Elevated Ornithine Research Articles

Chlorophyll biosynthesis under the control of arginine metabolism

In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen sources, a growth-limiting factor. For acclimation, the dynamic degradation/synthesisof tetrapyrrolic pigments, as well as of the amino acid arginine, is pivotal; however, there has been no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays, we found that in the cyanobacterium Synechocystis sp. PCC 6803, the arginine metabolism-related ArgD and CphB enzymes form protein complexes with Gun4, an essential protein for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the Gun4-ArgD complex accumulates in cells supplementedwith ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrroles, which arrested the recovery from nitrogen deficiency. Our data reveal a direct crosstalk between tetrapyrrole biosynthesis and arginine metabolism that highlights the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis.

Chronic sleep loss sensitizes Drosophila melanogaster to nitrogen stress

Chronic sleep loss profoundly impacts metabolic health and shortens lifespan, but studies of the mechanisms involved have focused largely on acute sleep deprivation.1,2 To identify metabolic consequences of chronically reduced sleep, we conducted unbiased metabolomics on heads of three adult Drosophila short-sleeping mutants with very different mechanisms of sleep loss: fumin (fmn), redeye (rye), and sleepless (sss).3,4,5,6,7 Common features included elevated ornithine and polyamines, with lipid, acyl-carnitine, and TCA cycle changes suggesting mitochondrial dysfunction. Studies of excretion demonstrate inefficient nitrogen elimination in adult sleep mutants, likely contributing to their polyamine accumulation. Increasing levels of polyamines, particularly putrescine, promote sleep in control flies but poison sleep mutants. This parallels the broadly enhanced toxicity of high dietary nitrogen load from protein in chronically sleep-restricted Drosophila, including both sleep mutants and flies with hyper-activated wake-promoting neurons. Together, our results implicate nitrogen stress as a novel mechanism linking chronic sleep loss to adverse health outcomes-and perhaps for linking food and sleep homeostasis at the cellular level in healthy organisms.

Late onset hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome, presenting as recurrent metabolic encephalopathy, A case report

and importance: Hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH) syndrome (OMIM 238970) seems to be an autosomal recessive disorder caused by a mitochondrial ornithine transporter 1 deficiency, which results in urea cycle dysfunction. HHH is the most uncommon of the urea cycle diseases, with less than 100 cases recorded. A previously healthy 29 year old male presented to the emergency department complaining of decreased level of consciousness. CT scan, Cerebro-spinal-fluid analysis and toxicology screen were non-significant. Extended serum analysis showed elevated levels of ammonia. Urgent amino acid level analysis showed elevated ornithine. Follow up genetic testing showed that the patient is homozygous for the mutation c.44delG in exon 3 of SLC25A15 gene. In this case, HHH syndrome presented as a late-onset metabolic encephalopathy. For diagnosis; elevated levels of ammonia, ornithine accompanied by the abovementioned genetic mutation confirms the diagnosis. Treatment focuses on reduction of the ammonia levels using sodium benzoat, citrulline or arginine, and low protein diet. HHH syndrome, which is a urea cycle disorder, can present as a late-onset metabolic encephalopathy. High suspicion for genetic causes of metabolic encephalopathy should be maintained even for older patients without prior diagnosis in childhood/adolescence.

