Implication of RNA-Binding Protein La in Proliferation, Migration and Invasion of Lymph Node-Metastasized Hypopharyngeal SCC Cells

The 5-year survival rate for oral cavity cancer is poorer than for breast, colon or prostate cancer, and has improved only slightly in the last three decades. Hence, new therapeutic strategies are urgently needed. Here we demonstrate by tissue micro array analysis for the first time that RNA-binding protein La is significantly overexpressed in oral squamous cell carcinoma (SCC). Within this study we therefore addressed the question whether siRNA-mediated depletion of the La protein may interfere with known tumor-promoting characteristics of head and neck SCC cells. Our studies demonstrate that the La protein promotes cell proliferation, migration and invasion of lymph node-metastasized hypopharyngeal SCC cells. We also reveal that La is required for the expression of beta-catenin as well as matrix metalloproteinase type 2 (MMP-2) within these cells. Taken together these data suggest a so far unknown function of the RNA-binding protein La in promoting tumor progression of head and neck SCC.

Crystallization and preliminary X-ray crystallographic studies of Protac (R), a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 angstrom resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b = 63.3 and c = 48.2 angstrom, alpha = gamma = 90.0 degrees and beta = 90.8 degrees. (C) 2005 Elsevier B.V. All rights reserved.

Macromolecular array patterns of silk gland secretion in social Hymenoptera larvae

The cocoon, produced by most holometabolous insects, is built with silk that is usually produced by the larval salivary gland. Although this silk has been widely studied in the Lepidoptera, its composition and macromolecular arrangement remains unknown in the Hymenoptera. The macromolecular array patterns of the silk in the larval salivary gland of some meliponids, wasps, and ants were analyzed with polarized-light microscopy, and they were compared with those of Bombyx mori (Lepidoptera). There is a birefringent secretion in the glandular lumen of all larvae, due to filamentous structural proteins that display anisotropy. The silk in the distal, middle and proximal regions of the secretory portion of Formicidae and Vespidae glands presented a lattice optical pattern. We found a different pattern in the middle secretory portion of the Meliponini, with a zigzag rather than a lattice pattern. This indicates that the biopolymer fibers begin their macromolecular reorganization at this glandular region, different from the Formicidae and the Vespidae, in which the zigzag optical pattern was only found at the lateral duct. Probably, the mechanism of silk production in the Hymenoptera is a characteristic inherited from a common ancestor of Vespoidea and Sphecoidea; the alterations in the pattern observed in the Meliponini could be a derived characteristic in the Hymenoptera. We found no similarity in the macromolecular reorganization patterns of the silk between the Hymenoptera species and the silkworm.

The Development of a Universal In Silico Predictor of Protein-Protein Interactions

Protein-protein interactions (PPIs) are essential for understanding the function of biological systems and have been characterized using a vast array of experimental techniques. These techniques detect only a small proportion of all PPIs and are labor intensive and time consuming. Therefore, the development of computational methods capable of predicting PPIs accelerates the pace of discovery of new interactions. This paper reports a machine learning-based prediction model, the Universal In Silico Predictor of Protein-Protein Interactions (UNISPPI), which is a decision tree model that can reliably predict PPIs for all species (including proteins from parasite-host associations) using only 20 combinations of amino acids frequencies from interacting and non-interacting proteins as learning features. UNISPPI was able to correctly classify 79.4% and 72.6% of experimentally supported interactions and non-interacting protein pairs, respectively, from an independent test set. Moreover, UNISPPI suggests that the frequencies of the amino acids asparagine, cysteine and isoleucine are important features for distinguishing between interacting and non-interacting protein pairs. We envisage that UNISPPI can be a useful tool for prioritizing interactions for experimental validation. © 2013 Valente et al.

S-100 protein: is it useful as a tumour marker in diagnostic immunocytochemistry

A heterogeneous group of 159 tumours was studied for the presence of S-100 protein by the immunoperoxidase technique in order to determine whether this marker may be of value in facilitating immunocytochemical diagnosis. Among cases of melanocytic and pigmented lesions, S-100 was widely distributed and demonstrated the strongest degrees of reactivity. S-100 protein was identified in virtually all nerve sheath tumours such as schwannomas, neurofibromas, myxoid sheath nerve tumour and also in some tumours of controversial histogenesis such as granular cell tumours. The great majority of carcinomas did not express S-100, with only two cases of breast carcinoma displaying focal S-100 staining. In a miscellaneous group of tumours S-100 was demonstrated in chordomas, myoepitheliomas and Wilms' tumour with Schwann cell differentiation. Despite its presence in a wide array of cell types, S-100 protein continues to be an extremely useful marker especially for soft tissue and peripheral nervous system tumours.

Morphological alterations and gene and protein expression profiling of bladder tumor cells after treatment with gemcitabine.

