Ornithine Decarboxylase Antizyme Research Articles

Ornithine Decarboxylase Antizyme Pseudoknot RNA Binding to Spermine Regulates Gene Expression

Ornithine Decarboxylase Antizyme Pseudoknot RNA Binding to Spermine Regulates Gene Expression

Correction to: Analysis of Cotranslational Polyamine Sensing During Decoding of ODC Antizyme mRNA.

Correction to: Chapter 26 in: Rubén Alcázar and Antonio F. Tiburcio (eds.), Polyamines: Methods and Protocols, Methods in Molecular Biology, vol. 1694, https://doi.org/10.1007/978-1-4939-7398-9_26.

Analysis of Cotranslational Polyamine Sensing During Decoding of ODC Antizyme mRNA.

Polyamines are essential poly-cations with vital functions in all cellular systems. Their levels are controlled by intricate regulatory feedback mechanisms. Abnormally high levels of polyamines have been linked to cancer. A rate-limiting enzyme in the biosynthesis of polyamines in fungi and higher eukaryotes is ornithine-decarboxylase (ODC). Its levels are largely controlled posttranslationally via ubiquitin-independent degradation mediated by ODC antizyme (OAZ). The latter is a critical polyamine sensor in a feedback control mechanism that adjusts cellular polyamine levels. Here, we describe an approach employing quantitative western blot analyses that provides in vivo evidence for cotranslational polyamine-sensing by nascent OAZ in yeast. In addition, we describe an in vitro method to detect polyamine binding by antizyme.

Characterization of an androgen-responsive, ornithine decarboxylase-related protein in mouse kidney.

We have investigated and characterized a novel ornithine decarboxylase (ODC) related protein (ODCrp) also annotated as gm853. ODCrp shows 41% amino acid sequence identity with ODC and 38% with ODC antizyme inhibitor 1 (AZIN1). The Odcrp gene is selectively expressed in the epithelium of proximal tubuli of mouse kidney with higher expression in males than in females. Like Odc in mouse kidney, Odcrp is also androgen responsive with androgen receptor (AR)-binding loci within its regulatory region. ODCrp forms homodimers but does not heterodimerize with ODC. Although ODCrp contains 20 amino acid residues known to be necessary for the catalytic activity of ODC, no decarboxylase activity could be found with ornithine, lysine or arginine as substrates. ODCrp does not function as an AZIN, as it neither binds ODC antizyme 1 (OAZ1) nor prevents OAZ-mediated inactivation and degradation of ODC. ODCrp itself is degraded via ubiquination and mutation of Cys363 (corresponding to Cys360 of ODC) appears to destabilize the protein. Evidence for a function of ODCrp was found in ODC assays on lysates from transfected Cos-7 cells where ODCrp repressed the activity of endogenous ODC while Cys363Ala mutated ODCrp increased the enzymatic activity of endogenous ODC.

OAZ1 knockdown enhances viability and inhibits ER and LHR transcriptions of granulosa cells in geese.

An increasing number of studies suggest that ornithine decarboxylase antizyme 1 (OAZ1), which is regarded as a tumor suppressor gene, regulates follicular development, ovulation, and steroidogenesis. The granulosa cells in the ovary play a critical role in these ovarian functions. However, the action of OAZ1 mediating physiological functions of granulosa cells is obscure. OAZ1 knockdown in granulosa cells of geese was carried out in the current study. The effect of OAZ1 knockdown on polyamine metabolism, cell proliferation, apoptosis, and hormone receptor transcription of primary granulosa cells in geese was measured. The viability of granulosa cells transfected with the shRNA OAZ1 at 48 h was significantly higher than the control (p<0.05). The level of putrescine and spermidine in granulosa cells down-regulating OAZ1 was 7.04- and 2.11- fold higher compared with the control, respectively (p<0.05). The CCND1, SMAD1, and BCL-2 mRNA expression levels in granulosa cells down-regulating OAZ1 were each significantly higher than the control, respectively (p<0.05), whereas the PCNA and CASPASE 3 expression levels were significantly lower than the control (p<0.05). The estradiol concentration, ER and LHR mRNA expression levels were significantly lower in granulosa cells down-regulating OAZ1 compared with the control (p<0.05). Taken together, our results indicated that OAZ1 knockdown elevated the putrescine and spermidine contents and enhanced granulosa cell viability and inhibited ER and LHR transcriptions of granulosa cells in geese.