TMET-34. RADIATION METABOLOMICS IN PRIMARY HUMAN MENINGIOMA AND SCHWANNOMA: EARLY EXPERIENCE AND INITIAL RESULTS

Abstract Introduction Meningiomas and schwannomas account for 45% of primary CNS tumors. Yet when surgery and radiation fail, no further treatments exist. Metabolomics has been used to discover new cancer therapies; however, to date few have used metabolomics to study meningiomas and schwannomas. Here we present initial results and lessons learned from this novel endeavor. METHODS Primary tumors were obtained from patients during surgery and immediately taken for culturing or xenograft implantation. Upon reaching >90% confluence, cultures were treated with 0gy, 3gy, 10gy, or 20gy gamma radiation, then flash frozen 6 or 72 hours post-treatment. Xenograft tumors were implanted in nude mice. MRI 4 weeks post-implantation confirmed tumor viability. Mice were then given 10gy, 20gy, or sham radiation treatment. Xenografts were harvested 72 hours post-treatment. Metabolites were measured with a ThermoISQ gas chromatography-mass spectrometer. RESULTS Eleven meningiomas and nine schwannomas were successfully cultured. Unsupervised hierarchical clustering of cultures demonstrated greater influence from tumor of origin than from radiation. Univariate analysis of schwannoma xenografts demonstrated elevated ornithine following radiation (fold change 1.62; P = 0.008). However, principal component analysis did not show significant between-group differentiation. Orthotopic meningioma xenografts did not produce sufficient tissue for metabolomics; however, subsequent subcutaneous implants have been successful (data forthcoming). CONCLUSION Standard cell cultures did not reveal significant metabolic changes following radiation; it is unclear whether this was due to culture technique or inter-tumor heterogeneity. In radiated schwannoma xenografts, elevated ornithine may implicate related pathways such as ornithine decarboxylase-mediated polyamide synthesis for DNA double-strand break repair. Compared to other ‘-omics’ studies, metabolomics requires more tissue per sample ( >10mg) and is more sensitive to environmental conditions. Thus, large sample sizes are needed to detect significant changes, and xenografts are likely superior to cell culture. Future plans include increased xenograft sample size and stable isotope tracing for pathway analysis.

CRISPR correction of the Finnish ornithine delta-aminotransferase mutation restores metabolic homeostasis in iPSC from patients with gyrate atrophy

Hyperornithinemia with gyrate atrophy of the choroid and retina (HOGA) is a severe recessive inherited disease, causing muscular degeneration and retinochoroidal atrophy that progresses to blindness. HOGA arises from mutations in the ornithine aminotransferase (OAT) gene, and nearly one-third of the known patients worldwide are homozygous for the Finnish founder mutation OAT c.1205 T > C p.(Leu402Pro). We have corrected this loss-of-function OAT mutation in patient-derived induced pluripotent stem cells (iPSCs) using CRISPR/Cas9. The correction restored OAT expression in stem cells and normalized the elevated ornithine levels in cell lysates and cell media. These results show an efficient recovery of OAT function in iPSC, encouraging the possibility of autologous cell therapy for the HOGA disease.

Gyrate Atrophy

Gyrate Atrophy is an autosomal recessively inherited rare disease of the choroid and retina. Deficiency of ornithine aminotransferase enzyme (OAT) leads to 10 to 20 times ornithine levels in plasma which is thought to be the cause for the ocular findings. OAT gene is located on the tenth chromosome and when a mutation occurs OAT enzyme activity is decreased in the body. In the first decade, areas of chorioretinal atrophy are seen in the mid-peripheral retinal areas. In the progressing years, the macula may be involved and myopia is an accompanying feature]. Patients initially describe night vision problems and progressive visual field defects leading to central vision loss and blindness later on in the disease. Diagnosis is evident with clinical findings and elevated ornithine levels in plasma and is confirmed with OAT enzyme immune assay. Treatment is dietary modification and treatment of complications that may develop in the retina. GA is reported in diverse ethnic populations and in many sites around the World. Finland is the most reported country (1/50,000). It is very rare in Turkey.

Lysinuric protein intolerance: an overlooked diagnosis

BackgroundLysinuric protein intolerance (LPI) is an autosomal recessively inherited inborn error of metabolism (IEM) caused by the defect in the dibasic cationic amino acid transporter found on the basolateral membrane of the lung, small intestine, and kidney due to mutations in the SLC7A7 gene, which encodes the y+LAT1 protein. LPI may present as an acute hyperammonemic episode or as chronic symptoms. Major clinical symptoms are feeding problems, vomiting and diarrhea, failure to thrive, hepatosplenomegaly, and cytopenia. We present a delayed diagnosis of symptomatic LPI with a homozygous mutation in the SLC7A7 gene.Case presentationA 15-year-old girl was referred to our clinic due to growth retardation and diarrhea. Physical examination showed short stature, retarded puberty, and hepatosplenomegaly. Laboratory tests showed normal complete blood count and biochemical analyses except elevated aspartate aminotransferase, triglyceride, total cholesterol, and ferritin. Peripheral blood smear and hemoglobin electrophoresis were within normal limits. Bone marrow analysis showed hemophagocytic cells. Postprandial ammonium level was found elevated. Low lysine, arginine, and ornithine and elevated glycine and alanine in plasma amino acid analysis and high amount of lysine and slightly elevated arginine and ornithine excretion in urine were detected. Molecular genetic analysis of the SLC7A7 gene showed a previously reported homozygous mutation. Low protein diet, sodium benzoate, l-carnitine, low-dose l-citrulline, and calcium replacement were initiated. The patient is now in good condition still being followed up in our department.ConclusionsLPI is a metabolic disorder with multi-systemic involvement that may have severe consequences if left untreated. Initiation of early treatment is essential for the prevention of severe chronic complications. Also, confirmation of the genetic defect may provide the parents to have healthy offsprings in the future with the help of genetic counselling and preimplantation genetics.

