Regulation Of Ornithine Decarboxylase Research Articles

Phase 1 study of high-dose DFMO, celecoxib, cyclophosphamide and topotecan for patients with relapsed neuroblastoma: a New Approaches to Neuroblastoma Therapy trial

BackgroundMYC genes regulate ornithine decarboxylase (Odc) to increase intratumoral polyamines. We conducted a Phase I trial [NCT02030964] to determine the maximum tolerated dose (MTD) of DFMO, an Odc inhibitor, with celecoxib, cyclophosphamide and topotecan.MethodsPatients 2–30 years of age with relapsed/refractory high-risk neuroblastoma received oral DFMO at doses up to 9000 mg/m2/day, with celecoxib (500 mg/m2 daily), cyclophosphamide (250 mg/m2/day) and topotecan (0.75 mg/m2/day) IV for 5 days, for up to one year with G-CSF support.ResultsTwenty-four patients (median age, 6.8 years) received 136 courses. Slow platelet recovery with 21-day courses (dose-levels 1 and 2) led to subsequent dose-levels using 28-day courses (dose-levels 2a-4a). There were three course-1 dose-limiting toxicities (DLTs; hematologic; anorexia; transaminases), and 23 serious adverse events (78% fever-related). Five patients (21%) completed 1-year of therapy. Nine stopped for PD, 2 for DLT, 8 by choice. Best overall response included two PR and four MR. Median time-to-progression was 19.8 months, and 3 patients remained progression-free at >4 years without receiving additional therapy. The MTD of DFMO with this regimen was 6750 mg/m2/day.ConclusionHigh-dose DFMO is tolerable when added to chemotherapy in heavily pre-treated patients. A randomized Phase 2 trial of DFMO added to chemoimmunotherapy is ongoing [NCT03794349].

A Genome-Wide Association Study of Age-Related Hearing Impairment in Middle- and Old-Aged Chinese Twins.

Background Age-related hearing impairment (ARHI) is considered an unpreventable disorder. We aimed to detect specific genetic variants that are potentially related to ARHI via genome-wide association study (GWAS). Methods A sample of 131 dizygotic twins was genotyped for single-nucleotide polymorphism- (SNP-) based GWAS. Gene-based test was performed using VEGAS2. Pathway enrichment analysis was conducted by PASCAL. Results The twins are with a median age of 49 years, of which 128 were females and 134 were males. rs6633657 was the only SNP that reached the genome-wide significance level for better ear hearing level (BEHL) at 2.0 kHz (P = 1.19 × 10−8). Totally, 9, 10, 42, 7, 17, and 5 SNPs were suggestive evidence level for (P < 1 × 10−5) BEHLs at 0.5, 1.0, 2.0, 4.0, and 8.0 kHz and pure tone average (PTA), respectively. Several promising genetic regions in chromosomes (near the C20orf196, AQPEP, UBQLN3, OR51B5, OR51I2, OR52D1, GLTP, GIT2, and PARK2) nominally associated with ARHI were identified. Gene-based analysis revealed 165, 173, 77, 178, 170, and 145 genes nominally associated with BEHLs at 0.5, 1.0, 2.0, 4.0, and 8.0 kHz and PTA, respectively (P < 0.05). For BEHLs at 0.5, 1.0, and 2.0 kHz, the main enriched pathways were phosphatidylinositol signaling system, regulation of ornithine decarboxylase, eukaryotic translation initiation factor (EIF) pathway, amine compound solute carrier (SLC) transporters, synthesis of phosphoinositides (PIPS) at the plasma membrane, and phosphatidylinositols (PI) metabolism. Conclusions The genetic variations reported herein are significantly involved in functional genes and regulatory domains that mediate ARHI pathogenesis. These findings provide clues for the further unraveling of the molecular physiology of hearing functions and identifying novel diagnostic biomarkers and therapeutic targets of ARHI.

Critical Factors in Human Antizymes that Determine the Differential Binding, Inhibition, and Degradation of Human Ornithine Decarboxylase.

