Low Basal Expression Research Articles

Oligodendroglia are limited in type I interferon induction and responsiveness in vivo.

Type I interferons (IFNα/β) provide a primary defense against infection. Nevertheless, the dynamics of IFNα/β induction and responsiveness by central nervous system (CNS) resident cells in vivo in response to viral infections are poorly understood. Mice were infected with a neurotropic coronavirus with tropism for oligodendroglia and microglia to probe innate antiviral responses during acute encephalomyelitis. Expression of genes associated with the IFNα/β pathways was monitored in microglia and oligodendroglia purified from naïve and infected mice by fluorescent activated cell sorting. Compared with microglia, oligodendroglia were characterized by low basal expression of mRNA encoding viral RNA sensing pattern recognition receptors (PRRs), IFNα/β receptor chains, interferon sensitive genes (ISG), as well as kinases and transcription factors critical in IFNα/β signaling. Although PRRs and ISGs were upregulated by infection in both cell types, the repertoire and absolute mRNA levels were more limited in oligodendroglia. Furthermore, although oligodendroglia harbored higher levels of viral RNA compared with microglia, Ifnα/β was only induced in microglia. Stimulation with the double stranded RNA analogue poly I:C also failed to induce Ifnα/β in oligodendroglia, and resulted in reduced and delayed induction of ISGs compared with microglia. The limited antiviral response by oligodendroglia was associated with a high threshold for upregulation of Ikkε and Irf7 transcripts, both central to amplifying IFNα/β responses. Overall, these data reveal that oligodendroglia from the adult CNS are poor sensors of viral infection and suggest they require exogenous IFNα/β to establish an antiviral state. © 2012 Wiley Periodicals, Inc.

Analysis of the role of Nrf2 in the expression of liver proteins in mice using two-dimensional gel-based proteomics

BackgroundThe transcription factor Nrf2 regulates expression of multiple cellular defence proteins through the antioxidant response element (ARE). Nrf2-deficient mice (Nrf2−/−) are highly susceptible to xenobiotic-mediated toxicity, but it is not known whether this reflects low basal expression or reduced inducibility of Nrf2-regulated genes in response to chemical insults. MethodsWild type and Nrf2−/− mice were fed diet supplemented with the established Nrf2 inducer butylated hydroxyanisole (BHA) [0.5% (w/w)] for 14 days. To define the range of Nrf2-regulated proteins, both basally and following exposure to BHA, a comparison of the liver proteomes of Nrf2−/− and wild type mice was conducted. The two-dimensional gel electrophoresis (2-DE) technique and MALDI mass spectrometry were utilized in the attempt to define Nrf2-regulated proteins. ResultsOverall, 24 proteins were identified, which were regulated either basally (3 proteins), inducibly (16 proteins), or both (5 proteins). These included several well-established Nrf2-driven gene products e.g., aldo-keto reductase and glutathione transferases. Multiple consensus ARE/ARE-like sequences were found in the Nrf2-regulated genes. ConclusionsThis study confirms the central role of Nrf2 in the induction of multiple defense proteins as well as its control in the constitutive expression of certain proteins.

Characterization and Expression of a Phytochelatin Synthase Gene in Birch-leaf Pear (Pyrus betulaefolia Bunge)

Phytochelatins play a major role in metal detoxification in plants. However, the molecular mechanisms of heavy metal detoxification remain unknown, and birch-leaf pear genes related to metal detoxification have not yet been identified. Here, we report the isolation of cDNA and genomic DNA sequences encoding a phytochelatin synthase (PCS) from birch-leaf pear (Pyrus betulaefolia Bunge). The PbPCS1 cDNA sequence contained 1,965 nucleotides of a 1,494 bp open reading frame (ORF), which encodes a 497-amino-acid protein with a molecular weight of 55.00 kDa. Its corresponding genomic DNA sequence consists of 3,820 nucleotides and eight exons separated by seven introns. The deduced amino acid sequence of PbPCS1 contains five Cys residues (56, 90, 91, 109 and 113 amino acids) that are highly conserved in the plant PCS1 family, and 12 cysteine residues putatively involved in heavy metal binding sites, which included three adjacent Cys–Cys components (331–332, 351–352 and 369–370 amino acids) in the C-terminal variable domain. Homology analysis of the deduced PbPCS1 amino acid sequence revealed that it shares a high sequence similarity amongst N-terminal amino acids and low similarity with C-terminal amino acids with plant PCS1 proteins deposited with NCBI. PbPCS1 was most related to PCS1 from legume plants Lotus japonicus (GenBank accession no. AAT80342) and soybean (Glycine max L.; AAL78384) as they were clustered into the same clade in a neighbor-joining phylogenetic tree. Quantitative real-time PCR (qPCR) expression analysis revealed that PbPCS1 had a very low basal expression level in untreated whole seedlings, and levels were higher in roots than in leaves and stems. After 24 h of exposure to 20 μM CdSO4, CuSO4 or ZnSO4, PbPCS1 expression increased significantly in different organs. In addition, L-buthionine-sulfo-ximine (BSO) can inhibit PbPCS1 expression in roots, stems and leaves, while L-glutathionereduced (GSH) stimulates PbPCS1 expression in different organs of birch-leaf pear.