Effect of Oaz1 overexpression on goose ovarian granulosa cells.

Ornithine decarboxylase antizyme 1 (OAZ1) regulates both polyamine biosynthesis and transport and acts as a tumor suppressor because of its functions in the regulation of cell proliferation, apoptosis and growth. However, the roles of OAZ1 in the regulation of polyamine metabolism, cell proliferation, apoptosis and hormone receptor expression in granulosa cells (GCs) from geese have not been determined. To define the roles of OAZ1 in primary GCs, we constructed and transfected an Oaz1 overexpression vector with a thymidine deletion into primary GCs. Oaz1 overexpression induced 65 and 30% decreases in the putrescine and spermidine contents, respectively, 225% increase in the spermine content and up-regulated the expression levels of genes associated with polyamine metabolism (but Oaz2). The Ccnd1, Pcna, Bax and Caspase 3 mRNA expression levels in GCs overexpressing Oaz1 were significantly higher than the expression levels in the vehicle group (p<0.05). Additionally, a slight increase in the E2 concentration in the culture medium and enhanced Er, Fshr and Lhr expression were observed in the GCs 24h after Oaz1 overexpression (p<0.05). These data suggested that Oaz1 up-regulated the expression of genes related to polyamine metabolic enzymes and reproductive hormone receptors, disturbed intracellular polyamine homeostasis, and affected the transcription of genes associated to cell proliferation and apoptosis in goose primary GCs. Taken together, our results indicate that modifying polyamine metabolism by Oaz1 may interfere with the expression of genes involved in cell proliferation, apoptosis and responsiveness of ovary toward hormones in goose GCs.

Antizyme inhibitor 1: a potential carcinogenic molecule.

Polyamines are multivalent and organic cations essential for cellular growth, proliferation, differentiation, and apoptosis. Increased levels of polyamines are closely associated with numerous forms of cancer. An autoregulatory circuit composed of ornithine decarboxylase (ODC), antizyme (AZ) and antizyme inhibitor (AZI) govern the intracellular level of polyamines. Antizyme binds with ODC to inhibit ODC activity and to promote the ubiquitin‐independent degradation of ODC. Antizyme inhibitor binds to AZ with a higher affinity than ODC. Consequently, ODC is released from the ODC–AZ complex to rescue its activity. Antizyme inhibitor increases the ODC activity to accelerate the formation of intracellular polyamines, triggering gastric and breast carcinogenesis as well as hepatocellular carcinoma and esophageal squamous cell carcinoma development. Antizyme inhibitor 1 (AZIN1), a primary member of the AZI family, has aroused more attention because of its contribution to cancer. Even though its conformation is changed by adenosine‐to‐inosine (A→I) RNA editing, it plays an important role in tumorigenesis through regulating intracellular polyamines. Encouragingly, AZIN1 has been revealed to have an additional function outside the polyamine pathway so as to bypass the deficiency of targeting the polyamine biosynthetic pathway, promising to become a critical target for cancer therapy. Here, we review the latest research advances into AZIN1 and its potential contribution to carcinogenesis.