Antizyme (AZ) is a protein that negatively regulates ornithine decarboxylase (ODC). AZ achieves this inhibition by binding to ODC to produce AZ-ODC heterodimers, abolishing enzyme activity and targeting ODC for degradation by the 26S proteasome. In this study, we focused on the biomolecular interactions between the C-terminal domain of AZ (AZ95–228) and ODC to identify the functional elements of AZ that are essential for binding, inhibiting and degrading ODC, and we also identified the crucial factors governing the differential binding and inhibition ability of AZ isoforms toward ODC. Based on the ODC inhibition and AZ-ODC binding studies, we demonstrated that amino acid residues reside within the α1 helix, β5 and β6 strands, and connecting loop between β6 and α2 (residues 142–178), which is the posterior part of AZ95–228, play crucial roles in ODC binding and inhibition. We also identified the essential elements determining the ODC-degradative activity of AZ; amino acid residues within the anterior part of AZ95–228 (residues 120–145) play crucial roles in AZ-mediated ODC degradation. Finally, we identified the crucial factors that govern the differential binding and inhibition of AZ isoforms toward ODC. Mutagenesis studies of AZ1 and AZ3 and their binding and inhibition revealed that the divergence of amino acid residues 124, 150, 166, 171, and 179 results in the differential abilities of AZ1 and AZ3 in the binding and inhibition of ODC.

Abstract 3854: Antizyme inhibitor editing modulates nuclear localization but not antizyme binding: Implications for prostate cancer

Abstract The endogenous antizyme inhibitor (AZIN) binds to, and is a key regulator of, the cell cycle suppressor antizyme. Antizyme itself is a key regulator of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine synthesis. We have measured the expression and localization of AZIN in 202 prostate cancer specimens, along with 26 adjacent benign samples, and found that nuclear localization of AZIN is dramatically associated with worse outcome. Upregulation and nuclear localization of AZIN have been observed in several cancers, as has editing of the AZIN1 mRNA. Other have hypothesized that the RNA- edited AZIN (edAZIN) may have an increased affinity to antizyme and that could explain the association of edAZIN to the various cancers. We have studied the mechanism behind the nuclear localization of AZIN and found the single base pair substitution caused by RNA editing is sufficient to result in nuclear localization of the protein in all cell types tested. To determine if the nuclear localization might result from increased antizyme-edAZIN affinity, we developed fluorescent protein FRET sensor for protein-protein interaction using Clover-AZIN and antizyme-mRuby2 fusion proteins. Unexpectedly, we found that the editing event decreases edAZIN affinity for antizyme, notwithstanding increased interaction in vivo. Thus, the data indicate that the change in protein localization to the nucleus may be more important to oncogenic function than the actual degree of binding to antizyme. Other functional differences between edAZIN and AZIN might be explained by altered kinetics of binding, by the contribution of an additional adapter protein that modulates the intracellular antizyme:AZIN complex, or by competition for AZIN binding by other partners whose interaction is affected by the editing event. Therefore, we plan to identify AZIN and edAZIN interacting proteins by using proteomic analysis and later study the interaction between AZIN or edAZIN with their binding partners in more detail. Furthermore, our validated FRET assays can be used to identify antagonists of AZIN-antizyme binding (no antagonists are currently known). Given the important role of AZIN in tumor growth, discovery of small-molecule inhibitors of AZIN should lead to antitumor therapies. Citation Format: James M. Rice, Aram Ghalali, Liangzhe Wang, Amanda Kusztos, Finith Jernigan, Chin Lee Wu, Bruce Zetter, Michael S. Rogers. Antizyme inhibitor editing modulates nuclear localization but not antizyme binding: Implications for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3854.

