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Embryonic diapause is an evolutionary strategy to ensure that offspring are born when maternal and environmental conditions are optimal for survival. In many species of carnivores, obligate embryonic diapause occurs in every gestation. In mustelids, the regulation of diapause and reactivation is influenced by photoperiod, which then acts to regulate the secretion of pituitary prolactin. Prolactin in turn regulates ovarian steroid function. Reciprocal embryo transplant studies indicate that this state of embryonic arrest is conferred by uterine conditions and is presumed to be due to a lack of specific factors necessary for continued development. Studies of global gene expression in the mink (Neovison vison) revealed reduced expression of a cluster of genes that regulate the abundance of polyamines in the uterus during diapause, including the rate-limiting enzyme in polyamine production, ornithine decarboxylase (ODC). In addition, in this species, in vivo inhibition of the conversion of ornithine to the polyamine, putrescine, induces a reversible arrest in embryonic development and an arrest in both trophoblast and inner cell mass proliferation in vitro. Putrescine, at 0.5, 2 and 1,000 μM concentrations induced reactivation of mink embryos in culture, indicated by an increase in embryo volume, observed within five days. Further, prolactin induces ODC1 expression in the uterus, thereby regulating uterine polyamine levels. These results indicate that pituitary prolactin acts on ovarian and uterine targets to terminate embryonic diapause. In summary, our findings suggest that the polyamines, with synthesis under the control of pituitary prolactin, are the uterine factor whose absence is responsible for embryonic diapause in mustelid carnivores., (© 2016 Blackwell Verlag GmbH.)

Most proteasome substrates are marked for degradation by ubiquitin conjugation, but some are targeted by other means. The properties of these exceptional cases provide insights into the general requirements for proteasomal degradation. Here the focus is on three ubiquitin-independent substrates that have been the subject of detailed study. These are Rpn4, a transcriptional regulator of proteasome homeostasis, thymidylate synthase, an enzyme required for production of DNA precursors and ornithine decarboxylase, the initial enzyme committed to polyamine biosynthesis. It can be inferred from these cases that proteasome association and the presence of an unstructured region are the sole prerequisites for degradation. Based on that inference, artificial substrates have been designed to test the proteasome's capacity for substrate processing and its limitations. Ubiquitin-independent substrates may in some cases be a remnant of the pre-ubiquitome world, but in other cases could provide optimized regulatory solutions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Induction of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, in ODC transgenic skin stimulates epidermal proliferation but not hyperplasia, activates underlying stromal cells and promotes skin tumorigenesis following a single subthreshold dose of a carcinogen. Because chronic wounds are a well-recognized risk factor for skin cancer, we investigated the response to a tissue remodeling event in normal skin that is abraded to remove only the epidermal layer in K6/ODC transgenic (follicular ODC expression) and in inducible ODCER transgenic mice (suprabasal ODC expression). When regenerative epidermal hyperplasia was resolved in normal littermates following abrasion, ODC transgenic mice exhibited progressive epidermal hyperplasia with formation of benign tumor growths and maintained an increased epidermal proliferation index and activation of translation-associated proteins at abrasion sites. The epidermal hyperplasia and tumor-like growth was accompanied by activation of underlying stromal cells and prolonged infiltration of inflammatory cells. Treatment with the anti-inflammatory agent dexamethasone did not reduce the high proliferative index in the regenerated epidermis but dramatically reduced the epidermal hyperplasia and prevented the wound-induced tumor growths in abraded ODCER skin. Treatment with α-difluoromethylornithine, a specific inhibitor of ODC activity, normalized the wound response in transgenic mice and decreased wound-induced inflammation if administered from the time of abrasion but not if initiated 4 days following abrasion. These results suggest a role for polyamines in prolonging wound-associated inflammation in addition to stimulating proliferation both of which are sufficient to sustain epidermal hyperplasia and benign tumor growth even in the absence of genetic damage.

Polyamines are organic cations shown to control gene expression at the transcriptional, posttranscriptional, and translational levels. Multiple cellular oncogenic pathways are involved in regulation of transcription and translation of polyamine-metabolizing enzymes. As a consequence of genetic alterations, expression levels and activities of polyamine-metabolizing enzymes change rapidly during tumorigenesis resulting in high levels of polyamines in many human epithelial tumors. This review summarizes the mechanisms of polyamine regulation by canonical tumor suppressor genes and oncogenes, as well as the role of eukaryotic initiation factor 5A (EIF5A) in cancer. The importance of research utilizing pharmaceutical inhibitors and cancer chemopreventive strategies targeting the polyamine pathway is also discussed.

Polyamines are small cationic molecules that in adult brain are connected to neuronal signaling by regulating inward-rectifier K(+)-channels and different glutamate receptors. Antizyme inhibitors (AZINs) regulate the cellular uptake of polyamines and activate ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis. Elevated levels of ODC activity and polyamines are detected in various brain disorders including stroke and Alzheimer's disease (AD). We originally reported a novel brain- and testis-specific AZIN, called AZIN2, the distribution of which we have now studied in normal and diseased human brain by in situ hybridization and immunohistochemistry. We found the highest accumulation of AZIN2 in a pearl-on-the-string-like distribution along the axons in both the white and gray matter. AZIN2 was also detected in a vesicle-like distribution in the somas of selected cortical pyramidal neurons. Double-immunofluorescence staining revealed co-localization of AZIN2 and N-methyl D-aspartate-type glutamate receptors (NMDARs) in pyramidal neurons of the cortex. Moreover, we found accumulation of AZIN2 in brains affected by AD, but not by other neurodegenerative disorders (CADASIL or Lewy body disease). ODC activity is mostly linked to cell proliferation, whereas its regulation by AZIN2 in post-mitotically differentiated neurons of the brain apparently serves different purposes. The subcellular distribution of AZIN2 suggests a role in vesicular trafficking.

Ornithine decarboxylase (ODC) catalyzes the first and rate limiting step in the biosynthesis of polyamines in most eukaryotes. Because polyamines have pleiotropic and often dramatic effects on cellular processes at both high and low concentrations, ODC expression is tightly controlled. ODC is regulated by a family of polyamine-induced proteins, antizymes, which bind to, and inactivate it. In mammals, and apparently most vertebrates, antizymes are in turn antagonized by proteins called antizyme inhibitors. Antizyme inhibitors are homologs of ODC that have lost their decarboxylation activity but have retained their ability to bind antizyme, in most cases even more tightly than ODC. We present a phylogenetic analysis of over 200 eukaryotic homologs of ODC and evaluate their potential to be either true ODCs or catalytically inactive proteins that might be analogs of the previously identified antizyme inhibitors. This analysis yielded several orthologous groups of putative novel antizyme inhibitors each apparently arising independently. In the process we also identify previously unrecognized ODC paralogs in several evolutionary branches, including a previously unrecognized ODC paralog in mammals, and we evaluate their biochemical potential based on their pattern of amino acid conservation.

