transcriptional level. This along with increased ODC expression in cancer all feed forward to further enhance the proliferative capacity of the cancer cell.

Ornithine decarboxylase and glutamate decarboxylase 65 as prognostic markers of gallbladder malignancy: A clinicopathological study in benign and malignant lesions of the gallbladder

Ornithine decarboxylase (ODC) plays a critical role in cell proliferation and is overexpressed in a variety of cancers. Furthermore, γ-aminobutyric acid (GABA) content and glutamate decarboxylase (GAD) activity are increased in neoplastic tissues in colon and breast cancer. However, few studies have examined these molecules in gallbladder cancer specimens. We observed the expression levels of ODC and GAD65 in benign and malignant lesions of the gallbladder and investigated their clinicopathological significance for the first time. The expression levels of ODC and GAD65 in specimens from gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenomatous polyps (n=15) and chronic cholecystitis (n=35) were detected using immunohistochemical methods. Kaplan-Meier survival and Cox regression analyses were carried out to explore the clinical and pathological correlations. The levels of positive staining of ODC and GAD65 were significantly higher in gallbladder adenocarcinoma than in peritumoral tissues, adenomatous polyps and chronic cholecystitis. The Kaplan‑Meier survival analysis and Cox regression analysis showed that the expression of ODC and GAD65 correlated significantly with the one-year survival rate and the mean survival time of the patients postoperatively. We conclude that the overexpression of ODC and GAD65 are significant in the carcinogenesis and progression of gallbladder adenocarcinoma. They may be important biological markers for the evaluation of biological behaviors and the prognosis of gallbladder adenocarcinoma.

S -adenosylmethionine decarboxylase overexpression inhibits mouse skin tumor promotion

Neoplastic growth is associated with increased polyamine biosynthetic activity and content. Tumor promoter treatment induces the rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (AdoMetDC), and targeted ODC overexpression is sufficient for tumor promotion in initiated mouse skin. We generated a mouse model with doxycycline (Dox)-regulated AdoMetDC expression to determine the impact of this second rate-limiting enzyme on epithelial carcinogenesis. TetO-AdoMetDC (TAMD) transgenic founders were crossed with transgenic mice (K5-tTA) that express the tetracycline-regulated transcriptional activator within basal keratinocytes of the skin. Transgene expression in TAMD/K5-tTA mice was restricted to keratin 5 (K5) target tissues and silenced upon Dox treatment. AdoMetDC activity and its product, decarboxylated AdoMet, both increased approximately 8-fold in the skin. This enabled a redistribution of the polyamines that led to reduced putrescine, increased spermine, and an elevated spermine:spermidine ratio. Given the positive association between polyamine biosynthetic capacity and neoplastic growth, it was somewhat surprising to find that TAMD/K5-tTA mice developed significantly fewer tumors than controls in response to 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate chemical carcinogenesis. Importantly, tumor counts in TAMD/K5-tTA mice rebounded to nearly equal the levels in the control group upon Dox-mediated transgene silencing at a late stage of tumor promotion, which indicates that latent viable initiated cells remain in AdoMetDC-expressing skin. These results underscore the complexity of polyamine modulation of tumor development and emphasize the critical role of putrescine in tumor promotion. AdoMetDC-expressing mice will enable more refined spatial and temporal manipulation of polyamine biosynthesis during tumorigenesis and in other models of human disease.

Abstract 4126: Synergism of polyamine synthesis inhibition and AMP kinase activation on human melanoma proliferation and migration