Abstract 1815: CRM-1 as a potential therapeutic target in pancreatic cancer

Abstract CRM-1 exportin protein encoded by the XPO-1 gene maintains cancer cells through nuclear export signal (NES)-dependent protein exclusion of tumor suppressor proteins (TSPs). High CRM-1 expression has been correlated with poor prognosis in pancreatic cancer making it an attractive therapeutic target. Numerous attempts have been made in the past to develop specific CRM-1 inhibitors; however, earlier inhibitors such as Leptomycin B failed in the clinic due to acute off target toxicity. Using structure based drug design; we have identified new class of selective inhibitors of nuclear export (SINE) that irreversibly inhibits CRM1 and lock TSPs in cancer cell nucleus leading to cancer cell selective apoptosis. The inhibitors have excellent pharmacokinetic parameters and minimal toxicity to normal tissue. Here, we tested SINEs against pancreatic cancer, a disease that urgently needs novel and effective therapies. CRM-1 inhibitors [(KPT-185 (most potent analog), KPT-127, KPT-205, KPT-227 as well as Leptomycin B (as positive control) and not its inactive analog KPT-Tran (as negative control)] induced growth inhibition (MTT) and apoptosis (Annexin V and Histone DNA ELISA) in five cell lines (IC50 ∼150 nM). KPT-SINEs did not have any effect on normal HPDE cells (IC50>5 µM). Mechanistically, KPT-185 mediated apoptosis was found to be through activation, phosphorylation and nuclear localization of a TSP prostate apoptosis response 4 (PAR-4). PAR-4 has earlier been established by our laboratory as a therapeutic target in pancreatic cancer. Co-immunoprecipitation studies demonstrated that KPT-185 dissociated CRM-1-PAR-4 and CRM-1-p-PAR-4 interaction. Further, PAR-4 siRNA knockdown abrogated KPT-SINEs ability to induce growth inhibition or apoptosis. In normal cells PAR-4 localization or phosphorylation was not observed and this was associated with low basal expression of PAR-4 and its phosphorylation inducer protein kinase A protein. Role of other TSPs such as p53 and p27 was also explored, however, our investigations directly point to a PAR-4 specific mechanism. KPT-251 (structurally very similar to KPT-185 with improved pharmacokinetics properties) was well tolerated in SCID mice upto 150 mg/kg, p.o. Oral administration of analog KPT-251 at 25 and 150 mg/kg daily for two weeks reduced pancreatic tumors significantly (p<0.01). Histological examination of remnant tumors showed enhanced staining of PAR-4 and reduced Ki67. Tumor tissue lysates were probed for protein expression using western blotting that revealed activation of PAR-4 signaling and co-immunoprecipitation assay demonstrated dissociation of CRM-1-PAR-4 in tumors as well. In conclusion, discovery of our novel non-toxic KPTs can significantly impact the advancement of the field of CRM-1 inhibitors and could become effective anti-cancer therapeutics for pancreatic cancer as well as other solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1815. doi:1538-7445.AM2012-1815

HDA19 is required for the repression of salicylic acid biosynthesis and salicylic acid‐mediated defense responses in Arabidopsis