We have previously observed that a chronic drinking water exposure to monomethylarsonous acid [MMA(III)], a cellular metabolite of inorganic arsenic, increases tumor frequency in the skin of keratin VI/ornithine decarboxylase (K6/ODC) transgenic mice. To characterize gene expression profiles predictive of MMA(III) exposure and mode of action of carcinogenesis, skin and papilloma RNA was isolated from K6/ODC mice administered 0, 10, 50, and 100 ppm MMA(III) in their drinking water for 26 weeks. Following RNA processing, the resulting cRNA samples were hybridized to Affymetrix Mouse Genome 430A 2.0 GeneChips(R). Micoarray data were normalized using MAS 5.0 software, and statistically significant genes were determined using a regularized t-test. Significant changes in bZIP transcription factors, MAP kinase signaling, chromatin remodeling, and lipid metabolism gene transcripts were observed following MMA(III) exposure as determined using the Database for Annotation, Visualization and Integrated Discovery 2.1 (DAVID) (Dennis et al., Genome Biol 2003;4(5):P3). MMA(III) also caused dose-dependent changes in multiple Rho guanine nucleotide triphosphatase (GTPase) and cell cycle related genes as determined by linear regression analyses. Observed increases in transcript abundance of Fosl1, Myc, and Rac1 oncogenes in mouse skin support previous reports on the inducibility of these oncogenes in response to arsenic and support the relevance of these genomic changes in skin tumor induction in the K6/ODC mouse model.

Although p53 is known to play a critical role in the proliferation of gastrointestinal epithelia, the role of the Mdm2/p53 pathway in response to inducers of apoptosis in intestinal epithelial cells is unknown. Our data show that camptothecin (CPT)-induced apoptosis correlated with increased p53, p21Cip1, and Mdm2 protein levels, with a simultaneous increase in ATR Ser428, p53 Ser15 and Mdm2 Ser166 phosphorylation in IEC-6 cells. Increased p53 levels and its phosphorylation increased Bax protein, caspase-9, -3 activation and apoptosis. However, TNF-alpha/CHX-mediated apoptosis was independent of p53 protein levels and phosphorylation. The translation inhibitor, cycloheximide (CHX), prevented CPT-induced apoptosis. CHX completely prevented CPT-induced p53 phosphorylation and synthesis of p21Cip1, Bax and Bcl-xL proteins without altering p53 levels. The p53 activator, RITA, augmented CPT-induced apoptosis. The Mdm2 antagonist, Nutlin-3, significantly increased apoptosis, which was accompanied by increased p53, Mdm2 and p21Cip1 protein levels. The ATM/ATR kinase inhibitor, CGK733, blocked CPT-induced p53 Ser15 phosphorylation and protected cells from CPT-induced apoptosis. Inhibition of ornithine decarboxylase (ODC) with alpha-difluromethylornithine (DFMO) and subsequent depletion of intracellular polyamines increased p53 protein, Mdm2 Ser166 phosphorylation and conferred resistance to CPT-induced apoptosis. However, polyamine depletion had no effect on p53 phosphorylation. Nutlin-3 reversed the protective effect of DFMO and sensitized cells to CPT-induced apoptosis. These results suggest that p53 stabilization and accumulation in response to polyamine depletion predominantly modulate cell cycle checkpoints via p21Cip1 expression and inhibit transcription of target genes responsible for apoptosis. In contrast, phosphorylation and stabilization of p53 in response to DNA-damage lead to apoptosis, which indicates different roles of p53 during DNA damage and polyamine depletion.

Protein function is mediated by different amino acid residues, both their positions and types, in a protein sequence. Some amino acids are responsible for the stability or overall shape of the protein, playing an indirect role in protein function. Others play a functionally important role as part of active or binding sites of the protein. For a given protein sequence, the residues and their degree of functional importance can be thought of as a signature representing the function of the protein. We have developed a combination of knowledge- and biophysics-based function prediction approaches to elucidate the relationships between the structural and the functional roles of individual residues and positions. Such a meta-functional signature (MFS), which is a collection of continuous values representing the functional significance of each residue in a protein, may be used to study proteins of known function in greater detail and to aid in experimental characterization of proteins of unknown function. We demonstrate the superior performance of MFS in predicting protein functional sites and also present four real-world examples to apply MFS in a wide range of settings to elucidate protein sequence-structure-function relationships. Our results indicate that the MFS approach, which can combine multiple sources of information and also give biological interpretation to each component, greatly facilitates the understanding and characterization of protein function.

Ornithine decarboxylase (ODC), a tumor promoter, provokes cell proliferation, and inhibits cell death; but the mechanism involved in cell differentiation remains unknown. Herein, we examine whether it functions during macrophage-like differentiation. Previous studies reveal that ODC, a rate-limiting enzyme of polyamine biosynthesis, and polyamines are involved in restraining immune response in activated macrophage. By using 12-O-tetradecanoylphorbol-13-acetate (TPA)-differentiated human promyelocytic HL-60 and promonocytic U-937 cells, we discover that polyamines block the expression, secretion and activation of MMP-9. Meanwhile conventional expression of ODC represses tumor necrosis factor-alpha (TNF-alpha) expression and nuclear factor-kappaB (NF-kappaB) activation as well as MMP-9 enzyme activity. Following stimulation by TNF-alpha, the secretion of MMP-9 is restored in ODC-overexpressed cells. In addition, the NF-kappaB inhibitors (pyrrolidinedithiocarbamate, BAY-11-7082 and lactacystin) suppress the TPA-induced MMP-9 enzyme activity. Concurrently, both the irreversible inhibitor of ODC, alpha-difluoromethylornithine, and TNF-alpha could not recover MMP-9 activation following NF-kappaB inhibitor treatment in parental cells. Furthermore, ODC could directly inhibit and attenuate NF-kappaB DNA binding and transcriptional activation. Therefore, we suggest that ODC inhibits the TNF-alpha-elevated MMP-9 activation via NF-kappaB as TPA-induced macrophage-like differentiation and this interrupting mechanism may provide a new conceivable resolution why leukemia is poorly differentiated besides atypical growth.

Polyamines are key regulators in cell growth and differentiation. It has been shown that ornithine decarboxylase (Odc) was essential for post-implantation embryo development, and overexpression of spermidine/spermine N1-acetyltransferase will lead to ovarian hypofunction and hypoplastic uteri. However, the expression and function of polyamine-related genes in mouse uterus during early pregnancy are still unknown. In this study we investigated the expression, regulation, and function of polyamine-related genes in mouse uterus during the peri-implantation period. Odc expression was strongly detected at implantation sites and stimulated by estrogen treatment. The expression of Odc antizyme 1 and spermidine/spermine N1-acetyltransferase was also highly shown at implantation sites and regulated by Odc or polyamine level in uterine cells. Embryo implantation was significantly inhibited by alpha-difluoromethylornithine, an Odc inhibitor. Moreover, the reduction of Odc activity caused by alpha-difluoromethylornithine treatment was compensated by the up-regulation of S-adenosylmethionine decarboxylase gene expression. Collectively, our results indicated that the coordinated expression of uterine polyamine-related genes may be important for embryo implantation.