Characterization of OAZ1 and its potential functions in goose follicular development

Background: Ornithine decarboxylase antizyme 1 (OAZ1) is an important regulator of polyamine synthesis and uptake. Our previous studies indicated that high OAZ1 expression in the ovaries of laying geese is responsible for poor egg production. In the present study, the molecular characterization of goose OAZ1 gene was analyzed, as well as the expression profile in various follicular tissues. Results: An 873-bp cDNA sequence of the OAZ1 gene (Accession No. KC845302) with a +1 frameshift site (+175T) was obtained. The sequence consisted of a 652-bp two overlapping open reading frames (a putative protein with 216 amino acids). The OAZ domain, OAZ signature and OAZ super family domain were prominent conserved regions among species. As the follicle size increased, OAZ1 abundance showed an increasing trend during follicular development, while it decreased during follicular regression. The level of OAZ1 mRNA expression was the lowest in the fifth largest preovulatory follicle, and was 0.65-fold compared to the small white follicle ( P OAZ1 mRNA expression in the largest preovulatory and postovulatory follicle was 2.11- and 2.49-fold compared to the small white follicle, respectively ( P Conclusions: The goose OAZ1 structure confirms that OAZ1 plays an important role in ornithine decarboxylase-mediated regulation of polyamine homeostasis. Our findings provide an evidence for a potential function of OAZ1 in follicular development, ovulation and regression. Normal 0 false false false ES-CL X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi; mso-ansi-language:ES-CL;}

Chronic periodontitis can affect the levels of potential oral cancer salivary mRNA biomarkers.

More than 100 salivary constituents have been found to show levels significantly different in patients with oral squamous cell carcinoma (OSCC) from those found in healthy controls, and therefore have been suggested to be potential salivary biomarkers for OSCC detection. However, many of these potential OSCC salivary biomarkers are also involved in chronic inflammation, and whether the levels of these biomarkers could be affected by the presence of chronic periodontitis was not known. The objective of this pilot study was therefore to measure the levels of seven previously reported potential OSCC salivary mRNA biomarkers in patients with chronic periodontitis and compare them to levels found in patients with OSCC and healthy controls. The seven salivary mRNAs were interleukin (IL)-8, IL-1β, dual specificity phosphatase 1, H3 histone family 3A, ornithine decarboxylase antizyme 1, S100 calcium-binding protein P (S100P) and spermidine/spermine N1-acetyltransferase 1. Unstimulated whole saliva samples were collected from a total of 105 human subjects from the following four study groups: OSCC; CPNS (chronic periodontitis, moderate to severe degree, non-smokers); CPS (chronic periodontitis, moderate to severe degree, smokers); and healthy controls. Levels of each mRNA in patient groups (OSCC or chronic periodontitis) relative to the healthy controls were determined by a pre-amplification reverse transcription-quantitative polymerase chain reaction approach with nested gene-specific primers. Results were recorded and analyzed by the Bio-Rad CFX96 Real-Time System. Mean fold changes between each pair of patient vs. control groups were analyzed by the Mann-Whitney U-test with Bonferroni corrections. Only S100P showed significantly higher levels in patients with OSCC compared to both patients with CPNS (p = 0.003) and CPS (p = 0.007). The difference in S100P levels between patients with OSCC and healthy controls was also marginally significant (p = 0.009). There was no significant difference in the levels of salivary IL-8, IL-1β and dual specificity phosphatase 1 mRNAs between patients with OSCC and patients with CPNS (p = 0.510, 0.058 and 0.078, respectively); no significant difference in levels of salivary ornithine decarboxylase antizyme 1 and spermine N1-acetyltransferase mRNAs between patients with OSCC and patients with CPS (p = 0.318 and 0.764, respectively); and no significant difference in levels of the H3 histone family 3A mRNA between patients with OSCC and either CPS (p = 0.449) or healthy controls (p = 0.107). Salivary S100P mRNA could be a reliable biomarker for OSCC detection, regardless of the presence of chronic periodontitis. The presence of chronic periodontitis could significantly affect the levels of the other six mRNAs, and negatively influence reliability for using them as biomarkers for oral cancer detection.

Expression of ODC Antizyme Inhibitor 2 (AZIN2) in Human Secretory Cells and Tissues.

Ornithine decarboxylase (ODC) antizyme inhibitor 2 (AZIN2), originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s) of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3) to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.

Structural basis of Ornithine Decarboxylase inactivation and accelerated degradation by polyamine sensor Antizyme1.

Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step of polyamine biosynthesis in humans. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Excessive accumulation of polyamines has a cytotoxic effect on cells and elevated level of ODC activity is associated with cancer development. To maintain normal cellular proliferation, regulation of polyamine synthesis is imposed by Antizyme1 (AZ1). The expression of AZ1 is induced by a ribosomal frameshifting mechanism in response to increased intracellular polyamines. AZ1 regulates polyamine homeostasis by inactivating ODC activity and enhancing its degradation. Here, we report the structure of human ODC in complex with N-terminally truncated AZ1 (cAZ1). The structure shows cAZ1 binding to ODC, which occludes the binding of a second molecule of ODC to form the active homodimer. Consequently, the substrate binding site is disrupted and ODC is inactivated. Structural comparison shows that the binding of cAZ1 to ODC causes a global conformational change of ODC and renders its C-terminal region flexible, therefore exposing this region for degradation by the 26S proteasome. Our structure provides the molecular basis for the inactivation of ODC by AZ1 and sheds light on how AZ1 promotes its degradation.

Interaction of polyamines and mTOR signaling in the synthesis of antizyme (AZ)

Tissue polyamine levels are largely determined by the activity of ornithine decarboxylase (ODC, EC 4.1.17), which catalyzes the conversion of ornithine to the diamine putrescine. The activity of the enzyme is primarily regulated by a negative feedback mechanism involving ODC antizyme (AZ). Our previous studies demonstrated that AZ synthesis is stimulated by the absence of amino acids, the levels of which are sensed by the mTOR complex containing TORC1, which is stimulated by amino acids and inhibited by their absence, and TORC2 the function of which is not well defined. Polyamines, which cause a +1 ribosomal frameshift during the translation of AZ mRNA are required to increase AZ synthesis in both the presence and absence of amino acids. Amino acid starvation increases TORC2 activity. We have demonstrated that mTORC2 activity is necessary for AZ synthesis in the absence of amino acids. Tuberous sclerosis protein (TSC), a negative regulator of mTOR function regulates the activities of both the TORC1 and TORC2. TSC2 knockdown increased mTORC1 activity with concomitant inhibition of mTORC2 activity eliminating AZ induction in the absence of amino acids as well as that induced by spermidine. Thus, these results clearly demonstrate that in addition to polyamines, mTORC2 activity is necessary for AZ synthesis. Moreover, our results support a role for mTORC2 in the synthesis of a specific protein, AZ, which regulates growth of intestinal epithelial cells.

Polyamines directly promote antizyme-mediated degradation of ornithine decarboxylase by the proteasome.

Ornithine decarboxylase (ODC), a ubiquitin-independent substrate of the proteasome, is a homodimeric protein with a rate-limiting function in polyamine biosynthesis. Polyamines regulate ODC levels by a feedback mechanism mediated by ODC antizyme (OAZ). Higher cellular polyamine levels trigger the synthesis of OAZ and also inhibit its ubiquitin-dependent proteasomal degradation. OAZ binds ODC monomers and targets them to the proteasome. Here, we report that polyamines, aside from their role in the control of OAZ synthesis and stability, directly enhance OAZ-mediated ODC degradation by the proteasome. Using a stable mutant of OAZ, we show that polyamines promote ODC degradation in Saccharomyces cerevisiae cells even when OAZ levels are not changed. Furthermore, polyamines stimulated the in vitro degradation of ODC by the proteasome in a reconstituted system using purified components. In these assays, spermine shows a greater effect than spermidine. By contrast, polyamines do not have any stimulatory effect on the degradation of ubiquitin-dependent substrates.