Maternal Amino Acid Profiles to Distinguish Constitutionally Small versus Growth-Restricted Fetuses Defined by Doppler Ultrasound: A Pilot Study.

Fetuses measuring below the 10th percentile for gestational age may be either constitutionally small for gestational age (SGA) or have pathologic fetal growth restriction (FGR). FGR is associated with adverse outcomes; however, identification of low-risk SGA cases is difficult. We performed a pilot study evaluating maternal markers of pathologic FGR, hypothesizing there are distinct amino acid signatures that might be used for diagnosis and development of new interventions. This was a cohort study of healthy women with sonographic fetal estimated fetal weight <5th percentile divided into two groups based upon umbilical artery (UmA) Doppler studies or uterine artery (UtA) Doppler studies. We collected maternal blood samples prior to delivery and used ion pair reverse phase liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry to assess 44 amino acids. Among 14 women included, five had abnormal UmA, and three had abnormal UtA Doppler results. Those with abnormal UmA showed elevated ornithine. Those with abnormal UtA had lower dimethylglycine, isoleucine, methionine, phenylalanine, and 1-methylhistidine. We found several amino acids that might identify pregnancies affected by pathologic FGR. These findings support the feasibility of future larger studies to identify maternal metabolic approaches to accurately stratify risk for small fetuses.

A pilot study on machine learning approach to delineate metabolic signatures in intellectual disability

Intellectual disability (ID) is a neurodevelopmental disorder characterized by cognitive delays. Inborn errors of metabolism constitute an important subgroup of ID for which various treatments options are available. We aimed to identify potential biomarkers of inherited metabolic disorders from the children with ID using tandem mass spectrometry and develop a novel machine learning algorithm to differentiate between the cases and the controls. All of the cases were having IQ score <70, gross motor delay, speech disorder and no recognizable symptoms of the condition. Metabolite profiling of ID individuals exhibited low tyrosine/large neutral amino acids, high citrulline/arginine ratios; elevated proline, alanine, phenylalanine, and ornithine, while a significant decrease in the level of amino acid arginine, and elevated C4 (butyrylcarnitine) and C4OH/C3DC (3-hydroxybutyrylcarnitine/malonylcarnitine). Machine learning algorithm differentiated cases and controls efficiently using specific thresholds of ornithine, arginine and C4OH/C3DC. Furthermore, ID cases were distinguished into mild, moderate, and severe based on specific thresholds of methionine, arginine, and C5OH/C4DC (3-hydroxyisovalerylcarnitine/methylmalonylcarnitine). The machine learning algorithm could successfully identify specific metabolite markers in ID and correlate the same with neurological features.

Interplay of Nitric Oxide Synthase (NOS) and SrrAB in Modulation of Staphylococcus aureus Metabolism and Virulence.

Staphylococcus aureus nitric oxide synthase (saNOS) is a major contributor to virulence, stress resistance, and physiology, yet the specific mechanism(s) by which saNOS intersects with other known regulatory circuits is largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory menaquinones, is a positive regulator of nos expression. Several SrrAB-regulated genes were also previously shown to be induced in an aerobically respiring nos mutant, suggesting a potential interplay between saNOS and SrrAB. Therefore, a combination of genetic, molecular, and physiological approaches was employed to characterize a nos srrAB mutant, which had significant reductions in the maximum specific growth rate and oxygen consumption when cultured under conditions promoting aerobic respiration. The nos srrAB mutant secreted elevated lactate levels, correlating with the increased transcription of lactate dehydrogenases. Expression of nitrate and nitrite reductase genes was also significantly enhanced in the nos srrAB double mutant, and its aerobic growth defect could be partially rescued with supplementation with nitrate, nitrite, or ammonia. Furthermore, elevated ornithine and citrulline levels and highly upregulated expression of arginine deiminase genes were observed in the double mutant. These data suggest that a dual deficiency in saNOS and SrrAB limits S. aureus to fermentative metabolism, with a reliance on nitrate assimilation and the urea cycle to help fuel energy production. The nos, srrAB, and nos srrAB mutants showed comparable defects in endothelial intracellular survival, whereas the srrAB and nos srrAB mutants were highly attenuated during murine sepsis, suggesting that SrrAB-mediated metabolic versatility is dominant in vivo.