Pseudomonas aeruginosa uses acyl-homoserine lactones to coordinate gene transcription in a process called quorum sensing (QS). The QS molecules C4-HSL and C12-oxo-HSL are synthesized from the universal precursor S-adenosyl methionine, which is also a precursor of polyamines in human cells. Polyamines are required for mitotic cell division and peak during this phase. The polyamine putrescine is synthesized by ornithine decarboxylase (ODC) as a rate-limiting step. The ODC enzyme concentration also peaks during the mitotic phase. This peak is mediated by translation of ODC mRNA by the ITAF45 protein, which translocates from the nuclear compartment to the cytoplasm in a phosphorylation-dependent manner. We observed that C12-HSL-treated human epidermal cells had a higher cytoplasm-to-nuclear ITAF45 protein concentration and this translocation was dependent on the dephosphorylation of ITAF45. Finally, C12-HSL-treated cells also had a time-course-dependent higher concentration of ODC mRNA. Based on these mitotic markers, more human cells were apparently trapped in the mitotic phase when treated with C12-HSL. This should normally imply higher levels of putrescine. However, C12-HSL-treated human cells had a significantly lower concentration of putrescine and displayed a lower cell proliferation rate. In conclusion, the P. aeruginosa autoinducer C12-oxo-HSL apparently arrests human cells in the mitotic phase by lowering the concentration of putrescine.

ODC (ornithine decarboxylase), the rate-limiting enzyme in polyamine biosynthesis, is regulated by specific inhibitors, AZs (antizymes), which in turn are inhibited by AZI (AZ inhibitor). We originally identified and cloned the cDNA for a novel human ODC-like protein called ODCp (ODC paralogue). Since ODCp was devoid of ODC catalytic activity, we proposed that ODCp is a novel form of AZI. ODCp has subsequently been suggested to function either as mammalian ADC (arginine decarboxylase) or as AZI in mice. Here, we report that human ODCp is a novel AZI (AZIN2). By using yeast two-hybrid screening and in vitro binding assay, we show that ODCp binds AZ1-3. Measurements of the ODC activity and ODC degradation assay reveal that ODCp inhibits AZ1 function as efficiently as AZI both in vitro and in vivo. We further demonstrate that the degradation of ODCp is ubiquitin-dependent and AZ1-independent similar to the degradation of AZI. We also show that human ODCp has no intrinsic ADC activity.

Michael Brownlee has proposed a 'Unifying Mechanism' of hyperglycemia-induced damage in diabetes mellitus. At the crux of this hypothesis is the generation of reactive oxygen species (ROS), and their impact on glycolytic pathways. Diabetes is the leading cause of chronic kidney failure. In the early phase of diabetes, prior to establishment of proteinuria or fibrosis, comes kidney growth and hyperfiltration. This early growth phase consists of an early period of hyperplasia followed by hypertrophy. Hypertrophy also contributes to cellular oxidative stress, and may precede the ROS perturbation of glycolytic pathways described in the Brownlee proposal. This increase in growth promotes hyperfiltration, and along with the hypertrophic phenotype appears required for hyperglycemia-induced cell damage and the progression of downstream diabetic complications. Here we will evaluate this growth phenomenon in the context of diabetes mellitus.

Purpose: Loss of the methylthioadenosine phosphorylase (MTAP) gene at 9p21 is observed frequently in a variety of human cancers. We have shown previously that MTAP can act as a tumor suppressor gene and that its tumor suppressor function is related to its effect on polyamine homeostasis. Ornithine decarboxylase is a key enzyme in the regulation of polyamine metabolism. The aim of this study is to analyze MTAP and ornithine decarboxylase (ODC) expression in primary pancreatic tumor specimens., Experimental Design: We measured MTAP and ODC activity in protein extracts derived from 30 surgically resected tumor samples and eight normal pancreas samples. In a subset of six samples, we also examined MTAP DNA using interphase fluorescence in situ hybridization. In addition, we examined the effect of the ODC inhibitor difluoromethylornithine on two pancreatic adenocarcinoma-derived cell lines., Result: MTAP activity was 2.8-fold reduced in adenocarcinomas and 6.3-fold reduced in neuroendocrine tumors compared with control pancreas. Conversely, ODC activity was 3.6-fold elevated in adenocarcinomas and 3.9-fold elevated in neuroendocrine tumors compared with control pancreas. Using interphase fluorescence in situ hybridization, we found in tumor samples that 43 to 75% of the nuclei had lost at least one copy of MTAP locus, indicating that loss of MTAP activity was at least partially because of deletion of the MTAP locus. We also show that inhibition of ODC by difluoromethylornithine caused decreased cell growth and increased apoptosis in two MTAP-deleted pancreatic adenocarcinoma-derived cell lines., Conclusions: MTAP activity is frequently lost, and ODC activity is frequently elevated in both pancreatic adenocarcinoma and neuroendocrine tumors. Inhibition of ODC activity caused decreased cell growth and increased apoptosis in pancreatic tumor-derived cell lines. These findings suggest that MTAP and polyamine metabolism could be potential therapeutic targets in the treatment of pancreatic cancer.

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

Background: L-Arginine is a nutritional supplement that may be useful for promoting intestinal repair. Arginine is metabolised by the oxidative deiminase pathway to form nitric oxide (NO) and by the arginase pathway to yield ornithine and polyamines., Aims: To determine if arginine stimulates restitution via activation of NO synthesis and/or polyamine synthesis., Methods: We determined the effects of arginine on cultured intestinal cell migration, NO production, polyamine levels, and activation of focal adhesion kinase, a key mediator of cell migration., Results: Arginine increased the rate of cell migration in a dose dependent biphasic manner, and was additive with bovine serum concentrate (BSC). Arginine and an NO donor activated focal adhesion kinase (a tyrosine kinase which localises to cell matrix contacts and mediates beta1 integrin signalling) after wounding. Arginine stimulated cell migration was dependent on focal adhesion kinase (FAK) signalling, as demonstrated using adenovirus mediated transfection with a kinase negative mutant of FAK. Arginine stimulated migration was dependent on NO production and was blocked by NO synthase inhibitors. Arginine dependent migration required synthesis of polyamines but elevating extracellular arginine concentration above 0.4 mM did not enhance cellular polyamine levels., Conclusions: These results showed that L-arginine stimulates cell migration through NO and FAK dependent pathways and that combination therapy with arginine and BSC may enhance intestinal restitution via separate and convergent pathways.

The role of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated polyamine biosynthesis in the development of metastatic squamous cell carcinoma (mSCC) in protein kinase C epsilon (PKC epsilon) transgenic mice was determined. TPA treatment induced epidermal ornithine decarboxylase (ODC) activity and putrescine levels approximately 3-4-fold more in PKC epsilon transgenic mice than their wild-type littermates. Development of mSCC by the 7,12-dimethylbenz(a)anthracene (100 nmol)-TPA (5 nmol) protocol in PKC epsilon transgenic mice was completely prevented by administration of the suicide inhibitor of ODC alpha-difluoromethylornithine (DFMO, 0.5% w/v) in the drinking water during TPA promotion. However, DFMO treatment led to marked hair loss in PKC epsilon transgenic mice. DFMO treatment-associated hair loss in PKC epsilon transgenic mice was accompanied by a decrease in the number of intact hair follicles. These results indicate that TPA-induced ODC activity and the resultant accumulation of putrescine in PKC epsilon transgenic mice are linked to growth and maintenance of hair follicles, and the development of mSCC. Severe hair loss observed in PKC epsilon transgenic mice on DFMO during skin tumor promotion has not been reported before in the prevention of cancer in other animal models or in human cancer prevention trials.