To cope with a lifetime of exposure to a variety of pathogens, plants have developed exquisite and refined defense mechanisms that vary depending on the type of attacking pathogen. Defense-associated transcriptional reprogramming is a central part of plant defense mechanisms. Chromatin modification has recently been shown to be another layer of regulation for plant defense mechanisms. Here, we show that the RPD3/HDA1-class histone deacetylase HDA19 is involved in the repression of salicylic acid (SA)-mediated defense responses in Arabidopsis. Loss of HDA19 activity increased SA content and increased the expression of a group of genes required for accumulation of SA as well as pathogenesis related (PR) genes, resulting in enhanced resistance to Pseudomonas syringae. We found that HDA19 directly associates with and deacetylates histones at the PR1 and PR2 promoters. Thus, our study shows that HDA19, by modifying chromatin to a repressive state, ensures low basal expression of defense genes, such as PR1, under unchallenged conditions, as well as their proper induction without overstimulation during defense responses to pathogen attacks. Thus, the role of HDA19 might be critical in preventing unnecessary activation and self-destructive overstimulation of defense responses, allowing successful growth and development.

Quantitative Expression Analysis of TaSOS1 and TaSOS4 Genes in Cultivated and Wild Wheat Plants Under Salt Stress

Salt stress is a mixture of ionic, osmotic, and oxidative stresses. The expression of TaSOS1 (a transmembrane Na(+)/H(+) antiporter) and TaSOS4 [a cytoplasmic pyridoxal (PL) kinase] genes were measured in four different salinity levels and different time courses of salinity exposure using qRT-PCR technique. Mahuti (salt tolerant) and Alamut (salt sensitive) cultivars were used as cultivated wheat, and T. boeticum and Aegilops crassa as wild wheat plants. Salt-induced expression of TaSOS1 in these wild wheat plants indicates the presence of active TaSOS1 gene on the genomes A and D. The TaSOS1 and TaSOS4 transcript levels were found to be downregulated after salt treatment in all cultivars except in A. crassa, which was in contrast with its expression pattern in roots that was being upregulated from a very low-basal expression, after salt treatments. Duncan's Multiple Range Test showed a significant difference between expression in the 200-mM NaCl concentration with the 50 and 100mM for the TaSOS1 gene, and no significant difference for TaSOS4. Lack of significant correlation between the TaSOS1 and TaSOS4 gene expressions confirms the theory that PLP has no significant effect on the expression of the TaSOS1 gene in wheat leaves.

Induction of COX-2 Enzyme and Down-regulation of COX-1 Expression by Lipopolysaccharide (LPS) Control Prostaglandin E2 Production in Astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E(2) (PGE(2)) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE(2) and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE(2) production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE(2), and suggest that the coordinated down-regulation of COX-1 facilitates PGE(2) production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.

Peroxiredoxin distribution in the mouse brain with emphasis on neuronal populations affected in neurodegenerative disorders

Redox changes are observed in neurodegenerative diseases, ranging from increased levels of reactive oxygen/nitrogen species and disturbance of antioxidant systems, to nitro-oxidative damage. By reducing hydrogen peroxide, peroxynitrite, and organic hydroperoxides, peroxiredoxins (Prdxs) represent a major potential protective barrier against nitro-oxidative insults in the brain. While recent works have investigated the putative role of Prdxs in neurodegenerative disorders, less is known about their expression in the healthy brain. Here we used immunohistochemistry to map basal expression of Prdxs throughout C57BL/6 mouse brain. We first confirmed the neuronal localization of Prdx2-5 and the glial expression of Prdx1, Prdx4, and Prdx6. Then we performed an in-depth analysis of neuronal Prdx distribution in the brain. Our results show that Prdx2-5 are widely detected in the different neuronal populations, and especially well expressed in the olfactory bulb, in the cerebral cortex, in pons nuclei, in the red nucleus, in all cranial nerve nuclei, in the cerebellum, and in motor neurons of the spinal cord. In contrast, Prdx expression is very low in the dopaminergic neurons of substantia nigra pars compacta and in the CA1/2 pyramidal cells of hippocampus. This low basal expression may contribute to the vulnerability of these neurons to nitro-oxidative attacks occurring in Parkinson's disease and Alzheimer's disease. In addition, we found that Prdx expression levels are unevenly distributed among neurons of a determined region and that distinct regional patterns of expression are observed between isoforms, reinforcing the hypothesis of the nonredundant function of Prdxs.