Control of Polyamine Biosynthesis by Antizyme Inhibitor 1 Is Important for Transcriptional Regulation of Arginine Vasopressin in the Male Rat Hypothalamus

The polyamines spermidine and spermine are small cations present in all living cells. In the brain, these cations are particularly abundant in the neurons of the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus, which synthesize the neuropeptide hormones arginine vasopressin (AVP) and oxytocin. We recently reported increased mRNA expression of antizyme inhibitor 1 (Azin1), an important regulator of polyamine synthesis, in rat SON and PVN as a consequence of 3 days of dehydration. Here we show that AZIN1 protein is highly expressed in both AVP- and oxytocin-positive magnocellular neurons of the SON and PVN together with antizyme 1 (AZ1), ornithine decarboxylase, and polyamines. Azin1 mRNA expression increased in the SON and PVN as a consequence of dehydration, salt loading, and acute hypertonic stress. In organotypic hypothalamic cultures, addition of the irreversible ornithine decarboxylase inhibitor DL-2-(difluoromethyl)-ornithine hydrochloride significantly increased the abundance of heteronuclear AVP but not heteronuclear oxytocin. To identify the function of Azin1 in vivo, lentiviral vectors that either overexpress or knock down Azin1 were stereotaxically delivered into the SON and/or PVN. Azin1 short hairpin RNA delivery resulted in decreased plasma osmolality and had a significant effect on food intake. The expression of AVP mRNA was also significantly increased in the SON by Azin1 short hairpin RNA. In contrast, Azin1 overexpression in the SON decreased AVP mRNA expression. We have therefore identified AZIN1, and hence by inference, polyamines as novel regulators of the expression of the AVP gene.

Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents

Fermentation inhibitors present in lignocellulose hydrolysates are inevitable obstacles for achieving economic production of biofuels and biochemicals by industrial microorganisms. Here we show that spermidine (SPD) functions as a chemical elicitor for enhanced tolerance of Saccharomyces cerevisiae against major fermentation inhibitors. In addition, the feasibility of constructing an engineered S. cerevisiae strain capable of tolerating toxic levels of the major inhibitors without exogenous addition of SPD was explored. Specifically, we altered expression levels of the genes in the SPD biosynthetic pathway. Also, OAZ1 coding for ornithine decarboxylase (ODC) antizyme and TPO1 coding for the polyamine transport protein were disrupted to increase intracellular SPD levels through alleviation of feedback inhibition on ODC and prevention of SPD excretion, respectively. Especially, the strain with combination of OAZ1 and TPO1 double disruption and overexpression of SPE3 not only contained spermidine content of 1.1mg SPD/g cell, which was 171% higher than that of the control strain, but also exhibited 60% and 33% shorter lag-phase period than that of the control strain under the medium containing furan derivatives and acetic acid, respectively. While we observed a positive correlation between intracellular SPD contents and tolerance phenotypes among the engineered strains accumulating different amounts of intracellular SPD, too much SPD accumulation is likely to cause metabolic burden. Therefore, genetic perturbations for intracellular SPD levels should be optimized in terms of metabolic burden and SPD contents to construct inhibitor tolerant yeast strains. We also found that the genes involved in purine biosynthesis and cell wall and chromatin stability were related to the enhanced tolerance phenotypes to furfural. The robust strains constructed in this study can be applied for producing chemicals and advanced biofuels from cellulosic hydrolysates.

Influence of ornithine decarboxylase antizymes and antizyme inhibitors on agmatine uptake by mammalian cells.

Agmatine (4-aminobutylguanidine), a dicationic molecule at physiological pH, exerts relevant modulatory actions at many different molecular target sites in mammalian cells, having been suggested that the administration of this compound may have therapeutic interest. Several plasma membrane transporters have been implicated in agmatine uptake by mammalian cells. Here we report that in kidney-derived COS-7 cell line, at physiological agmatine levels, the general polyamine transporter participates in the plasma membrane translocation of agmatine, with an apparent Km of 44 ± 7 µM and Vmax of 17.3 ± 3.3 nmol h(-1) mg(-1) protein, but that at elevated concentrations, agmatine can be also taken up by other transport systems. In the first case, the physiological polyamines (putrescine, spermidine and spermine), several diguanidines and bis(2-aminoimidazolines) and the polyamine transport inhibitor AMXT-1501 markedly decreased agmatine uptake. In cells transfected with any of the three ornithine decarboxylase antizymes (AZ1, AZ2 and AZ3), agmatine uptake was dramatically reduced. On the contrary, transfection with antizyme inhibitors (AZIN1 and AZIN2) markedly increased the transport of agmatine. Furthermore, whereas putrescine uptake was significantly decreased in cells transfected with ornithine decarboxylase (ODC), the accumulation of agmatine was stimulated, suggesting a trans-activating effect of intracellular putrescine on agmatine uptake. All these results indicate that ODC and its regulatory proteins (antizymes and antizyme inhibitors) may influence agmatine homeostasis in mammalian tissues.