Polyamines are ubiquitous cellular components that are involved in normal and neoplastic growth. Polyamine biosynthesis is very highly regulated in mammalian cells by the activities of two key decarboxylases acting on ornithine and S-adenosylmethionine. Recent studies, which include crystallographic analysis of the recombinant human proteins, have provided a detailed knowledge of their structure and function. Ornithine decarboxylase is a PLP-requiring decarboxylase, whereas S-adenosylmethionine decarboxylase (AdoMetDC) contains a covalently bound pyruvate prosthetic group. Both enzymes have a key cysteine residue, which is involved in protonation of the Schiff base intermediate C(alpha) to form the product. These residues, Cys360 in ornithine decarboxylase (ODC) and Cys82 in AdoMetDC, react readily with nitric oxide (NO), which is therefore a potent inactivator of polyamine synthesis. The inactivation of these enzymes may mediate some of the antiproliferative actions of NO.

Nicotine is one of the most representative components in cigarette smoke leading to gastric ulceration. Both ornithine decarboxylase and potassium ion (K(+)) channels are essential for cell growth and wound repair. The aim of the present study is to elucidate the causative relationship of these two factors during wound healing and the influence of nicotine on this healing process in rat gastric mucosal epithelial cells (RGM-1). Nicotine markedly inhibited cell migration and proliferation in RGM-1 cells. The latter effect was significantly antagonized by a nicotinic receptor blocker, mecamylamine. Nicotine also suppressed ornithine decarboxylase activity significantly. Our data showed that inhibition of cell proliferation and ornithine decarboxylase activity by nicotine was accompanied with a reduction in K(+) channel protein expression, all of which were significantly alleviated by spermidine pretreatment. These results suggested that there was a cause/effect link between ornithine decarboxylase and K(+) channel on wound repair. Nicotine in cigarette smoke inhibited this healing process and delayed wound repair in gastric epithelial cells.

Angiogenesis, an essential process for tumor growth, is regulated by endothelial proliferation factors and their inhibitors such as endostatin. Endostatin, a carboxyl-terminal fragment of type XVIII collagen, inhibits endothelial proliferation, angiogenesis, and tumor growth. Ornithine decarboxylase (ODC), a molecule that is overexpressed in various cancers, is associated with promoting tumor growth and angiogenesis. We found that ODC-overexpressing human cancer cells and breast cancer specimens showed suppressed expression of type XVIII collagen and endostatin. We hypothesized that ODC overexpression may facilitate angiogenesis in tumors by suppressing endostatin expression. ODC-overexpressing COS cells, which showed suppressed type XVIII collagen and endostatin expression, were established. Conditioned media derived from these cells, containing decreased levels of endostatin, induced significant endothelial proliferation. ODC-overexpressing cells, when transplanted into nude mice, suppressed type XVIII collagen expression and promoted neovascularization in vivo. Thus, overexpression of ODC facilitates endothelial proliferation by suppressing endostatin expression.

Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene. ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation.

Myc is a transcriptional activator whose deregulated expression not only promotes proliferation but also induces or sensitizes cells to apoptosis. Here we demonstrate that c-myc plays a role in triggering apoptosis in CEM T leukaemia cells exposed to progressive medium exhaustion. Indeed starved cells undergo apoptosis in the presence of constitutively elevated c-myc expression and the phorbol ester, phorbol 12-miristate 13-acetate (PMA), which rescues cells from apoptosis, induces complete c-myc down-regulation. We also investigate the hypothesis that ornithine decarboxylase (ODC), a transcriptional target of c-myc, is a down-stream mediator of c-myc driven apoptosis. We demonstrate that PMA induces in starved cells an earlier and larger decrease in ODC expression (mRNA and activity) and intracellular polyamine content, compared to untreated starved cells. Moreover we show that alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzymatic activity, effectively reduces, while exogenous added polyamines enhance apoptosis in starved cells. All these data indicate that ODC and polyamines may act as facilitating factors in triggering apoptosis induced by growth/survival factors withdrawal.

We have generated a hybrid transgenic mouse line overexpressing both ornithine decarboxylase (ODC) and spermidine/spermine N(1)-acetyltransferase (SSAT) under the control of the mouse metallothionein (MT) I promoter. In comparison with singly transgenic animals overexpressing SSAT, the doubly transgenic mice unexpectedly displayed much more striking signs of activated polyamine catabolism, as exemplified by a massive putrescine accumulation and an extreme reduction of hepatic spermidine and spermine pools. Interestingly, the profound depletion of the higher polyamines in the hybrid animals occurred in the presence of strikingly high ODC activity and tremendous putrescine accumulation. Polyamine catabolism in the doubly transgenic mice could be enhanced further by administration of zinc or the polyamine analogue N(1),N(11)-diethylnorspermine. In tracer experiments with [(14)C]spermidine we found that, in comparison with syngenic animals, both MT-ODC and MT-SSAT mice possessed an enhanced efflux mechanism for hepatic spermidine. In the MT-ODC animals this mechanism apparently operated in the absence of measurable SSAT activity. In the hybrid animals, spermidine efflux was stimulated further in comparison with the singly transgenic animals. In spite of a dramatic accumulation of putrescine and a profound reduction of the spermidine and spermine pools, only marginal changes were seen in the level of ODC antizyme. Even though the hybrid animals showed no liver or other organ-specific overt toxicity, except an early and permanent loss of hair, their life span was greatly reduced. These results can be understood from the perspective that catabolism is the overriding regulatory mechanism in the metabolism of the polyamines and that, even under conditions of severe depletion of spermidine and spermine, extremely high tissue pools of putrescine are not driven further to replenish the pools of the higher polyamines.