Abstract Introduction: Metformin has been shown to inhibit human cancer cell growth via activation of MAP kinase with resultant inhibition of mTOR signaling pathway. We have reported the over-expression of ornithine decarboxylase (ODC) in human melanoma cell lines. ODC is a key enzyme in polyamine biosynthesis. We determined the effect of metformin in combination with DMFO, an ODC inhibitor, on tumor proliferation and migration, and the possible mechanisms, using human melanoma cells. Methods: Human melanoma cells were treated with Metformin, DFMO, or combination. Cell proliferation assay was performed using Cell Titer Blue proliferation assay. Cell migration assay was performed by seeding melanoma cells in 24-well plate. After overnight incubation, an artificial homogenous wound was created onto the monolayer with a plastic micropipette tip. After wounding, the migration of cells into the wound was observed at 0- and 48-hour time points. Quantitative RT-PCR for ODC1 detection was performed using Assay-on-Demand Hs00159739-ml. GAPDH served as housekeeping control. Western blotting was performed to determine the expression of AMP kinase, mTOR, p70S6K and 4E-BP1, Raf-1 and Raf-B. Data were presented as means ± SD for the three separate experiments. For comparison between groups, the student's t test was used and p&amp;lt; 0.05 was considered to be statically significant. Results: There was dose-dependent inhibition of melanoma cell proliferation and migration with either metformin or DFMO. Synergistic inhibition was observed with the combination treatment on both cell migration and proliferation. There was synergistic inhibition of ODC expression with the combined treatment compared with DFMO alone (p&amp;lt;0.01). The combination treatment also lead to significant enhancement of AMPK activation and increased phosphorylation of AMPKΨ at Thr-172 compared with metformin alone (p&amp;lt;0.01). The combination treatment resulted in inhibition of mTOR signaling with decreased phosphorylation of mTOR, p70S6K and 4E-BP1, Raf-1 and Raf-B in treated cancer cells, when compared with individual drug treatments. Conclusion: We have observed a synergistic effect of AMP kinase activation and polyamine synthesis inhibition on suppressing human melanoma proliferation and migration. The synergism is due to enhanced inhibition of both polyamine synthesis and mTOR signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4126. doi:1538-7445.AM2012-4126

Abstract 1522: Elevated polyamine levels generate an immunosuppressive oncogenic microenvironment

Abstract Increased suprabasal epidermal activity of ornithine decarboxylase (ODC), a key regulatory enzyme in polyamine biosynthesis, stimulates cellular proliferation and vascularization in the underlying dermis, and promotes skin tumorigenesis after treatment with a single, subthreshold dose of a carcinogen in ODCER transgenic mice. Interestingly, skin tumor formation is also induced in ODCER transgenic mice by wounding and does not require prior carcinogen treatment. This wound-induced tumor formation is dependent on the polyamine-induction of sustained recruitment of inflammatory cells to the dermal wound site. Moreover, when weakly transformed (Ras activation) primary keratinocytes (Ker/Ras) were injected subcutaneously in syngeneic ODCER transgenic mice, tumor growth occurred while it did not in normal littermates. These data suggest the generation of an oncogenic stromal microenvironment in mice with elevated epidermal polyamine levels. Polyamine-Based Therapy (PaBT: combination of the well-characterized ODC inhibitor, difluoromethylornithine (DFMO), and an inhibitor of cellular polyamine transport, AMXT 1501) significantly inhibited tumor growth in wildtype FVB mice that were orthotopically injected with either syngeneic Ker/Ras cells or neu02 mammary tumor cells. Flow cytometry analyses of tumor inflammatory infiltrates revealed that PaBT significantly increased the percentage of CD3+ cell population (includes T cells and NK cells) and decreased the percentage of both F4/80+ (macrophage) and Gr-1+ (neutrophil and monocyte) subpopulations of CD45+ cells compared to tumors in control, non-treated mice. When PaBT treatment was stopped and tumors were resected, we re-challenged two weeks later with a second injection of neu02 tumor cells. Mice that had been previously treated with PaBT were protected from secondary tumor growth (demonstrating no or very little tumor growth) compared to mice that never received PaBT. In order to examine whether elevated polyamine biosynthesis suppresses a T-cell mediated adaptive immune response, we examined the effect of ODC overexpression in keratinocytes on a classic contact hypersensitivity (CHS) response test using ODCER transgenic mice. These mice showed reduced ear swelling in comparison to normal littermates together with reduced neutrophil infiltration and decreased migration of FITC-loaded dendritic cells to draining lymph nodes following hapten elicitation of CHS. Collectively, these data suggest that the immunosuppression mediated by increased epidermal ODC activity may contribute to an environment conducive to tumor growth, and, conversely, that depletion of tumor polyamines will block tumorigenesis in part by normalizing the stromal microenvironment and restoring tumor immune surveillance. (Supported by NIH grant CA070739 and funding from Aminex Therapeutics Inc) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1522. doi:1538-7445.AM2012-1522