Effects of ornithine decarboxylase antizyme 1 on the proliferation and differentiation of human oral cancer cells.

Ornithine decarboxylase antizyme 1 (OAZ1) is an antizyme targeting ornithine decarboxylase for degradation, subsequently inhibiting polyamine production to prevent cell proliferation. OAZ1 is also involved in other major cellular events, including differentiation and apoptosis. Recent studies have shown that OAZ1 has tumor suppressor activities and its effects on cell proliferation and differentiation have been reported in several cancer cell lines. To explore the role of OAZ1 in human oral cancer, the effects of OAZ1 were studied on the proliferation and differentiation of human malignant oral cancer cell line, SCC15. MTT assay and flow cytometry analysis showed that stable OAZ1 expression in SCC15 significantly inhibited cell proliferation (P<0.001) and induced G0/G1 arrest with the G1‑phase cells increased from 55.83 to 65.26%. Morphological observation revealed the increased formation of epithelial islands. Further results from quantitative reverse transcription‑polymerase chain reaction and western blot analysis proved the upregulation of several terminal differentiation marker genes (K10, FLG and LOR) in OAZ1‑expressed SCC15 cells. To elucidate the possible mechanism of LOR upregulation by OAZ1, further experiments were performed and it was found that the OAZ1 expression inhibited Smad nuclear interacting protein 1 (SNIP1) at the protein level and RNA interference of SNIP1 in SCC15 cells, which increased the expression of LOR. These results show that OAZ1 simultaneously inhibits the proliferation and induces the differentiation of oral cancer cells in humans. The effects on cellular differentiation depend partly on the degradation of SNIP1.

Abstract 681: Heat shock-mediated suppression of ODC protein expression in colorectal cancer cells

Abstract Polyamines are small, positively charged metabolites that have important roles in several cancer hallmarks, including cell proliferation, invasion, and angiogenesis. Difluoromethylornithine (DFMO) is an FDA-approved drug that is in clinical trials for treatment of colorectal cancer. DFMO functions by inhibiting ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine biosynthesis. By reducing polyamine levels, DFMO has shown much promise as an anti-cancer chemotherapeutic. One potential limitation to DFMO is that it causes a profound increase in ODC expression, which we suspect may counteract the intended use of the drug. However, strategies to suppress ODC expression would theoretically increase DFMO efficacy. We have discovered that transient hyperthermia (heat shock, 42°C) causes a profound and rapid suppression in ODC protein levels in RKO colorectal cancer cells. To investigate the responsible mechanism, we began by examining ODC mRNA expression and stability. Hyperthermia caused only a moderate reduction in ODC transcript levels. Also, mRNA stability, which was assessed using actinomycin D, was not reduced by heat shock. We next examined the effect of hyperthermia on the control of ODC protein turnover, which is known to be mediated by ODC antizyme (OAZ1). We performed real-time PCR to assess the effect of heat shock on OAZ1 expression. Data reveal a significant increase in the level of OAZ1 transcript. Since heat shock is known to activate the transcription factor HSF1, we next investigated the role of HSF1 in OAZ1 induction. Using siRNA to silence HSF1, we found that the induction of OAZ1 by heat shock is fully dependent on HSF1 expression. In addition to heat shock, HSF1 is also activated by various chemotherapeutics, but most notably by Hsp90 inhibitors. We therefore tested if Hsp90 inhibitors, geldanamycin and 17-AAG could be used to induce OAZ1 and promote ODC turnover. These results show that both Hsp90 inhibitors caused a significant activation of HSF1, an induction in OAZ1 expression, and decrease in ODC protein levels. We therefore propose that Hsp90 inhibitors may be effective in attenuating ODC expression and polyamine metabolism. We suggest that they may be used in combination with DFMO to prevent the compensatory increase in ODC levels and increase anti-cancer effects. Citation Format: Christina T.K. Wales, Aaron T. Jacobs. Heat shock-mediated suppression of ODC protein expression in colorectal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 681. doi:10.1158/1538-7445.AM2014-681