Poly(C)-binding protein 1 (PCBP1) mediates housekeeping degradation of mitochondrial antiviral signaling (MAVS)

Mitochondrial antiviral signaling (MAVS) is a key adaptor in cellular antiviral innate immunity. We previously identified poly(C)-binding protein 2 (PCBP2) as a feedback inhibitor of MAVS that facilitates its degradation after viral infection, but little is known about the regulatory potential of poly(C)-binding protein 1 (PCBP1), which highly resembles PCBP2. Here we report that PCBP1 mediates housekeeping degradation of MAVS using the same mechanism as PCBP2 employs. Overexpression of PCBP1 impairs MAVS-mediated antiviral responses, while knockdown of PCBP1 exerts the opposite effect. The suppression is due to PCBP1-induced MAVS degradation. We observe that PCBP1 and PCBP2 show synergy in MAVS inhibition, but their expression patterns are distinct: PCBP1 is stably and abundantly expressed, while PCBP2 shows low basal expression with rapid induction after infection. Individual knockdown and subcellular fractionation analyses reveal that unlike the postinfection inhibitor PCBP2, PCBP1 continuously eliminates cellular MAVS. Our findings unravel a critical role of PCBP1 in regulating MAVS for both fine-tuning the antiviral immunity and preventing inflammation.

Abstract B221: Clopidogrel augments cisplatin cytotoxicity by enhancing delivery and direct receptor mediated effects.

Abstract Clopidogrel, a well known P2Y12 (purinergic receptor) inhibitor is widely used for its role in preventing thrombosis by platelet aggregation inhibition. Tumor vasculature is critically dependent on platelet mediated hemostasis and disruption of the same can promote its leakiness. This can increase the intra-tumoral delivery of chemotherapeutic nanoformulations, which depend on the Endothelial permeability and retention (EPR) effect. Also, Expression of P2Y12 receptors has been documented in some cancer cell lines like C6 glioma, kidney carcinoma and colon carcinoma. However, its direct role in altering response to chemotherapeutics has not been studied. In this project we evaluated the role of P2Y12 receptor inhibitors (Clopidogrel and 2-Methylthioadenosine 5-Monophosphate) in potentiating the tumor cytotoxicity of cisplatin based chemotherapeutics in vitro and in vivo. Western blot studies in 4T1 breast cancer cell lines showed a low basal expression of P2Y12 receptors with significant up regulation by cisplatin treatment in a time and dose dependent fashion. Co-administration of P2Y12 inhibitor (MeSAMP) with cisplatin resulted in significantly increased upregulation of caspase dependent apoptotic pathway when compared to cisplatin alone. This was mediated by downstream inhibition of PI3K-pAKT-mTOR pathway by the P2Y12 inhibitor. In-vitro cell proliferation assay showed that co-administration of 2-MeSAMP significantly reduced the IC-50 of cisplatin from 3.9 μM to 1.8μM. In-vivo studies in murine syngenic 4T1 breast cancer model showed significantly decreased tumour growth and increased survival in treatment groups using combination of clopidogrel and cisplatin (free and nanoformulation forms). This was more pronounced with cisplatin nano-formulation combination therapy. Further analysis of tumour tissue using Inductive Coupled Plasma-spectrophotometry confirmed that co-treatment with clopidogrel increased the delivery of free and nanoformulation form of cisplatin to tumor tissue by 1.8 and 4 folds respectively. Taken together our study findings suggest that clopidogrel can enhance therapeutic efficiency of cytotoxic agents by increasing delivery and by direct receptor level effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B221.

Oxidative Damage Targets Complexes Containing DNA Methyltransferases, SIRT1, and Polycomb Members to Promoter CpG Islands

Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an invivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

Gene–environment interactions: The potential role of contaminants in somatic growth and the development of the reproductive system of the American alligator

Developing organisms interpret and integrate environmental signals to produce adaptive phenotypes that are prospectively suited for probable demands in later life. This plasticity can be disrupted when embryos are impacted by exogenous contaminants, such as environmental pollutants, producing potentially deleterious and long-lasting mismatches between phenotype and the future environment. We investigated the ability for in ovo environmental contaminant exposure to alter the growth trajectory and ovarian function of alligators at five months after hatching. Alligators collected as eggs from polluted Lake Apopka, FL, hatched with smaller body masses but grew faster during the first five months after hatching, as compared to reference-site alligators. Further, ovaries from Lake Apopka alligators displayed lower basal expression levels of inhibin beta A mRNA as well as decreased responsiveness of aromatase and follistatin mRNA expression levels to treatment with follicle stimulating hormone. We posit that these differences predispose these animals to increased risks of disease and reproductive dysfunction at adulthood.