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UVB component, to human skin is the major cause for more than a million new cases of cutaneous malignancies diagnosed annually in the United States. Photocarcinogenesis, like other cancers, is a multistep process that includes initiation and promotion. A proper understanding of the molecular events occurring during the tumor promotion phase of photocarcinogenesis could lead to the development of novel approaches for the management of skin cancer. Using a transgenic mouse model (K5/ODC mice), which overexpresses the enzyme ornithine decarboxylase (ODC) in hair follicle keratinocytes, we studied the role of this gene in photocarcinogenesis. A single UVB-exposure of 180 mJ/cm(2) to the transgenic mice resulted in a minimal increase in bifold skin thickness and ODC activity. However, in SKH-1 hairless mice, the most common and highly sensitive model for photocarcinogenesis, and in littermate nontransgenic mice, increases in skin thickness and ODC activity were substantial. In long-term experiments, mice were exposed to 180 mJ/cm(2) of UVB radiation three times a week for 2 weeks (tumor-initiating dose). At 30 weeks after this treatment, in two independent experiments, 40% of the K5/ODC transgenic mice exposed to UVB were found to develop epidermal tumors. The tumors were histologically verified as benign papillomas and squamous cell carcinomas. Interestingly, 100% of the transgenic mice also developed >20 pigmented cysts/mouse, which contained keratinocyte material with increased keratinocytic melanization. Under similar UVB-exposure protocol, the nontransgenic littermates or SKH-1 hairless mice did not develop tumors or pigmented cysts for up to 50 weeks. Oral consumption of alpha-difluoromethylornithine, an irreversible specific inhibitor of ODC, in the drinking water (1% w/v) to the transgenic mice resulted in complete prevention of UVB-mediated tumorigenesis and a substantial decrease in the formation of pigmented cysts (<10 per mouse). These data establish a definitive role of ODC in the promotion phase of photocarcinogenesis.

The level of expression of oxyR, the gene that protects Escherichia coli against oxidative stress, was enhanced by physiological concentrations of the biogenic amine putrescine. This level was directly proportional to the degree of negative DNA supercoiling. 1,4-Diamino-2-butanone (DAB), a specific inhibitor of ornithine decarboxylase, the key enzyme of polyamine synthesis, produced an inhibitory effect on the level of oxyR expression under oxidative stress, which was relieved by the addition of putrescine. The direct relationship between putrescine concentration and the degree of negative DNA supercoiling suggests that the mechanism of action of putrescine as the modulator of oxyR transcription activity is based on both its direct influence on the gene expression level and its indirect effect mediated by topological DNA changes. Putrescine was shown to produce a protective effect if the DNA is damaged by reactive oxygen species.

Under optimal conditions of growth, senescence, a terminal phase of development, sets in after a certain physiological age. It is a dynamic and closely regulated developmental process which involves an array of changes at both physiological and biochemical levels including gene expression. A large number of biotic and abiotic factors accelerate the process. Convincing evidence suggests the involvement of polyamines (PAs) and ethylene in this process. Although the biosynthetic pathways of both PAs and ethylene are interrelated, S-adenosylmethionine (SAM) being a common precursor, their physiological functions are distinct and at times antagonistic, particularly during leaf and flower senescence and also during fruit ripening. This provides an effective means for regulation of their biosynthesis and also to understand the mechanism by which the balance between the two can be established for manipulating the senescence process. The present article deals with current advances in the knowledge of the interrelationship between ethylene and PAs during senescence which may open up new vistas of investigation for the future.

Ornithine decarboxylase (ODC), an important enzyme in the polyamine biosynthetic pathway, is aberrantly regulated in many epithelial tumors of rodents and humans. In murine skin, it has been shown that ODC overexpression provides a sufficient condition for tumor promotion. Therefore, we hypothesized that K6/ODC transgenic mice in which ODC overexpression was targeted to hair follicle keratinocytes might provide a sensitive model for identifying genotoxic carcinogens. Ten known carcinogens or noncarcinogens have been tested in the model so far and results are highly concordant with 2-year rodent bioassays (100% concordant). More importantly, each of two chemicals tested that is recognized as a human carcinogen was identified as a carcinogen in K6/ODC transgenic mice. In addition, 7, 12-dimethylbenz(a)anthracene (DMBA) dose response studies indicated that even at a very low dose, 2 nmol, a high percentage of mice (50%) had already developed tumors 8 weeks after treatment. We conclude that the K6/ODC transgenic mouse model is very sensitive to topical application of genotoxic carcinogens and could therefore be a useful mouse model for carcinogen identification and chemical risk assessment.

Ornithine decarboxylase (ODC) overexpressed from a heterologous promoter drives the tumorigenic transformation of NIH 3T3 cells and provides a model to investigate the underlying molecular mechanisms. These transformed cells, designated NODC cells, exhibit elevated levels of epidermal growth factor receptor (EGFR) tyrosine kinase (Tyr-k) activity relative to control transfected cells and inhibition of EGFR Tyr-k activation suppresses the transformed growth phenotype of these cells. Thus, ODC-induced transformation of NIH 3T3 cells appears to be mediated, at least in part, by enhanced signaling through the EGFR pathway. Here we extend these studies by evaluating: (i) the effects on growth regulation of overexpressing ODC in EGFR-deficient NIH 3T3 cells; (ii) the potential role of TGFalpha in mediating the EGFR-dependent transformation of NIH 3T3 cells by ODC. Disruption of EGFR-TGFalpha interactions either by deleting EGFR, by treatment with anti-TGFalpha neutralizing antibody or by transfection with a TGFalpha antisense expression vector suppressed acquisition of the full transformed growth phenotype. Specifically, the loss of contact inhibition and the capacity for clonogenic growth appear more dependent on EGFR-TGFalpha interactions than anchorage-independent growth in ODC-overexpressing cells. ODC overexpression does not alter the amount, localization or secretion of TGFalpha. Thus, TGFalpha is not the ODC-responsive component of the EGFR signaling pathway but appears to be critically involved in development of the transformed phenotype of NODC cells.

Overexpression of ornithine decarboxylase (ODC) has been shown to be characteristic of tumor development and progression in humans and experimental animals. Therefore, we have examined the effects of 1, 3-diaminopropane dihydrochloride (DAP), a potent inhibitor of ODC, on rat two-stage urinary bladder carcinogenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In experiment 1 (36 weeks), 6-week-old F344 male rats were administered 0.05% BBN in drinking water for 4 weeks and then divided into four groups. Animals of groups 1 and 2 received basal diet and drinking water supplemented with or without DAP (2 g/l). Groups 3 and 4 were given diet containing 5% sodium L-ascorbate (NaAsA), a typical urinary bladder tumor promoter, and drinking water with or without DAP. Administration of DAP to group 1 significantly reduced tumor size, multiplicity and incidence, particularly of papillomas, when compared with group 2 values. DAP together with NaAsA (group 3) also decreased tumor size relative to the group 4 case. To determine the effects of DAP on the early stages of bladder carcinogenesis and its mechanisms, a similar protocol was conducted (experiment 2) with death after 20 weeks. DAP treatment caused complete inhibition (0% incidence) of papillary and/or nodular hyperplasia in group 1 but was without influence in group 3, as compared with the respective controls. Moreover, the ODC activity, bromodeoxyuridine labeling indices and mRNA expression levels of cyclin D1 in the urinary bladder mucosa, determined by northern blotting, were markedly lower in group 1 than in group 2, but values were comparable for both groups administered NaAsA. Assessment of mRNA expression levels of the angiogenic vascular endothelial growth factor suggested no involvement in the inhibitory effects of DAP on urinary bladder carcinogenesis. The results indicate that inhibition of ODC could reduce urinary bladder carcinogenesis in rats, particularly in the early stages, through antiproliferative mechanisms.