Ornithine decarboxylase prevents dibenzoylmethane‐induced apoptosis through repressing reactive oxygen species generation

Dibenzoylmethane (DBM) belongs to the flavonoid family and is a minor constituent of the root extract of licorice and the β-diketone analogue of curcumin. It exhibits antimutagenic, anticancer, and chemopreventive effects. Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway, plays an important role in growth, proliferation, and transformation. Our previous studies showed ODC overexpression prevented etoposide-, paclitaxel-, and cisplatin-induced apoptosis. Here, we investigated one mechanism of DBM-induced apoptosis and the antiapoptotic effects of ODC during DBM treatment. We found that DBM induced apoptosis, promoted reactive oxygen species (ROS) generation, and disrupted the mitochondrial membrane potential (Δψ(m). N-acetylcysteine, a ROS scavenger, reduced DBM-induced apoptosis, which led to the loss of Δψ(m) due to reduced ROS. Overexpression of ODC in parental cells had the same effects as the ROS scavenger. The results demonstrated that DBM-induced apoptosis was a ROS-dependent pathway and ODC overexpression blocked DBM-induced apoptosis by inhibiting intracellular ROS production.

Hydroxydibenzoylmethane induces apoptosis through repressing ornithine decarboxylase in human promyelocytic leukemia HL-60 cells

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Δψ(m)), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.

Elevated Epidermal Ornithine Decarboxylase Activity Suppresses Contact Hypersensitivity

Previous reports have shown that elevated polyamine biosynthesis is sufficient to promote skin tumorigenesis in susceptible mouse strains. We hypothesized that increased activity of epidermal ornithine decarboxylase (ODC), a key regulatory enzyme in polyamine biosynthesis, may suppress the cutaneous immune response in addition to stimulating proliferation. Using an ODCER transgenic mouse model in which ODC is targeted to the epidermis, we examined the effect of ODC overexpression in keratinocytes on a classic contact hypersensitivity (CHS) response. Compared with normal littermate mice, ODCER transgenic mice showed reduced ear swelling, reduced neutrophil infiltration, and decreased migration of fluorescein isothiocyanate-loaded dendritic cells (DCs) to draining lymph nodes following hapten elicitation of CHS. In addition, elevated epidermal ODC activity suppressed the levels of cytokines keratinocyte-derived chemokine, monocyte chemoattractant protein-1, interleukin-6 (IL-6), and IL-10. Adoptive transfer of lymphocytes from sensitized ODCER transgenic or normal littermate mice to naive ODCER transgenic or wild-type mice indicated that elevated epidermal ODC activity suppresses both the sensitization and elicitation phases of CHS. The specific ODC inhibitor, α-difluoromethylornithine, abrogated all suppressive effects of ODC in CHS reactions. Collectively, these data suggest that the immunosuppression promoted by increased epidermal ODC is mediated by a reduction in cytokine levels, which suppresses DC migration and reduces immune cell infiltration to the site of hapten application.

Overexpression of ornithine decarboxylase suppresses thapsigargin-induced apoptosis.