Abstract 691: DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation

Abstract Background: Neuroblastoma (NB) is the most common extracranial solid pediatric tumor and is associated with MYCN amplification. MYCN is a regulator of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis. Inhibition of this pathway in MYCN-amplified NB tumors has been shown to be a target for treatment. Alpha-difluoromethylornithine (DFMO) inhibits ODC and is currently being used in a Phase II clinical trial for NB. BET inhibitors JQ1 and OTX-015 have been shown to be effective against MYCN-amplified cancers; it is hypothesized that they downregulate MYCN as well as cancer stem cell (CSC) signaling. We propose that inhibiting MYCN with a BET inhibitor, coupled with inhibition of ODC with DFMO, will result in enhanced inhibition of NB growth and tumor-initiating properties. Methods: Single and combination drug treatments were conducted on BE(2)-C, SMS-KCNR, CHLA90, and one patient-derived cell line. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability in 96-well plates. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. Drug combination studies were conducted in MYCN amplified tumors BE(2)-C and SMS-KCNR using ray designs to evaluate for synergism. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity and apoptosis in NB cells. Western blots measured protein levels of apoptosis markers (cleaved caspase 3 and cleaved PARP) and CSC markers (Nanog, Sox2, NF-kB, CXCR4, Lin28B, and MYCN). Neurosphere assays were used to evaluate tumor initiation via sphere formation. Results: Cell viability of MYCN amplified NB showed an IC50 range of 1.48-1.69 μM for JQ1 and 839.3 nM-2.36 μM for OTX-015; MYCN non-amplified NB showed an IC50 range of 4.22-11.58 μM for JQ1 and 2.99-11.03 uM for OTX-015. Combination treatment in MYCN NB had a synergistic effect, based on Loewe-additivity as the null hypothesis, for cell viability suppression by ray design experiments for BE(2)-C and SMS-KCNR. Western blots showed greater expression of apoptosis markers and decreased expression of CSC markers in combination relative to single drug treatments. A greater than 50% decrease in neurosphere formation with combination treatment in BE(2)-C, SMS-KCNR, and the patient cell line provides evidence for reduced CSC activity. IncuCyte imaging showed an increase in cell death with time post-treatment. Conclusion: The combination of DFMO with BET inhibitors has a synergistic effect in treating MYCN amplified NBs as shown by a decrease in cell viability. This combination targets CSC pathways and decreases tumor-initiating ability. Given the lack of effective treatment options for children with relapsed or refractory high-risk NB, this combination may be a promising novel therapy. Citation Format: Sarah DeCou, Ping Zhao, Tracey Avequin, Abhinav Nagulapally, Jeffrey Bond, Giselle Saulnier Sholler. DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 691. doi:10.1158/1538-7445.AM2017-691

Multifaceted interactions and regulation between antizyme and its interacting proteins cyclin D1, ornithine decarboxylase and antizyme inhibitor.

Ornithine decarboxylase (ODC), cyclin D1 (CCND1) and antizyme inhibitor (AZI) promote cell growth. ODC and CCND1 can be degraded through antizyme (AZ)-mediated 26S proteasomal degradation. This paper describes a mechanistic study of the molecular interactions between AZ and its interacting proteins. The dissociation constant (Kd) of the binary AZ-CCND1 complex and the respective binding sites of AZ and CCND1 were determined. Our data indicate that CCND1 has a 4-fold lower binding affinity for AZ than does ODC and an approximately 40-fold lower binding affinity for AZ than does AZI. The Kd values of AZ-CCND1, AZ-ODC and AZ-AZI were 0.81, 0.21 and 0.02 μM, respectively. Furthermore, the Kd values for CCND1 binding to the AZ N-terminal peptide (AZ34-124) and AZ C-terminal peptide (AZ100-228) were 0.92 and 8.97 μM, respectively, indicating that the binding site of CCND1 may reside at the N-terminus of AZ, rather than the C-terminus. Our data also show that the ODC-AZ-CCND1 ternary complex may exist in equilibrium. The Kd values of the [AZ-CCND1]-ODC and [AZ-ODC]-CCND1 complexes were 1.26 and 4.93 μM, respectively. This is the first paper to report the reciprocal regulation of CCND1 and ODC through AZ-dependent 26S proteasomal degradation.