Molecular cloning and mRNA expression analysis of ornithine decarboxylase antizyme 2 in ovarian follicles of the Sichuan white goose (Anser cygnoides).

The ornithine decarboxylase antizyme 2 (OAZ2) gene is a member of the antizyme gene family. Antizymes play pivotal roles in various cellular pathways, including polyamine anabolism and apoptosis. The molecular structure and expression profile of the OAZ2 in goose ovarian follicles have not been reported. In this study, the OAZ2 cDNA sequence of the Sichuan white goose was cloned (Anser cygnoides), and phylogenetic and structural analyses of the OAZ2 were performed. The expression profiling of OAZ2 mRNA in goose ovarian follicles was examined using quantitative real-time PCR. The sequence analysis showed that the 756 bp OAZ2 sequence contained two overlapping open reading frames (ORF). ORF1 was 99 bp in length, and encoded a 32 aa polypeptide. ORF2 was 477 bp in length, and encoded a 158 aa polypeptide. The frameshift site that initiates the translation of ORF2 was located at nucleotide position 97 in the OAZ2. The analysis of OAZ2 mRNA expression in hierarchical follicles showed that the level of OAZ2 mRNA was higher in the SWF and F2 follicular stages than that in the ovarian stroma (P<0.05). The lowest level of OAZ2 expression was detected in the ovarian stroma. These results suggest that the highly conserved frameshift region plays an important role in sustaining the function of OAZs. Furthermore, the significantly higher level of OAZ2 mRNA in the SWF stage indicates that OAZ2 may be involved in recruiting hierarchical follicles. Our results also suggest that OAZ2 may augment the effects of OAZ1 in follicle development.

Dense spermatozoa in stallion ejaculates contain lower concentrations of mRNAs encoding the sperm specific calcium channel 1, ornithine decarboxylase antizyme 3, aromatase, and estrogen receptor alpha than less dense spermatozoa

Stallions are unique among livestock in that, like men, they commonly receive medical treatment for subfertility. In both species, about 15% of individuals have normal semen parameters but are subfertile, indicating a need for novel analyses of spermatozoa function. One procedure for improving fertilizing capability of stallions and men is isolation of dense spermatozoa from an ejaculate for use in artificial insemination. In the current study, dense and less dense spermatozoa were purified by density gradient centrifugation from individual ejaculates from seven reproductively normal adult stallions. The RNA isolated from the spermatozoa seemed to be naturally fragmented to an average length of 250 bases, consistent with reports of spermatozoa RNA from other species. The DNAse treatment of RNA prepared from spermatozoa removed any genomic DNA contamination, as assessed by PCR with intron spanning primers for the protamine 1 (PRM1) gene. Concentrations of seven mRNAs in spermatozoa, correlated with the fertility of men and bulls, were quantified by reverse transcription polymerase chain reaction in dense and less dense spermatozoa. Concentrations of four mRNAs were two- to four-fold lower in dense spermatozoa compared with less dense spermatozoa: Encoding the spermatozoa-specific calcium channel (P < 0.03), ornithine decarboxylase antizyme 3 (P < 0.02), aromatase (P < 0.02), and estrogen receptor alpha (P < 0.08). In contrast, concentrations of three other mRNAs, encoding PRM1 and heat shock proteins HSPA8 and DNAJC4, were not different (P > 0.1). These results identify new differences in mRNA concentrations in populations of spermatozoa with dissimilar densities.