Plasma metabolic profiling on postoperative colorectal cancer patients with different traditional Chinese medicine syndromes

ObjectivesThis study aims to investigate the metabolic profiles of postoperative colorectal cancer (PCRC) patients with different traditional Chinese medicine (TCM) syndromes and to discuss the metabolic mechanism under PCRC progression and TCM syndrome classification. MethodsFifty healthy controls (HC) and 70 PCRC patients, including 10 Dampness and heat syndrome (DHS), 33 Spleen deficiency syndrome (SDS), 19 Liver and kidney Yin deficiency syndrome (LKYDS) and 8 with non-TCM syndrome (NS) were enrolled. Plasma metabolic profiles were detected by Gas chromatography-mass spectrometry (GC–MS) and analyzed by principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA). Furthermore, pathway enrichment was analyzed based on KEGG and DAVID databases and metabolic network was constructed via metaboanalyst and cytoscape. ResultsThe top-3 metabolites with higher abundance in PCRC compared with HC were terephthalic acid (165.417-fold), ornithine (24.484-fold) and aminomalonic acid (21.346-fold). And the cholesterol (0.588-fold) level was decreased in PCRC. l-Alanine, 1, 2-ethanediamine, urea, glycerol, glycine, aminomalonic acid, creatinine and palmitic acid were specifically altered in the DHS, while d-tryptophan was exclusively changed in SDS, and l-proline, 1, 2, 3-propanetricarboxylic acid, d-galactose and 2-indolecarboxylic acids in LKYDS. ConclusionsThe plasma metabolic profiles were perturbed in PCRC patients. Increased levels of terephthalic acid might indicate high risk of relapse and elevated ornithine may contribute to the post-operational recovery or may raise the susceptibility to PCRC recurrence. The metabolic profiles of DHS, SDS, LKYDS and NS were almost separately clustered, indicating the possibility of explaining TCM syndromes classification using metabolomics. Furthermore, creatinine and aminomalonic acid alternation might correlate with the formation of DHS, while d-tryptophan may associate with SDS and d-galactose and 1, 2, 3-propanetricarboxylic acid may relate to LKYDS. As numbers of patients in each TCM syndrome are small, further study is needed to verify those results.

Opposite Associations of Plasma Homoarginine and Ornithine with Arginine in Healthy Children and Adolescents

Homoarginine, a non-proteinogenic amino acid, is formed when lysine replaces ornithine in reactions catalyzed by hepatic urea cycle enzymes or lysine substitutes for glycine as a substrate of renal arginine:glycine amidinotransferase. Decreased circulating homoarginine and elevated ornithine, a downstream product of arginase, predict adverse cardiovascular outcome. Our aim was to investigate correlates of plasma homoarginine and ornithine and their relations with carotid vascular structure in 40 healthy children and adolescents aged 3–18 years without coexistent diseases or subclinical carotid atherosclerosis. Homoarginine, ornithine, arginine, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) were measured by liquid chromatography-tandem mass spectrometry with stable isotope-labeled internal standards. Intima-media thickness (IMT) and extra-medial thickness (EMT) of common carotid arteries were estimated by B-mode ultrasound. Homoarginine correlated with arginine (r = 0.43, p = 0.005), age (r = 0.42, p = 0.007) and, weakly, with an increased arginine-to-ornithine ratio, a putative measure of lower arginase activity (r = 0.31, p = 0.048). Ornithine correlated inversely with arginine (r = −0.64, p < 0.001). IMT, EMT or their sum were unrelated to any of the biochemical parameters (p > 0.12). Thus, opposite associations of plasma homoarginine and ornithine with arginine may partially result from possible involvement of arginase, an enzyme controlling homoarginine degradation and ornithine synthesis from arginine. Age-dependency of homoarginine levels can reflect developmental changes in homoarginine metabolism. However, neither homoarginine nor ornithine appears to be associated with carotid vascular structure in healthy children and adolescents.