Association between the ornithine decarboxylase G316A polymorphism and breast cancer survival.

Ornithine decarboxylase (ODC) is a significant rate-limiting enzyme in polyamine synthesis, required for normal cell growth, and is highly expressed in various malignancies, including colorectal and breast cancer. In the present study, the associations between the ODC G316A single nucleotide polymorphism (SNP) and breast cancer-specific survival were investigated. In addition, the functional effects of this SNP were examined in the MCF-7 human breast cancer cell line. The present study recruited 300 stage I-III breast cancer cases, which were diagnosed at the Affiliated Cancer Hospital of Zhengzhou University (Zhengzhou, China) between 2002 and 2003, with follow-up visits conducted until May 2013. ODC G316A was genotyped (ODC GG vs. ODC AG/AA) in the 300 cases and the association of the genotypes with cancer-specific survival was analyzed. In the MCF-7 cell line, the ODC allele-specific binding of E-box transcription factors was determined using western blot and chromatin immunoprecipitation assays. Survival differences were observed between the two genotypes: Compared with the ODC GG genotype, patients with ODC GA/AA exhibited significantly higher survival rates (P<0.05). In cultured cells, the ODC SNP, which is flanked by two E-boxes, appeared to predict ODC promoter activity. Furthermore, the E-box activator c-MYC and repressor MAX interactor 1 were found to preferentially bind to ODC minor A-alleles compared with major G-alleles, in cultured MCF-7 cells. In conclusion, the results of the current study suggest that the regulation of ODC may affect survival in breast cancer patients and indicate a model in which the ODC SNP may be protective for breast adenoma recurrence and detrimental for survival following a diagnosis of breast cancer.

Molecular cloning and mRNA expression analysis of antizyme inhibitor 1 in the ovarian follicles of the Sichuan white goose

Antizyme inhibitor 1 (Azin1) plays critical roles in various cellular pathways, including ornithine decarboxylase regulation, polyamine anabolism and uptake and cell proliferation. However, the molecular characteristics of the AZIN1 gene and its expression profile in goose tissues and ovarian follicles have not been reported. In this study, the AZIN1 cDNA of the Sichuan white goose (Anser cygnoides) was cloned, and analyzed for its phylogenetic and physiochemical properties. The expression profile of AZIN1 mRNA in geese tissues and ovarian follicles were examined using quantitative real-time PCR. The results showed that the open reading frame of the AZIN1 cDNA is 1353bp in length, encoding a 450 amino acid protein with a molecular weight of 50kDa. Out of all tissues examined, AZIN1 expression was highest in the adrenal gland and lowest in breast muscle. There was also a high expression of AZIN1 in the cerebellum and isthmus of oviduct. With follicular development, AZIN1 gene expression gradually increased, and its expression in F1 was significantly higher than in F5 (P<0.05). AZIN1 expression was also significantly higher in the POF1 than in the other follicles (P<0.05), and there was a low mRNA expression of AZIN1 in atretic follicles. The results of AZIN1 expression profiling in ovarian follicles suggest that AZIN1 may play an important role in the progression of follicular development, potentially through regulating polyamine levels.

Adult Atlantic salmon (Salmo salarL.) adapts to long-term surplus dietary arginine supplementation