Acute exercise stress activates Nrf2/ARE signaling and promotes antioxidant mechanisms in the myocardium

Oxidative stress has been implicated in the pathogenesis of cardiovascular diseases, including myocardial hypertrophy and infarction. Although impairment of antioxidant defense mechanisms has been thought to provoke oxidative stress-induced myocardial dysfunction, it has been difficult to clearly demonstrate. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive, basic leucine zipper protein that regulates the transcription of several antioxidant genes. We previously reported that sustained activation of Nrf2 upregulates transcription of a number of endogenous antioxidants in the heart. Here, we show that acute exercise stress (AES) results in activation of Nrf2/ARE (antioxidant response element) signaling and subsequent enhancement of antioxidant defense pathways in wild-type (WT) mouse hearts, while oxidative stress, along with blunted defense mechanisms, was observed in Nrf2−/− mice. We also find that AES is associated with increased trans-activation of ARE-containing genes in exercised animals when compared to age-matched sedentary WT mice. However, enhanced oxidative stress in response to AES was observed in Nrf2−/− mice due to lower basal expression and marked attenuation of the transcriptional induction of several antioxidant genes. Thus, AES induces ROS and promotes Nrf2 function, but disruption of Nrf2 increases susceptibility of the myocardium to oxidative stress. Our findings suggest the basis for a nonpharmacological approach to activate Nrf2/ARE signaling, which might be a potential therapeutic target to protect the heart from oxidative stress-induced cardiovascular complications.

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

BackgroundThe strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies.ResultsThe hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells.ConclusionThe hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

Post-transcriptionally Regulated Expression System in Human Xenogeneic Transplantation Models

Cells have developed a mechanism to discriminate between premature termination codons (PTCs) and normal stop codons during translation, sparking vigorous research to develop drugs promoting readthrough at PTCs to treat genetic disorders caused by PTCs. It was posed that this concept could also be applied to regulated gene therapy protocols by incorporating a PTC into a therapeutic gene, so active protein would only be made after administration of a readthrough agent. The strengths of the system are highlighted here by results demonstrating: (i) background expression levels were reduced to 0.01% to 0.0005% of wild type in unselected mass populations of cells depending upon the specific stop codon utilized and its position within the gene; (ii) expression levels responded well to multiple "On" and "Off" regulation cycles in vivo in human xenograft systems; (iii) the level of induction approached three logs using aminoglycoside activators including NB54, a newly synthesized aminoglycoside with significantly reduced toxicity; and (iv) expression levels could be appreciably altered when employing different promoters in a variety of cell types. These results strongly support the contention that this system should have important clinical applications when tight control of gene expression is required.

A PET Brain Reporter Gene System Based on Type 2 Cannabinoid Receptors

PET of gene expression in the brain may greatly facilitate neuroscience research and potential clinical implementation of gene or cell therapy of central nervous system diseases. To date, no adequate PET reporter system is available for the central nervous system because available tracers either do not cross the intact blood-brain barrier or have high background signals. Here we report the first, to our knowledge, PET reporter system for imaging gene expression in the intact brain. We selected the human type 2 cannabinoid receptor (hCB(2)) as a reporter because of its low basal expression in the brain. An inactive mutant (D80N) was chosen so as not to interfere with signal transduction. As a reporter probe we used the (11)C-labeled CB(2) ligand, (11)C-GW405833, which readily crosses the blood-brain barrier. Dual-modality imaging lentiviral and adeno-associated viral vectors encoding both hCB(2)(D80N) and firefly luciferase or enhanced green fluorescent protein were engineered and validated in cell culture. Next, hCB(2)(D80N) was locoregionally overexpressed in rat striatum by stereotactic injection of lentiviral and adeno-associated viral vectors. Kinetic PET revealed specific and reversible CB(2) binding of (11)C-GW405833 in the transduced rat striatum. hCB(2) and firefly luciferase expression was followed until 9 mo and showed similar kinetics. Both hCB(2) expression and enhanced green fluorescent protein expression were confirmed by immunohistochemistry. Dual-modality imaging viral vectors encoding hCB(2)(D80N) were engineered, and the reporter system was validated in different animal species. The results support the potential future clinical use of CB(2) as a PET reporter in the intact brain.