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, has paradoxical roles in apoptosis. Our published papers show overexpression of ODC prevents the apoptosis induced by many cytotoxic drugs. Thapsigargin (TG) is an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) ATPase (SERCA) pumps and causes ER stress-induced apoptosis. We used ODC overexpressing cell lines to examine whether overexpression of ODC inhibits TG-induced apoptosis. Our results indicated overexpression of ODC attenuated TG-induced apoptosis. Overexpression of ODC blocked procaspase-4 cleavage and phosphorylation of protein kinase-like ER-resident kinase (PERK), triggered by TG. It also attenuated the increase in CAAT/enhancer binding protein homologous protein (CHOP). Cells with overexpressed ODC had greater Bcl-2 expression. Overexpression of ODC preserved the expression of Bcl-2, inhibited the increase in Bak and stabilized mitochondrial membrane potential without the influences of TG. Cytochrome c release and down-stream caspase activation were blocked. That is, overexpression of ODC inhibits the mitochondria-mediated apoptotic pathway, induced by TG. Finally, overexpression of ODC maintains the protein and mRNA expression of SERCA. In conclusion, overexpression of ODC suppresses TG-induced apoptosis by blocking caspase-4 activation and PERK phosphorylation, attenuating CHOP expression and inhibiting the mitochondria-mediated apoptotic pathway.

Abstract 57: Inhibition of human melanoma proliferation by combination treatment with ODC inhibitor and mTOR inhibitor

Abstract Recent reports have demonstrated that polyamine synthesis is critical for tumor cell growth. Furthermore, mTOR was also shown to be critical for tumor cell growth. We hypothesized that inhibition of key polyamine synthesis enzyme, ornithine decarboxylase (ODC), and mTOR would have growth inhibitory effect on human melanoma cells. ODC expression was evaluated in human melanoma and human melanoma cell lines by Western blot using polyclonal goat anti-ODC and quantitative real-time PCR using TaqMan Gene Expression Assay Applied Biosystems). The siRNA duplexes targeting ODC and a scrambled control siRNA were designed and synthesized (Santa Cruz, CA) and transient transfections were performed. Cell proliferation assay was performed using Cell Titer Blue cell proliferation assay according to the manufacturer's protocol. The cells were treated with or without various concentrations of DFMO or rapamycin or combination then incubated up to 96 hours. For comparison between groups, the Student's t test were used and p&amp;lt;0.05 was considered to be statistically significant. Over-expression of ODC was observed in human melanoma cells compared with tumor asssociated fibroblasts as well as human melanoma cells. Over-expression of ODC was observed in six of the ten human melanoma cell lines on Western blot analysis and RT-PCR analysis. Dose-dependent growth inhibition of human melanoma cells were observed following DFMO treatment (p&amp;lt;0.05) in melanoma cell lines with over expression of ODC and correlated with suppression of ODC expression levels. Grow inhibition was also observed folllowing Rapamycin treatment in the melanoma cell lines. Treatment with ODC-siRNA also resulted in significant growth inhibition of melanoma cell lines. Effects of DFMO induced growth inhibition of melanoma cells were further enhanced with combined treatment with rapamycin. Overexpression of ODC was observed in human melanoma cells. Inhibition of polyamine synthesis and mTOR can have significant growth inhibitory effects on human melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 57.

Post-transcriptional regulation of MEK-1 by polyamines through the RNA-binding protein HuR modulating intestinal epithelial apoptosis

MEK-1 [MAPK (mitogen-activated protein kinase) kinase-1] is an important signal transducing enzyme that is implicated in many aspects of cellular functions. In the present paper, we report that cellular polyamines regulate MEK-1 expression at the post-transcriptional level through the RNA-binding protein HuR (Hu-antigen R) in IECs (intestinal epithelial cells). Decreasing the levels of cellular polyamines by inhibiting ODC (ornithine decarboxylase) stabilized MEK-1 mRNA and promoted its translation through enhancement of the interaction between HuR and the 3'-untranslated region of MEK-1 mRNA, whereas increasing polyamine levels by ectopic ODC overexpression destabilized the MEK-1 transcript and repressed its translation by reducing the abundance of HuR-MEK-1 mRNA complex; neither intervention changed MEK-1 gene transcription via its promoter. HuR silencing rendered the MEK-1 mRNA unstable and inhibited its translation, thus preventing increases in MEK-1 mRNA and protein in polyamine-deficient cells. Conversely, HuR overexpression increased MEK-1 mRNA stability and promoted its translation. Inhibition of MEK-1 expression by MEK-1 silencing or HuR silencing prevented the increased resistance of polyamine-deficient cells to apoptosis. Moreover, HuR overexpression did not protect against apoptosis if MEK-1 expression was silenced. These results indicate that polyamines destabilize the MEK-1 mRNA and repress its translation by inhibiting the association between HuR and the MEK-1 transcript. Our findings indicate that MEK-1 is a key effector of the HuR-elicited anti-apoptotic programme in IECs.