The current study aimed to investigate the effect of surplus dietary arginine on polyamine and amino acid metabolism and accretion of proteins and lipids, as previous studies have demonstrated a lipid-reducing effect following surplus arginine supplementation in viscera as well as increased muscle growth. Four plant protein-based diets were given increasing concentrations of L-arginine, from 21.1 to 36.1 g kg-1 DM, and fed to quadruplicate tanks of adult Atlantic salmon (1.1 kg) for 12 weeks. No effects on growth or deposition of fat or protein were observed. Free amino acid concentrations and related metabolites were unaffected in the liver, except for urea, while concentrations in muscle and plasma reflected production of arginine metabolites. Polyamine concentrations were unaffected in liver, muscle and white adipose tissue (WAT), as were the abundance and activity of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in polyamine turnover. Gene expression demonstrated differential regulation of ornithine decarboxylase (ODC) in liver and WAT, although overall little effects were observed on gene expression. Liver S-adenosylmethionine (SAM) concentrations decreased with arginine supplementation. We suggest that adult Atlantic salmon have adapted to surplus arginine, and the main long-term effect appears to be increased concentrations of arginine metabolites.

Hairless and the polyamine putrescine form a negative regulatory loop in the epidermis

Hairless (HR) is a nuclear protein with corepressor activity that is highly expressed in the skin and hair follicle. Mutations in Hairless lead to hair loss accompanied by the appearance of papules (atrichia with papular lesions), and similar phenotypes appear when the key polyamine enzymes ornithine decarboxylase (ODC) and spermidine/spermine N(1) -acetyltransferase (SSAT) are overexpressed. Both ODC and SSAT transgenic mice have elevated epidermal levels of putrescine, leading us to investigate the mechanistic link between putrescine and HR. We show here that HR and putrescine form a negative regulatory network, as epidermal ODC expression is elevated when HR is decreased and vice versa. We also show that the regulation of ODC by HR is dependent on the MYC superfamily of proteins, in particular MYC, MXI1 and MXD3. Furthermore, we found that elevated levels of putrescine lead to decreased HR expression, but that the SSAT-TG phenotype is distinct from that found when HR is mutated. Transcriptional microarray analysis of putrescine-treated primary human keratinocytes demonstrated differential regulation of genes involved in protein-protein interactions, nucleotide binding and transcription factor activity, suggesting that the putrescine-HR negative regulatory loop may have a large impact on epidermal homeostasis and hair follicle cycling.

The induction of cardiac ornithine decarboxylase by β2‐adrenergic agents is associated with calcium channels and phosphorylation of ERK1/2

The role that the induction of cardiac ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by beta-adrenergic agents may have in heart hypertrophy is a controversial issue. Besides, the signaling pathways related to cardiac ODC regulation have not been fully elucidated. Here we show that in Balb C mice the stimulation of cardiac ODC activity by adrenergic agents was mainly mediated by β2 -adrenergic receptors, and that this induction was lower in the hypertrophic heart. Interestingly, this stimulation was abolished by the L-calcium channel antagonists verapamil and nifedipine. In addition, whereas the treatment with β2 -adrenergic agents was associated to both the increases in ODC, ODC-antizyme inhibitor 1 (AZIN1), c-fos and c-myc mRNA levels and the phosphorylation of CREB and MAP kinases ERK1 and ERK2 (ERK1/2), the co-treatment with L-calcium channel blockers differentially prevented most of these changes. These results suggest that the stimulation of cardiac ODC by β2 -adrenergic agents is associated with the activation of MAP kinases through the participation of L-calcium channels, and that by itself p-CREB does not appear to be sufficient for the transcriptional activation of ODC. In addition, post-translational mechanisms related with the induction of AZIN1 appear to be related to the increase of cardiac ODC activity.

Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells

Our previous studies demonstrated that specific polyamine analogues, oligoamines, down-regulated the activity of a key polyamine biosynthesis enzyme, ornithine decarboxylase (ODC), and suppressed expression of estrogen receptor alpha (ERα) in human breast cancer cells. However, the mechanism underlying the potential regulation of ERα expression by polyamine metabolism has not been explored. Here, we demonstrated that RNAi-mediated knockdown of ODC (ODC KD) down-regulated the polyamine pool, and hindered growth in ERα-positive MCF7 and T47D and ERα-negative MDA-MB-231 breast cancer cells. ODC KD significantly induced the expression and activity of the key polyamine catabolism enzymes, spermine oxidase (SMO) and spermidine/spermine N (1)-acetyltransferase (SSAT). However, ODC KD-induced growth inhibition could not be reversed by exogenous spermidine or overexpression of antizyme inhibitor (AZI), suggesting that regulation of ODC on cell proliferation may involve the signaling pathways independent of polyamine metabolism. In MCF7 and T47D cells, ODC KD, but not DFMO treatment, diminished the mRNA and protein expression of ERα. Overexpression of antizyme (AZ), an ODC inhibitory protein, suppressed ERα expression, suggesting that ODC plays an important role in regulation of ERα expression. Decrease of ERα expression by ODC siRNA altered the mRNA expression of a subset of ERα response genes. Our previous analysis showed that oligoamines disrupt the binding of Sp1 family members to an ERα minimal promoter element containing GC/CA-rich boxes. By using DNA affinity precipitation and mass spectrometry analysis, we identified ZBTB7A, MeCP2, PARP-1, AP2, and MAZ as co-factors of Sp1 family members that are associated with the ERα minimal promoter element. Taken together, these data provide insight into a novel antiestrogenic mechanism for polyamine biosynthesis enzymes in breast cancer.

Helicobacter pyloriCagA induces ornithine decarboxylase upregulation via Src/MEK/ERK/c-Myc pathway: implication for progression of gastric diseases

Helicobacter pylori (H. pylori) dysregulates the expression of various genes resulting in gastric precursor lesions and cancer. Meanwhile, ornithine decarboxylase (ODC) is a key enzyme that catalyzes the formation of polyamines which are critical for cell growth. So far, the possible regulation of ODC by H. pylori and its virulence factors, and the associated mechanism in gastric epithelial cells remains undefined. In the present study, we found that cellular ODC protein was upregulated by wild-type H. pylori infection and ectopic expression of a cytotoxin-associated gene A (CagA). As a negative control, there was no such effect by cagA-mutant H. pylori infection. Results of signal protein inhibitor treatment demonstrated that the Src, MEK (mitogen-activated protein kinase kinase) and ERK (extracellular signal-regulated kinase) pathway was involved. Moreover, when c-Myc was inhibited, the stimulatory effect of CagA on ODC expression was abolished. Clinically, a positive correlation between c-Myc and ODC expression was observed in patient-derived abnormal gastric tissues. These results implied that the Src/MEK/ERK/c-Myc pathway was required for CagA-mediated ODC induction. Finally, inhibition of ODC expression led to decreased foci formation of gastric epithelial cells before and after H. pylori infection, and ODC protein was over-expressed in precancerous gastric lesions and primary gastric cancer. Collectively, our findings provide new insights into the mechanism behind H. pylori-infection-associated gastric diseases.

Minimal Antizyme Peptide Fully Functioning in the Binding and Inhibition of Ornithine Decarboxylase and Antizyme Inhibitor

Antizyme (AZ) is a protein with 228 amino acid residues that regulates ornithine decarboxylase (ODC) by binding to ODC and dissociating its homodimer, thus inhibiting its enzyme activity. Antizyme inhibitor (AZI) is homologous to ODC, but has a higher affinity than ODC for AZ. In this study, we quantified the biomolecular interactions between AZ and ODC as well as AZ and AZI to identify functional AZ peptides that could bind to ODC and AZI and inhibit their function as efficiently as the full-length AZ protein. For these AZ peptides, the inhibitory ability of AZ_95-228 was similar to that of AZ_WT. Furthermore, AZ_95-176 displayed an inhibition (IC50: 0.20 µM) similar to that of AZ-95-228 (IC50: 0.16 µM), even though a large segment spanning residues 177–228 was deleted. However, further deletion of AZ_95-176 from either the N-terminus or the C-terminus decreased its ability to inhibit ODC. The AZ_100-176 and AZ_95-169 peptides displayed a noteworthy decrease in ability to inhibit ODC, with IC50 values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC50 values comparable to that of AZ_WT and formed AZ-ODC complexes with K d,AZ-ODC values of 1.5, 5.3 and 5.6 µM, respectively. Importantly, our data also indicate that AZI can rescue AZ peptide-inhibited ODC enzyme activity and that it can bind to AZ peptides with a higher affinity than ODC. Together, these data suggest that these truncated AZ proteins retain their AZI-binding ability. Thus, we suggest that AZ_95-176 is the minimal AZ peptide that is fully functioning in the binding of ODC and AZI and inhibition of their function.