DNA methylation‐mediated silencing of nonsteroidal anti‐inflammatory drug‐activated gene (NAG‐1/GDF15) in glioma cell lines

Nonsteroidal anti-inflammatory drug-activated gene, NAG-1, a transforming growth factor-β member, is involved in tumor progression and development. The association between NAG-1 expression and development and progression of glioma has not been well defined. Glioblastoma cell lines have lower basal expression of NAG-1 than other gliomas and normal astrocytes. Most primary human gliomas have very low levels of NAG-1 expression. NAG-1 basal expression appeared to inversely correlate with tumor grade in glioma. Aberrant promoter hypermethylation is a common mechanism for silencing of tumor suppressor genes in cancer cells. In glioblastoma cell lines, NAG-1 expression was increased by the demethylating agent, 5-aza-2'-deoxycytidine. To investigate whether the NAG-1 gene was silenced by hypermethylation in glioblastoma, we examined DNA methylation status using genomic bisulfite sequencing. The NAG-1 promoter was densely methylated in several glioblastoma cell lines as well as in primary oligodendroglioma tumor samples, which have low basal expression of NAG-1. DNA methylation at two specific sites (-53 and +55 CpG sites) in the NAG-1 promoter was strongly associated with low NAG-1 expression. The methylation of the NAG-1 promoter at the -53 site blocks Egr-1 binding and thereby suppresses Nag-1 induction. Treatment of cells with low basal NAG-1 expression with NAG-1 inducer also did not increase NAG-1. Incubation with a demethylation chemical increased Nag-1 basal expression and subsequent incubation with a NAG-1 inducer increased NAG-1 expression. We concluded from these data that methylation of specific promoter sequences causes transcriptional silencing of the NAG-1 locus in glioma and may ultimately contribute to tumor progression.

Abstract 1195: EphA4 is a novel tumor suppressor in lung adenocarcinoma

Abstract Lung cancer is the leading cause of death from cancer, but the mechanisms behind its initiation and progression are not completely understood. Receptor tyrosine kinases (RTKs) are important in normal cellular physiology as well as in the pathogenesis of a variety of tumors. In this study, we explored the roles of the human Eph kinase family in lung adenocarcinoma. The Eph fmaily is the largest RTK superfamily with 14 receptors and 8 ligands (ephrins). Eph receptors have been implicated both as tumor suppressors and oncogenes. To explore the effect of Eph receptors and ephrins on lung cancer, we analyzed gene expression and survival data from 432 lung adenocarcinomas from Director's Challenge Consortium by univariate Cox proportional hazards model. Of the 11 Eph receptors and 8 ephrins analyzed, only the expression of EphA4, EphA5, EphB2, ephrinA1, and ephrinB3 correlated with overall survival. Analysis of an independent dataset confirmed that EphA4 and ephrinA1 expression negatively correlated with survival in lung adenocarcinoma. After adjusting for age, gender and stage, low EphA4 expression remained significantly associated with a poor outcome for both overall and relapse-free survival with a hazard ratio of 1.57. To validate the gene expression profile data, we measured EphA4 expression in 9 NSCLC cell lines and found that the expression levels of EphA4 by microarray were well correlated with expression levels measure by qPCR and Western blotting (R2=0.89, R2=0.79 respectively), which indicate that the tissue microarray probesets are of high quality. To investigate the role of EphA4 as a putative tumor suppressor, we overexpressed EphA4 in NSCLC cell lines with low basal expression and found that the cell migration was inhibited by 38% and E-cadherin expression was increased. Likewise, E-cadherin expression was decreased with EphA4 knockdown in NSCLC cell lines with high basal EphA4 expression. These data are consistent with a role for EphA4 as a tumor suppressor via increased expression of the cell surface adhesion protein E-cadherin and concomitant decreased cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1195. doi:10.1158/1538-7445.AM2011-1195