Elevated ornithine decarboxylase activity promotes skin tumorigenesis by stimulating the recruitment of bulge stem cells but not via toxic polyamine catabolic metabolites

Elevated expression of ornithine decarboxylase (ODC), the regulatory enzyme in polyamine biosynthesis, targeted to the epidermis is sufficient to promote skin tumor development following a single subthreshold dose of dimethylbenz(a)anthracene (DMBA). Since skin tumor promotion involves recruitment of hair follicle bulge stem cells harboring genetic lesions, we assessed the effect of increased epidermal ODC on recruitment of bulge stem cells in ODC-ER transgenic mice in which ODC activity is induced de novo in adult skin with 4-hydroxytamoxifen (4OHT). Bromodeoxyuridine-pulse labeling and use of K15.CrePR1;R26R;ODC-ER triple transgenic mice demonstrated that induction of ODC activity is sufficient to recruit bulge stem cells in quiescent skin. Because increased ODC activity not only stimulates proliferation but also increases reactive oxygen species (ROS) generation via subsequent induction of polyamine catabolic oxidases, we used an inhibitor of polyamine catabolic oxidase activity, MDL72527, to investigate whether ROS generation by polyamine catabolic oxidases contributes to skin tumorigenesis in DMBA-initiated ODC-ER transgenic skin. Newborn ODC-ER transgenic mice and their normal littermates were initiated with a single topical dose of DMBA. To assess tumor development originating from dormant bulge stem cells that possess DMBA-initiated mutations, epidermal ODC activity was induced in ODC-ER mice with 4OHT 5weeks after DMBA initiation followed by MDL72527 treatment. MDL72527 treatment resulted in a shorter tumor latency time, increased tumor burden, increased conversion to carcinomas, and lower tumor levels of p53. Thus, elevated epidermal ODC activity promotes tumorigenesis by stimulating the recruitment of bulge stem cells but not via ROS generation by polyamine catabolic oxidases.

Abstract 2471: Immunosuppressive effect of elevated epidermal ornithine decarboxylase activity

Abstract Tumor promoting agents, such as phorbol esters, promote tumorigenesis via induction of inflammation and hyperplasia. However, the immunosuppressive/immunomodulating properties of tumor promoting agents may play a relevant role in tumor promotion as well. Because elevated polyamine biosynthesis is sufficient to promote skin tumorigenesis, 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. To investigate the role of ODC in the control of cutaneous inflammation, a well-known model of T cell mediated inflammation was used. A contact hypersensitivity (CHS) response was elicited in the ears of both ODC transgenic mice, in which ODC is targeted to the epidermis, and their normal littermates by topical sensitization and challenge with oxazolone. Unlike in normal littermate mice, ear swelling was dramatically reduced in ODC transgenic mice after elicitation of CHS. Flow cytometric and immunohistochemical analyses of the infiltrating immune cells revealed fewer infiltrating immune cells, especially neutrophils, in the elicited ears of ODC transgenic mice. The specific ODC inhibitor, α-difluoromethylornithine, abrogated the suppressive effects of ODC in CHS reactions. Adoptive transfer of sensitized lymphocytes from either ODC transgenic or normal littermate mice to naïve ODC transgenic or wild-type mice indicated that elevated epidermal ODC activity suppress both the sensitization and elicitation phases of CHS. Cytokines are central mediators of immune functions associated with inflammatory reactions. The cytokine profile of ex vivo stimulated lymphocytes isolated from draining lymph nodes showed reduced levels of IL-6 and IL-10 in sensitized and challenged ODC transgenic mice. In vivo labeling of dendritic cells by topical application of fluorescein isothiocyanate (FITC) showed fewer FITC-labeled dendritic cells in the draining lymph nodes of ODC transgenic mice compared to normal littermates. Vascular permeability was assessed using Evans blue extravasation assays during the elicitation phase of CHS. At 24 hours after induction of CHS, there was 45% less plasma extravasation of Evans blue dye into the ear skin of ODC transgenic mice compared to normal littermates. Collectively these data indicate elevated epidermal ODC activity can suppress a number of functions associated with cutaneous inflammatory reactions. In addition to promoting cellular proliferation, the immune suppression mediated by increased epidermal ODC activity may provide an environment conducive to tumor growth. This work was supported by NIH grant CA070739. 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 2471.