Correlation between antizyme 1 and differentiation of vascular smooth muscle cells cultured in honeycomb-like type-I collagen matrix

Vascular smooth muscle cells (SMC) are able to proliferate when cultured on plates, but become differentiated when maintained in three-dimensional type I collagen matrices (honeycombs). SMC grown in honeycombs contained a low level of polyamines due to the presence of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase (ODC) and of polyamine uptake. To clarify the role of AZ1 in differentiation of SMC in honeycombs, an ODC gene was stably transfected into SMC (ODC-SMC). Although proliferation of ODC-SMC on plates was accelerated together with an increase in phosphorylated focal adhesion kinase (FAK) and a decrease in α-actin and myosin, maker proteins of differentiation, growth of ODC-SMC ceased in honeycombs similarly to normal SMC with a low level of phosphorylated FAK and a high level of α-actin and myosin. AZ1 expression in ODC-SMC on plates was low, but that in honeycombs was high. Antizyme in ODC-SMC in honeycombs not only decreased the level of ODC but also inhibited polyamine uptake activity. These results taken together suggest that low levels of polyamines caused by AZ1 in SMC in honeycombs inhibit phosphorylation of FAK and enhance expression of α-actin and myosin, resulting in differentiation through inhibition of focal adhesions.

Plant ornithine decarboxylase is not post-transcriptionally feedback regulated by polyamines but can interact with a cytosolic ribosomal protein S15 polypeptide

The formation of putrescine by ornithine decarboxylase (ODC) is a key regulatory step in polyamine biosynthesis in metazoa and fungi. Excess polyamines post-transcriptionally induce the synthesis of a unique non-competitive protein inhibitor of ODC, termed antizyme. Binding of antizyme to an ODC monomer subunit results in enzymatic inhibition, rapid ubiquitin-independent degradation of ODC by the 26S proteasome and recycling of antizyme. Plants possess an additional route for synthesizing putrescine via arginine decarboxylase (ADC). No homologue of ODC antizyme has been detected in plant genomes but several biochemical studies have reported plant ODC antizyme proteins of 9 and 16 kDa. Here we show that plant cells grown in liquid culture do not exhibit any substantial post-transcriptional, polyamine-responsive feedback regulation of ODC or ADC. However, using the yeast two hybrid system, a plant ODC-binding polypeptide was detected: the C-terminal 84-87 amino acids of cytosolic ribosomal protein (rp) S15. The Arabidopsis rpS15 polypeptide interacted specifically with plant ODC but not with human or Saccharomyces cerevisiae ODCs. Co-expression of either the full length or C-terminal rpS15 polypeptides with a plant ODC in yeast did not reduce ODC enzymatic activity. Only the full length mRNA encoding rpS15 was detected in Arabidopsis cells, suggesting that the C-terminal rpS15 polypeptide is encoded by a low abundance mRNA or the polypeptide is not physiologically relevant in plants. These results confirm the primacy of S-adenosylmethionine decarboxylase as the key regulatory enzyme in plant polyamine biosynthesis.