The polyamine metabolism genes ornithine decarboxylase and antizyme 2 predict aggressive behavior in neuroblastomas with and without MYCN amplification

High polyamine (PA) levels and ornithine decarboxylase (ODC) overexpression are well-known phenomena in many aggressive cancer types. We analyzed the expression of ODC and ODC-activity regulating genes antizymes 1-3 (OAZ1-3) and antizyme inhibitors 1-2 (AZ-IN1-2) in human neuroblastoma (NB) tumors and correlated these with genetic and clinical features of NB. Since ODC is a known target gene of MYCN, the correlation between ODC and MYCN was of special interest. Data were obtained from Affymetrix micro-array analysis of 88 NB tumor samples. In addition, mRNA expression levels of ODC, OAZ2 and MYCN in a MYCN-inducible NB cell line were determined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). ODC mRNA expression in NB tumors was significantly predictive of decreased overall survival probability and correlated with several unfavorable clinical NB characteristics (all p < 0.005). Interestingly, high ODC mRNA expression also showed significant correlation with poor survival prognosis in Kaplan-Meier analyses stratified for patients without MYCN amplification, suggesting an additional role for ODC independent of MYCN. Conversely, high OAZ2 mRNA expression correlated with increased survival and with several favorable clinical NB characteristics (all p < 0.003). In addition, we provide first evidence of a role for MYCN-associated transcription factors MAD2 and MAD7 in ODC regulation. In NB cell cultures, ectopic overexpression of MYCN altered ODC but not OAZ2 mRNA levels. In conclusion, these data suggest that elevated ODC and low OAZ2 mRNA expression levels correlate with several unfavorable genetic and clinical features in NB, offering new insights into PA pathways and PA metabolism-targeting therapy in NB.

Overexpression of ornithine decarboxylase increases myogenic potential of H9c2 rat myoblasts

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines-such as putrescine, spermidine and spermine-are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion-by means of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase-abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells.

Augmented bladder urothelial polyamine signaling and block of BK channel in the pathophysiology of overactive bladder syndrome

Overactive bladder syndrome (OAB) is an idiopathic condition characterized by urinary urgency and urge incontinence. Detrusor overactivity has been traditionally described as the physiologic mechanism for OAB. However, the bladder urothelium (BU) may also be involved in the pathophysiology. This study measured polyamine signaling and its downstream effects on membrane conductivity in bladder urothelial cells (BUC) obtained from asymptomatic and OAB subjects. Immunohistofluorescence was used to measure ornithine decarboxylase (ODC) expression in BU. BUC, cultured from BU biopsies, were used for electrophysiologic studies. dl-alpha-Difluoromethylornithine (DFMO), spermine, or spermidine was used to modulate polyamine signaling in BUC. Results showed ODC overexpression in OAB BU. In OAB BUC, whole cell and cell-attached configuration showed significantly decreased currents. Using inside-out patches, outward currents increased significantly, suggesting a cytoplasmic source of the outward current block in OAB BUC. In control BUC, outward currents were mediated by the large-conductance calcium-activated potassium (BK) channel due to calcium dose-dependence and block by iberiotoxin. Spermidine and spermine blocked the outward current in normal BUC in dose-dependent fashion. Conversely, DFMO significantly increased (P < 0.01) outward currents in OAB BUC both in cell-attached and in whole cell configuration. The outward currents in DFMO-treated-OAB BUC could be significantly reduced (P < 0.05) by adding back spermidine and spermine. These data suggest that polyamine signaling is upregulated in OAB urothelium and OAB BUC. Furthermore, polyamines in BUC block the BK channel. Targeting of bladder urothelial polyamine signaling may represent a novel approach for OAB treatment based on pathophysiologic mechanisms.