Can ornithine accumulation modulate abiotic stress tolerance in Arabidopsis?

The arginine biosynthetic pathway represents an area of plant biochemistry that has been poorly investigated. Recently, the first enzyme of the arginine pathway, encoded by the N-acetyl-L-glutamate synthase gene (SlNAGS1), was isolated and characterised in tomato, and was found to be structurally similar to other predicted NAGS. SlNAGS1 accumulation patterns suggest a possible role of this gene in hypoxia-induced responses. The 35S::SlNAGS1 Arabidopsis plants accumulated ornithine at high levels and exhibited increased tolerance to salt and drought stresses. Ornithine is the intermediate compound in the arginine biosynthesis where the pathway divaricates to the production of compounds, such as proline and polyamines that are known to serve osmoprotective functions. It is therefore likely that the elevated ornithine accumulation in the SlNAGS1-overexpressing plants be coupled with the production of a pool of osmoprotectants that end up to the improved stress tolerance. The possible implications of ornithine accumulation are discussed.

Diabetes‐induced arginase activity contributes to vascular and renal fibrosis and dysfunction

Impaired endothelial cell dependent (ECD) vasodilation and vascular fibrosis (VF) are prominent complications in diabetes. We previously reported that increased arginase activity in diabetic rats competes with NOS for L-arginine decreasing NO production and ECD vasodilation. Treatment with simvastatin (Sim) or L-citrulline (L-cit) prevents these effects. Arginase catabolizes L-arginine into ornithine and urea. Elevated ornithine leads to increased polyamine and proline production, which are involved in cell growth and collagen deposition. Our present study indicates that increased arginase activity in diabetic rats is associated with coronary VF as preventing arginase activation with Sim blocks these effects. Sim or L-cit treatment also prevents renal (glomerular and interstitial) fibrosis and proteinuria in diabetic rats. We examined the role of arginase (A) isoforms in AI +/+/AII −/−or AI+/−/AII−/− knockout (KO) and wild type (WT) mice in diabetes-induced impairment of ECD vasodilation and VF. Arginase activity is increased by 160% and 120% in aortas of diabetic WT and AI +/+/AII −/− KO mice respectively, compared to controls. Partial deletion of AI prevents this effect. Moreover, ECD vasodilation was less impaired in the AI+/−/AII−/− KO than in the WT diabetic mice. These results correlate with less VF in AI+/−/AII−/− KO vs WT diabetic mice. Our results suggest that diabetes-induced increases in arginase I activity are associated with vascular and renal fibrosis. Supported by NIH grant RO1 HL70215.

Elevated Ornithine Decarboxylase Levels Activate Ataxia Telangiectasia Mutated–DNA Damage Signaling in Normal Keratinocytes

We examined the effect of increased expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on cell survival in primary cultures of keratinocytes isolated from the skin of K6/ODC transgenic mice (Ker/ODC) and their normal littermates (Ker/Norm). Although elevated levels of ODC and polyamines stimulate proliferation of keratinocytes, Ker/ODC undergo apoptotic cell death within days of primary culture unlike Ker/Norm that continue to proliferate. Phosphorylation of ataxia telangiectasia mutated (ATM) and its substrate p53 are significantly induced both in Ker/ODC and in K6/ODC transgenic skin. Chromatin immunoprecipitation analyses show that the increased level of p53 in Ker/ODC is accompanied by increased recruitment of p53 to the Bax proximal promoter. ATM activation is polyamine dependent because alpha-difluoromethylornithine, a specific inhibitor of ODC activity, blocks its phosphorylation. Ker/ODC also displays increased generation of H(2)O(2), acrolein-lysine conjugates, and protein oxidation products as well as polyamine-dependent DNA damage, as measured by the comet assay and the expression of the phosphorylated form of the histone variant gamma H2AX. Both reactive oxygen species generation and apoptotic cell death of Ker/ODC may, at least in part, be due to induction of a polyamine catabolic pathway that generates both H(2)O(2) and cytotoxic aldehydes, because spermine oxidase (SMO) levels are induced in Ker/ODC. In addition, treatment with MDL 72,527, an inhibitor of SMO, blocks the production of H(2)O(2) and increases the survival of Ker/ODC. These results show a novel activation of the ATM-DNA damage signaling pathway in response to increased ODC activity in nontumorigenic keratinocytes.