Knockdown of ornithine decarboxylase antizyme 1 causes loss of uptake regulation leading to increased N 1, N 11-bis(ethyl)norspermine (BENSpm) accumulation and toxicity in NCI H157 lung cancer cells

Ornithine decarboxylase antizyme 1 (AZ1) is a major regulatory protein responsible for the regulation and degradation of ornithine decarboxylase (ODC). To better understand the role of AZ1 in polyamine metabolism and in modulating the response to anticancer polyamine analogues, a small interfering RNA strategy was used to create a series of stable clones in human H157 non-small cell lung cancer cells that expressed less than 5-10% of basal AZ1 levels. Antizyme 1 knockdown clones accumulated greater amounts of the polyamine analogue N (1),N (11)-bis(ethyl)norspermine (BENSpm) and were more sensitive to analogue treatment. The possibility of a loss of polyamine uptake regulation in the knockdown clones was confirmed by polyamine uptake analysis. These results are consistent with the hypothesis that AZ1 knockdown leads to dysregulation of polyamine uptake, resulting in increased analogue accumulation and toxicity. Importantly, there appears to be little difference between AZ1 knockdown cells and cells with normal levels of AZ1 with respect to ODC regulation, suggesting that another regulatory protein, potentially AZ2, compensates for the loss of AZ1. The results of these studies are important for the understanding of both the regulation of polyamine homeostasis and in understanding the factors that regulate tumor cell sensitivity to the anti-tumor polyamine analogues.

Cytoplasmic Accumulation of the RNA-binding Protein HuR Stabilizes the Ornithine Decarboxylase Transcript in a Murine Nonmelanoma Skin Cancer Model

Ornithine decarboxylase (ODC) is the first and usually rate-limiting enzyme in the polyamine biosynthetic pathway. Under normal physiological conditions, polyamine content and ODC enzyme activity are highly regulated. However, the induction of ODC activity is an early step in neoplastic transformation. The studies described here use normal mouse keratinocytes (C5N cells), and spindle carcinoma cells (A5 cells) to explore the regulation of ODC in nonmelanoma skin cancer development. Previous results have shown that induction of ODC activity is both necessary and sufficient for the promotion of skin tumors. We see a marked increase in ODC enzyme activity in A5 cells compared with C5N keratinocytes, which correlates with a 4-fold stabilization of ODC mRNA. These data suggest that ODC is post-transcriptionally regulated in skin tumor development. Thus, we sought to investigate whether the ODC transcript interacts with the RNA-binding protein HuR, which is known to bind to and stabilize its target mRNAs. We show that HuR is able to bind to the ODC 3'-UTR in A5 cells but not in C5N cells. Immunofluorescence results reveal that HuR is present in both the nucleus and cytoplasm of A5 cells, whereas C5N cells exhibit strictly nuclear localization of HuR. Knockdown experiments in A5 cells showed that when HuR is depleted, ODC RNA becomes less stable, and ODC enzyme activity decreases. Together, these data support the hypothesis that HuR plays a causative role in ODC up-regulation during nonmelanoma skin cancer development by binding to and stabilizing the ODC transcript.

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

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

High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads

High activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, is typically present in rapidly proliferating normal and malignant cells. The mitotically inactive steroidogenic cells in rodent testis and ovaries, however, also display high ODC activity. The activity of ODC in these cells responds to luteinizing hormone, and inhibition of ODC reduces the production of steroid hormones. Polyamines and ODC also control proliferation of germ cells and spermiogenesis. The activity of ODC, especially in proliferating cells, is regulated by antizyme inhibitor (AZIN). This protein displaces ODC from a complex with its inhibitor, antizyme. We have previously identified and cloned a second AZIN, i.e. antizyme inhibitor 2 (AZIN2), which has the highest levels of expression in brain and in testis. In the present study, we have used immunohistochemistry and in situ hybridization to localize the expression of AZIN2 in human gonads. We found a robust expression of AZIN2 in steroidogenic cells: testicular Leydig cells and Leydig cell tumors, in ovarian luteinized cells lining corpus luteum cysts, and in hilus cells. The results suggest that AZIN2 is not primarily involved in regulating the proliferation of the germinal epithelium, indicating a different role for AZIN1 and AZIN2 in the regulation of ODC. The localization of AZIN2 implies possible involvement in the gonadal synthesis and/or release of steroid hormones.