Objective: TGF-beta stimulation of cardiac myocytes induces a hypertrophic responsiveness to beta-adrenoceptor stimulation. This study investigates whether this beta-adrenoceptor mediated effect depends on induction of ornithine decarboxylase (ODC)., Methods: Isolated adult ventricular cardiomyocytes from rats were used as an experimental model. Cells were either cultured in 20% (v/v) FCS to activate autocrine released TGF-beta or used without pre-treatment. The hypertrophic response was characterized by an increased 14C-phenylalanine incorporation, RNA and protein mass or by an increased expression of atrionatriurectic factor and ODC. The results on cell cultures were compared to those achieved by isoprenaline perfused mice hearts from transgenic mice overexpressing TGF-beta 1., Results: ODC activity and expression increased within 2 h in TGF-beta 1 pre-treated cells under isoprenaline. In the presence of ODC inhibitors (alpha-methylornithine or difluoromethylornithine) this increase remained absent and the increases in 14C-phenylalanine incorporation, protein and RNA mass under isoprenaline were abolished. In cells not exposed to TGF-beta no induction of ODC was observed. Isoprenaline also induced ODC in isolated perfused ventricles from transgenic mice overexpressing TGF-beta 1, but not in ventricles from their nontransgenic counterparts., Conclusions: This study shows first, a pivotal role for ODC induction in the hypertrophic response of cardiomyocytes to beta-adrenoceptor stimulation and second, that ODC induction in vivo and in vitro requires pre-treatment of cardiomyocytes with TGF-beta. It is concluded that TGF-beta induces a hypertrophic responsiveness to beta-adrenoceptor stimulation that is characterized by ODC induction.

Interferon regulatory factor-1 (IRF-1) has been recognized as an important tumor suppressor and growth regulatory transcription factor, which is also involved in cell differentiation. In this study we investigated the role of IRF-1 in phorbol 12-myristate 13-acetate (PMA)-induced monocyte/macrophage differentiation of human monoblastic U937 cells. For this purpose U937 cells were stably transfected with a vector overexpressing IRF-1 antisense mRNA (U937 IRF-1A cells) and with the SV-40 empty vector (U937-SV40 e.v. cells). We report here that U937 and U937-SV40 e.v. cells differentiated into macrophage-like cells upon PMA stimulation and showed IRF-1 up-regulation. On the contrary, U937 IRF-1A cells stimulated with PMA kept an undifferentiated phenotype and proliferated actively. A direct correlation between induction of IRF-1 and up-regulation of IRF-1 gene targets such as ornithine decarboxylase (ODC) and WAF-1/CIP-1 was also observed in U937 cells. On the other hand U937 IRF-1A cells down-regulated ODC and did not express WAF-1. Results show that IRF-1 plays a pivotal role in PMA-induced monocyte/macrophage differentiation.

Candida albicans, the most common fungal pathogen, regulates its cellular morphology in response to environmental conditions. The ODC gene, which encodes ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was isolated and disrupted. Homozygous null Candida mutants behaved as polyamine auxotrophs and grew exclusively in the yeast form at low polyamine levels (0.01 mM putrescine) under all conditions tested. An increase in the polyamine concentration (10 mM putrescine) restored the capacity to switch from the yeast to the filamentous form. The strain with a deletion mutation also showed increased sensitivity to salts and calcofluor white. This Candida odc/odc mutant was virulent in a mouse model. The results suggest a model in which polyamine levels exert a pleiotrophic effect on transcriptional activity.

We have shown previously (W. Löscher et al., Cancer Lett., 71: 75-81, 1993; M. Mevissen et al., Carcinogenesis (Lond.), 17: 903-910, 1996) that 50-Hz magnetic fields (MFs) of low [50 or 100 microTesla (T)] flux density enhance mammary gland tumor development and growth in the 7,12-dimethylbenz[a]anthracene (DMBA) model of breast cancer in female Sprague Dawley rats. In these previous experiments, groups of rats were given 20 mg of DMBA (four weekly gavage doses of 5 mg each) and were MF- or sham-exposed for 13 weeks. The objective of the present study was to examine whether the use of a lower dose of DMBA (10 instead of 20 mg per rat), MF exposure of the rats before DMBA injection, and the increase of the MF exposure period after DMBA application to 26 weeks enhance the effect of MF on tumor development and growth. A group 99 rats was exposed to a homogeneous, horizontally polarized 100-microT MF of 50-Hz for 24 h/day for 7 days/week; another group of 99 rats was sham-exposed under the same environmental conditions as the MF-exposed rats. The exposure chambers were identical for MF-exposed and sham-exposed animals. The age of the rats was 45-49 days at the onset of exposure; duration of MF or sham exposure was 27 weeks. DMBA was administered p.o. at a dose of 10 mg/rat after 1 week of MF or sham exposure. The animals were palpated once weekly from week 6 onwards to assess the development of mammary tumors. At the end of the exposure period, the animals were killed for the determination of number and volume and histological verification of mammary tumors. All of the recordings were done in a blinded fashion; i.e., the investigators were not aware which animals were MF- or sham-exposed. Mammary tumor development and growth was significantly enhanced by MF exposure, the most marked effect on tumor incidence (190% above sham control) being observed 13 weeks after DMBA administration. At the time of necropsy, i.e., 26 weeks after DMBA administration, the incidence of histologically verified mammary tumors was 50.5% in controls and 64.7% in MF-exposed rats, the difference being statistically significant. More marked intergroup differences were recorded when tumor incidence was separately evaluated for each of the six mammary complexes, the most pronounced MF effect on tumor incidence being seen in the cranial thoracic complex. The data substantiate that, at least under the experimental conditions used in our laboratory, 50-Hz, 100-microT MF exposure significantly facilitates the development and growth of mammary tumors in the DMBA rat model of breast cancer.

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been recently identified to be a potent inducer of apoptosis in human non-small cell lung carcinoma (NSCLC) cells through a nuclear retinoic acid receptor independent mechanism. To approach the mechanism by which CD437 induces apoptosis in NSCLC cells, we investigated the involvement of c-Myc in CD437-induced apoptosis. CD437 (1 microM) up-regulated the expression of c-Myc and of its downstream target genes ornithine decarboxylase (ODC) and cdc25A in all three NSCLC cell lines (i.e., H460, SK-MES-1 and H1792) used. These effects were correlated with cellular susceptibilities to induction of apoptosis by CD437. Furthermore, CD437-induced apoptosis could be blocked by the ODC inhibitor difluoromethylornithine, the caspase inhibitors Z-VAD FMK and Z-DEVD FMK, and c-Myc antisense oligodeoxynucleotide, respectively. These data indicate that c-Myc gene plays an important role in mediating CD437-induced apoptosis in human NSCLC cells.