Loss of Methylthioadenosine Phosphorylase and Elevated Ornithine Decarboxylase Is Common in Pancreatic Cancer

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. 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. 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. 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.

Antitumor promotional effects of a novel intestinal bacterial metabolite (IH-901) derived from the protopanaxadiol-type ginsenosides in mouse skin

Epidemiological studies have demonstrated that ginseng intake decreases the risk of cancer. Ginseng saponins (ginsenosides) have been regarded as principal components responsible for the majority of pharmacological activities exerted by ginseng. IH-901 [20-O-beta-d-glucopyranosyl-20(S)-protopanaxadiol], an intestinal bacterial metabolite derived from protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess antitumor effects, including inhibition of invasion, metastasis and angiogenesis and induction of tumor cell apoptosis. Tumor promotion often accompanies an elevated ornithine decarboxylase (ODC) activity, acute inflammation and induction of cyclooxygenase-2 (COX-2) activity. Here we examined the effects of IH-901 on tumor promotion and related molecular events in mouse skin in vivo. Mouse ear edema induced by the prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was repressed by IH-901 pre-treatment in a dose-dependent manner. Topical application of IH-901 onto shaven backs of female ICR mice led to the inhibition of TPA-induced expression of COX-2 and production of prostaglandin E(2). The eukaryotic transcription factor NF-kappaB has been involved in intracellular signaling pathways associated with inflammation and carcinogenesis. IH-901 pre-treatment inhibited TPA-induced epidermal NF-kappaB DNA binding in mouse skin, which appeared to be mediated by blocking phosphorylation and subsequent degradation of IkappaBalpha. In an attempt to elucidate the molecular mechanisms by which IH-901 inactivates NF-kappaB, its effects on activation of upstream signaling kinases were explored. IH-901 also inhibited the activation of ERK1/2 and Akt signaling. When IH-901 was treated topically prior to TPA, expression and activity of ODC were inhibited dose-dependently. In addition, IH-901 given prior to each topical dose of TPA markedly lowered the number of papillomas in mouse skin induced by 7,12-dimethylbenz[a]anthracene. Taken together, these findings suggest that IH-901 exerts anti-inflammatory effects by inhibiting TPA-induced COX-2 expression, which may contribute to its antitumor-promoting effects on mouse skin carcinogenesis.

Lupeol, a triterpene, inhibits early responses of tumor promotion induced by benzoyl peroxide in murine skin

The modulating effect of Lupeol [lup-20(29)-en-3 β -ol], a triterpene found in many fruits and medicinal plants, on benzoyl peroxide-induced tumor promotion responses or tumor promotion in murine skin is described. Benzoyl peroxide is an effective cutaneous tumor promoter acting through the generation of oxidative stress, the induction of ornithine decarboxylase activity and the enhancement of DNA synthesis. Benzoyl peroxide treatment increases cutaneous microsomal lipid peroxidation and hydrogen peroxide generation. The activity of the cutaneous antioxidant enzymes, namely catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase, is decreased and levels of cutaneous glutathione are depleted. Benzoyl peroxide treatment also induces ornithine decarboxylase activity and enhances [3H]thymidine uptake in DNA synthesis. Prophylactic treatment of mice with lupeol (0.75 and 1.5 mg per animal) 1 hour before benzoyl peroxide treatment resulted in a diminution of benzoyl peroxide-mediated damage. The susceptibility of cutaneous microsomal membrane to lipid peroxidation and hydrogen peroxide generation was significantly reduced (P< 0.01 and P< 0.01, respectively). In addition, depleted levels of glutathione and inhibited activity of antioxidant enzymes were recovered to a significant level (P< 0.01, P< 0.05 and P< 0.01, respectively). Similarly, the elevated ornithine decarboxylase activity and enhanced thymidine uptake in DNA synthesis were inhibited significantly (P< 0.05) in a dose-dependent manner. The protective effect of lupeol was dose dependent in all parameters. The results suggest that lupeol is an effective skin chemopreventive agent that may suppress benzoyl peroxide-induced cutaneous toxicity.