Abstract 667: Anticancer activity and tumor selectivity of LOR-253, a novel drug candidate, in lung carcinoma

Abstract LOR-253 is a novel drug candidate entering a first in man dose escalation Phase I clinical trial in patients with advanced solid tumors. LOR-253 was well tolerated in single and repeat-dose GLP-toxicology studies conducted in rats and dogs at significantly higher doses than equivalent efficacious doses determined in xenograft models in mice, which indicates a potential wide therapeutic window. Previously we reported that LOR-253 reduced the levels of intracellular labile zinc leading to cell cycle arrest, through the down regulation of the metal-responsive transcription factor 1 (MTF-1) and the induction of the Krüppel like factor 4 (KLF4). KLF4 is a G1/S cell cycle checkpoint gene with tumor suppressor function in a variety of cancers. A recent study demonstrated dramatically decreased levels of KLF4 expression in most primary lung tumors (Hu et al. Clin Cancer Res 2009; 15 (18): 5688-95). Anti-tumor activity of KLF4 was demonstrated when KLF4 expression was enforced in cancer cell lines with low basal KLF4 expression levels. In addition, tumor growth in animals was highly compromised when KLF4 gene was stably transfected. The aim of this study was to investigate differential effects of LOR-253 on normal lung fibroblasts vs lung cancer cell lines with different basal levels of KLF4 expression. We found an inverse correlation between LOR-253 sensitivity and basal KLF4 expression. LOR-253 was highly active in three different NSCLC cell lines (H1299, H322 and H226) as shown by much lower IC50 values. KLF4 expression levels in these cell lines were significantly lower than that in normal cells. In contrast, tumor cell lines with KLF4 expression levels similar or higher than normal cells were less sensitive to LOR-253 treatment. Similar results were obtained in in vivo xenograft models, which showed higher in vivo efficacy of LOR-253 in treated mice harboring low basal KLF4-expressing tumors. A time-course gene and protein expression of KLF4 and cyclin D1 in normal lung fibroblast (HFL1) and in NSCLC H226 cells treated with LOR-253 demonstrated a differential effect between normal vs cancer cells. A maximum expression of KLF4 and suppression of cyclin D1 in H226 cells were observed between 16 and 24 hours of treatment while there was no significant effect on HFL1 cells. Based on these results the cell line H226 was selected for further LOR-253 target validation and efficacy biomarker expression studies. A dose-response effect of LOR-253 treatment on tumor growth and KLF4 and cyclin D1 expression was observed in the H226 xenograft model using RT-PCR, Western blot and immunohistochemical analysis. Studies are underway to determine the expression levels of other biomarkers such as MTF-1, HIF-1 a and b-catenin in response to LOR-253 treatment, as well as the effect of in vivo gene knock down and over-expression of MTF-1, KLF4 and other biomarkers on the antitumor activity of LOR-253. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 667. doi:10.1158/1538-7445.AM2011-667

Blunted hepcidin response to inflammation in the absence of Hfe and transferrin receptor 2

AbstractThe induction of the iron-regulatory peptide hepcidin by proinflammatory cytokines is thought to result in the withholding of iron from invading pathogens. Hfe and transferrin receptor 2 (Tfr2) are involved in the homeostatic regulation of hepcidin and their disruption causes hereditary hemochromatosis (HH). To determine whether either Hfe or Tfr2 is involved in the inflammatory pathway regulating hepcidin, we analyzed the effect of inflammation in 3 mouse models of HH. The inflammatory response and indicators of iron homeostasis were measured in wild-type, Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− mice injected with lipopolysaccharide (LPS). The administration of LPS significantly reduced serum iron in wild-type and Hfe−/− mice, with smaller reductions in Tfr2−/− and Hfe−/−/Tfr2−/− mice. Low basal levels of hepcidin in the Hfe−/−/Tfr2−/− mice were increased in response to LPS, but remained significantly lower than in the other strains of mice. These results suggest that despite the absence of Hfe and Tfr2, hepcidin is responsive to inflammation; however, the low basal expression and subsequent low levels of circulating hepcidin are insufficient to reduce serum iron effectively. This suggests that in HH, the iron-withholding response to invading pathogens may be inadequate, and this is especially the case in the absence of both Hfe and Tfr2.