Nitric oxide (NO) is a molecule involved in several signal transduction pathways leading either to proliferation or to cell death. Induction of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, represents an early event preceding DNA synthesis. In some cell types increased ODC activity seems to be involved in cytotoxic response. We investigated the role of NO and ODC induction on the events linked to cell proliferation or to cell death in cultured chick embryo cardiomyocytes. Exposure of cardiomyocytes to tumor necrosis factor (TNF) and lipopolysaccharide (LPS) caused NO synthase (NOS) and ODC induction as well as increased incorporation of [3H]-thymidine. This last effect was blocked by a NOS inhibitor and was strongly reduced by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Sodium nitroprusside (SNP), an exogenous NO donor, inhibited the increases of NOS and ODC activities and abolished the mitogenic effect of TNF and LPS. Moreover, SNP alone caused cell death in a dose dependent manner. The cytotoxicity of SNP was not affected by DFMO while it was prevented by antioxidants. The results suggest that different pathways would mediate the response of cardiomyocytes to NO: they can lead either to ODC induction and DNA synthesis when NO is formed through NOS induction or to growth inhibition and cell death, when NO is supplied as NO donor. Increased polyamine biosynthesis would mediate the proliferative response of NO, while the cytotoxicity of exogenous NO seems to involve some oxidative reactions and to depend on the balance between NO availability and cellular redox mechanisms.

Ornithine decarboxylase (ODC) is a key enzyme of polyamine biosynthesis essential for growth-related cellular functions. Apart from its physiological role in cell proliferation, ODC also contributes to the induction of apoptosis under certain conditions, e.g. following growth factor withdrawal. The rate of cell death is a function of its enzyme activity, ODC activity is inhibited by a regulatory protein antizyme, also known to suppress polyamine uptake. We report that forced expression of antizyme prevents ODC-mediated cell death in human gingival fibroblasts under very low serum conditions. These data suggest an important antiapoptotic role for antizyme in cell survival.

Purpose: To study the possible role of 50 Hz magnetic fields (MF) in UV-induced skin tumourigenesis using a sensitive animal model., Materials and Methods: Transgenic mice (line K2) over-expressing the human ornithine decarboxylase (ODC) gene and their non-transgenic littermates were exposed for 10.5 months to UV-only or a combination of UV and a continuous (100 microT) or an intermittent MF with varying intensity (1.3-130 microT)., Results: Both MF exposure and transgenicity enhanced the onset rate of macroscopically detectable tumours, but the effect was statistically significant only for the MF exposure (p < 0.015). The number of animals bearing malignant tumours was low and similar in all exposure groups. Epidermal cysts (EC) appeared to be strongly associated with both MF exposure and high ODC activity (transgenic animals). However, EC are not known to be associated with carcinogenesis. The UV-only or combined UV and MF exposure did not affect the ODC activities measured at the end of the exposure., Conclusions: These results support the proposed tumour-promoting effect of MF, but do not suggest an important role for increased ODC activity in this process.

The effects of congenital absence of visual input on daily rhythms of feeding behavior and tissue ornithine decarboxylase activity were examined in hereditary blind rats. Food intake of normal and hereditary microphthalmic rats was determined every 3 h over 48 h under a 12 h-12 h light-dark cycle. Normal rats took their daily food mainly during the dark period, with two clear peaks in the evening and morning, but hereditary microphthalmic rats ate continuously, suggesting that the feeding rhythm of the hereditary microphthalmic rats is not entrained by the light-dark cycle due to a lack of their optic nerve. The hepatic and renal ornithine decarboxylase activities in normal rats showed a circadian rhythm with a peak at 2000 hours in the dark period as reported previously. In hereditary blind rats, however, the hepatic and renal enzyme activities were found not to undergo any remarkable diurnal change through the light-dark period. These results mean that the feeding rhythm and hepatic and renal ornithine decarboxylase activity rhythm in hereditary blind rats disappeared.

Patients with xeroderma pigmentosum (XP), a DNA repair disorder, run a large risk of developing skin cancer in sun-exposed areas. Cancer proneness in these patients correlates with a mammalian SOS-like response, "enhanced reactivation (ER) of viruses." Here, we report that radiation-induced activation of the ornithine decarboxylase (ODC) gene, a putative proto-oncogene, is required for this response. Various diploid fibroblast strains derived from a non-cancer-prone subclass of XP patients, which lack the ER response, were irradiated with 2 J/m2 and assessed for gene induction. In these fibroblasts, an absence of induction of ODC by UV-C was observed at the levels of mRNA, protein, and enzyme activity. This lack of induction is quite specific because the genes for fos and collagenase were induced as they were in normal XP cells. The apparent linkage between non-cancer proneness and a lack of ER and ODC induction was confirmed in a fibroblast strain derived from a patient with another DNA repair disorder, trichothiodystrophy, which does not lead to cancer proneness: in these cells, no induction of the ER response nor of ODC occurs after UV-C irradiation. Repair deficiency, however, is not essential because the simultaneous lack of ODC and ER induction after 10 J/m2 UV-C was found in at least one repair-proficient fibroblast. Next, a specific inhibitor of ODC, difluoromethylornithine, at a dose of 10 mM, completely blocked the ER response in cultured normal skin fibroblasts, suggesting that the ODC enzyme is in fact essential for the ER response. Difluoromethylornithine, although it did not affect other processes such as DNA repair, leads to a block in the cell division cycle at the G1-S transition. Interestingly, other blockers of this transition, wortmannin (500 nM) and mimosine (100 mM), also decreased the ER response. Finally, the ER and ODC responses also seem to be linked after treatment with X-irradiation (3 Gy), suggesting that both are part of a general response to DNA damage, at least in human skin fibroblasts. Apart from the abnormal ER and ODC responses, fibroblasts from non-tumor-prone XP patients react in the same way to radiation as do fibroblasts from tumor-prone XP patients with respect to other parameters. Thus, the lack of ODC induction after radiation may help to protect XP patients against skin carcinogenesis.

The 5' untranslated region (UTR) has an inhibitory role in the translatability of ornithine decarboxylase (ODC) mRNA and of hybrid mRNA species, whereas the ODC 3' UTR causes a partial release of this inhibition. We designed experiments to explore whether the co-operation between ODC 5' UTR and 3' UTR in the translational regulation is due to a direct interaction of those sequences or whether it is mediated by their interaction with cellular factor(s). We stably transfected Chinese hamster ovary (CHO)-K1 cells and transiently transfected COS-1 cells with expression vectors carrying different chimaeric DNAs having the luciferase (LUC) coding sequence as reporter gene, the ODC 5' UTR or the ODC 3' UTR, or both, in the appropriate positions. We compared the results obtained by assaying the LUC activities of both transfected cell lines with each chimaeric DNA with those observed by translating the hybrid RNAs in a translation system in vitro. When the ODC 3' UTR was present, we observed a partial release of the translation inhibition owing to the ODC 5' UTR only in vivo. The releasing effect was restored in vitro by the addition of cytoplasmic extracts from wild-type CHO-K1 or COS-1 cells, prepared 2 and 8 h after their release from serum starvation. We also observed a partial inhibition of the translatability of the hybrid RNA owing to the presence of the ODC 3' UTR itself; the translational efficiency could be rescued by cell extract from 8 h serum-stimulated cells. The co-operation between the ODC-UTRs might be mediated by factors expressed by cells during particular phases of the cell cycle. Excess copies of the ODC-UTRs, expressed in trans, could compete in binding limited amounts of such regulatory factors and remove them from interaction with the endogenous ODC mRNA. This phenomenon should be reflected by modifications of the kinetics of ODC and/or LUC activities during serum stimulation. The overexpression of the ODC 3' UTR determined an increase in both endogenous ODC activity and LUC activity. Moreover, in the transfectants expressing the hybrid RNA species bearing the ODC 3' UTR the basal ODC activity is higher than that observed in control cells. We suggest that excess copies of the ODC 3' UTR mis-regulate the endogenous ODC translatability, probably by tying up regulatory molecules expressed by cells in limited amounts and sequestering them from the ODC mRNA species they should interact with.

Overexpression of human ornithine decarboxylase (ODC) under the control of strong promoters induces morphological transformation of immortalized NIH3T3 and Rat-1 fibroblasts [M. Auvinen et al., Nature (Lond.), 360: 355-358, 1992]. We demonstrate here that ODC-overproducing NIH3T3 cells are tumorigenic in nude mice, giving rise to rapidly growing, large fibrosarcomas at the site of inoculation. The tumors are capable of invading host fat and muscle tissues and are vascularized abundantly. To disclose the molecular mechanism(s) driving the tumorigenic, invasive, and angiogenic phenotype of the tumors, the ODC-overproducing cell lines and tumor tissues were analyzed for the expression of various potential regulators and mediators of cell proliferation, matrix degradation, and angiogenesis. The tumorigenicity of ODC transformants was associated with elevated polyamine levels and down-regulated growth factor receptors. The invasiveness of the ODC-induced tumors could not be attributed to overexpression of various known extracellular matrix-degrading proteases or matrix metalloproteinases. The induction of the tumor neovascularization proved not to be elicited by vascular endothelial growth factor or basic fibroblast growth factor. Instead, the ODC-overexpressing cells appeared to secrete a novel angiogenic factor(s) that was able to promote migration of bovine capillary endothelial cells in collagen gels and increase the proliferation of human endothelial cells in vitro. In parallel, ODC-transformed cells displayed down-regulation of thrombospondin-1 and -2, the negative regulators of angiogenesis. Thus, the induction of the angiogenic phenotype of the ODC transformants is likely due both to increased expression and secretion of the new angiogenesis-stimulating factor(s) and decreased production and release of the antiangiogenic thrombospondins.

In these experiments we tested the hypothesis that constitutive activation of polyamine(PA) biosynthesis may contribute to mammary carcinogenesis. Spontaneously immortalized normal human MCF-10A breast epithelial cells were infected with the retroviral vector pLOSN containing a cDNA which codes for a truncated and more stable ornithine decarboxylase (ODC), the rate-limiting enzyme in PA synthesis. Upon chronic selective pressure with alpha-difluoromethyl-ornithine (DFMO) (an irreversible inhibitor of ODC), infected MCF-10A cells exhibited an approximately 250-fold increase in ODC activity, which persisted despite discontinuation of DFMO. ODC-over-expressing MCF-10A cells showed a modest decrease in S-adenosylmethionine decarboxylase and an increase in spermidine/spermineN1-acetyltransferase. Analysis of cellular PA profile revealed a selective accumulation of putrescine without alterations in spermidine and spermine contents. Lesser degrees of increased ODC activity were obtained reproducibly by re-exposing the cells to incremental small doses of DFMO. We observed a bell-shaped dose-related positive effect of ODC activity on clonogenicity in soft agar of MCF-10A cells. Since anchorage-dependent growth was actually reduced, such positive influence on this feature of transformation was not a non-specific consequence of a growth advantage provided by ODC over-expression. In addition, we observed a close parallelism between the dose-dependent effects of ODC expression on clonogenicity and activity of the ERK-2 kinase, a central element of the MAPK cascade. Our data demonstrate an interaction between PA and the MAPK signalling pathway and suggest that the latter may be involved in ODC-induced transformation of mammary epithelial cells.

The role of polyamines in the expression of cricket oviposition, a juvenile hormone-dependent behavior, was investigated using a specific inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (alpha-DFMO). The fat body of treated female house crickets (Acheta domesticus) did not show any putrescine and presented reduced levels of spermidine, whereas spermine titres were significantly enhanced. In nervous tissue, alpha-DFMO did not affect spermine titres but induced a severe drop in spermidine levels. In polyamine depleted females, the expression of egg-laying behavior was delayed and was expressed less frequently compared with controls. As drug treatment did not seem to affect juvenile hormone titres, the data suggest that juvenile hormone might act on behavior by way of polyamine metabolism. These results support the view that, in insects, as in vertebrates, the ornithine decarboxylase-polyamine system is involved in the maturation of complex behaviors.

Mice that overexpress a mutated ornithine decarboxylase (ODC) transgene in outer root sheath keratinocytes of the hair follicle were used to study the role of this enzyme in regulating hair follicle structure and function. These transgenic mice have a normal first hair cycle, but lose their hair completely beginning 2-3 wk after birth. Transgene overexpression in follicular keratinocytes is first detected at day 12 after birth, coincident with the development of follicular cysts in the upper portion of the dermis. The onset of keratin 6 expression also begins around day 12; because the promoter/regulatory region of the bovine keratin 6 gene was used to target ODC transgene expression of hair follicle keratinocytes, these data demonstrate the faithful temporal and cell type-specific expression of the K6 -driven transgene. The ODC inhibitor 2-difluoromethylornithine could prevent hair loss and partially normalize skin histology if administered before the onset of ODC overexpression. 2-Difluoromethylornithine could also reactivate hair growth in animals with complete hair loss. Our results suggest that ODC is an important regulatory gene for the mouse hair follicle.

After jejunectomy, a rapid and sustained increase in the abundance of proglucagon mRNA occurs in residual ileum and is accompanied by increases in plasma intestinal proglucagon-derived peptides. This response may be a component of adaptive growth, or proglucagon-derived peptides may regulate adaptive growth. To distinguish these possibilities, rats were treated with difluoromethylornithine, blocking ornithine decarboxylase activity and thereby adaptive bowel growth. Three groups fed ad libitum were compared: (1) resect: rats with 80% proximal small bowel resection; (2) resect + difluoromethylornithine: resected rats given difluoromethylornithine in drinking water; and (3) transect: transected controls. Six days after surgery, the resect + difluoromethylornithine group demonstrated inhibition of adaptive bowel growth. Abundance of ileal proglucagon mRNA in resect and resect + difluoromethylornithine groups was double that in the transect group (P < 0.02), whereas ornithine decarboxylase mRNA levels did not differ. Plasma enteroglucagon and glucagon-like peptide-I levels were greater in resect than transect groups (P < 0.002) and did not differ between resect and resect + difluoromethylornithine groups. The rise in ileal proglucagon mRNA after proximal small bowel resection is not inhibited by difluoromethylornithine despite blocking bowel growth and, therefore, is not merely a component of adaptive growth. Proglucagon-derived peptides are possible modulators of adaptive bowel but cannot stimulate growth when ornithine decarboxylase activity is inhibited.