Chemical agents used in cancer therapy are associated with cell cycle arrest, activation or deactivation of mechanisms associated to DNA repair and apoptosis. However, due to the complexity of biological systems, the molecular mechanisms responsible for these activities are not fully understood. Thus, studies about gene and protein expression have shown promising results for understanding the mechanisms related to cellular responses and regression of cancer after chemotherapy. This study aimed to evaluate the gene and protein expression profiling in bladder transitional cell carcinoma (TCC) with different TP53 status after gemcitabine (1.56 μM) treatment. The RT4 (grade 1, TP53 wild type), 5637 (grade 2, TP53 mutated) and T24 (grade 3, TP53 mutated) cell lines were used. PCR arrays and mass spectrometry were used to analyze gene and protein expression, respectively. Morphological alterations were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results of PCR array showed that gemcitabine activity was mainly related to CDKN1A, GADD45A and SERTDA1 overexpression, and BAX overexpression only in the wild type TP53 cells. Mass spectrometry demonstrated that gemcitabine modulated the protein expression, especially those from genes related to apoptosis, transport of vesicles and stress response. Analyses using SEM and TEM showed changes in cell morphology independently on the cell line studied. The observed decreased number of microvillus suggests low contact among the cells and between cell and extracellular matrix; irregular forms might indicate actin cytoskeleton deregulation; and the reduction in the amount of organelles and core size might indicate reduced cellular metabolism. In conclusion, independently on TP53 status or grade of bladder tumor, gemcitabine modulated genes related to the cell cycle and apoptosis, that reflected in morphological changes indicative of future cell death.

Funktionelle Charakterisierung der Protein-Kinase CK2 in der Suppression Th2-vermittelter Immunantworten durch regulatorische T-Zellen

Regulatorische T-Zellen (Tregs) leisten durch ihre suppressiven Eigenschaften einen essenziellen Beitrag zur Aufrechterhaltung der immunologischen Toleranz. Sie verhindern schädliche Immunreaktionen gegen Autoantigene, kommensale Bakterien, sowie harmlose Nahrungsmittel-bestandteile. Gleichzeitig gewährleisten sie die Entwicklung effektiver Immunantworten gegen eindringende Pathogene, wie z.B. Parasiten, Bakterien und Viren. Damit haben Tregs direkten Einfluss auf das Gleichgewicht zwischen Immunität und Toleranz. Fehler in der suppressiven Funktionsweise von Tregs begünstigen daher auf der einen Seite die Entstehung zahlreicher autoimmuner Erkrankungen und Allergien. Auf der anderen Seite können Tregs Immunreaktionen bei chronischen Infektionen reduzieren, sowie die Entstehung effektiver Immunantworten gegen Tumore hemmen. Ihre Beteiligung an der Ätiologie all dieser Krankheiten macht Tregs zu einem bedeutenden potenziellen Zielobjekt, um diese Krankheiten effektiv zu therapieren. Die Erweiterung des Grundwissens um die molekularen Mechanismen der Treg-vermittelten Suppression ist daher ein notwendiger Schritt bei der Entwicklung Treg-basierter Theraphieansätze. 2003 konnte mit Foxp3 ein Transkriptionsfaktor identifiziert werden, der maßgeblich die suppressiven Funktionen von Tregs steuert. Um weiteren Einblick in die der Suppression zugrundeliegenden Signalwege zu erhalten, wurde im Institut für Immunologie ein komparativer Kinomarray durchgeführt, anhand dessen die Casein Kinase 2 (CK2) als eine der aktivsten Kinasen in Tregs identifiziert wurde (Daten freundlicherweise von Prof. Dr. Tobias Bopp bereitgestellt). rnBasierend auf den Ergebnissen des Kinomarrays wurde in dieser Arbeit die Funktion der CK2 in Tregs untersucht. Dabei konnte in in vitro Experimenten die Treg-vermittelte Suppression durch den pharmakologische CK2 Inhibitor DMAT aufgehoben werden. Weil derartige Inhibitoren jedoch nicht absolut spezifisch die Aktivität nur einer Kinase supprimieren, wurden außerdem Mäuse mit konditionalem „knockout“ der CK2β Untereinheit spezifisch in Tregs gekreuzt (CK2βTreg-/- Mäuse). Die Analyse dieser Tiere offenbarte eine essenzielle Beteiligung der CK2 an den suppressiven Funktionen von Tregs. So entwickeln CK2βTreg-/- Mäuse mit zunehmendem Alter Splenomegalien und Lymphadenopathien, von denen in besonderem Maße die Mukosa-assoziierten Lymphknoten betroffen sind. Eine Analyse des Aktivierungsstatus der T-Zellen in den Tieren konnte zudem einen erhöhten Anteil sogenannter Effektor-Gedächtnis T-Zellen aufdecken, die charakteristische Merkmale eines Th2 Phänotyps zeigten. Erhöhte Titer des Antikörperisotyps IgE in den Seren von CK2βTreg-/- Mäusen suggerieren zusätzlich eine fehlerhafte Suppression speziell Th2-vermittelter Immunantworten durch CK2β-defiziente Tregs. In Th2-vermittelten Asthma Experimenten in vivo konnte der Verdacht der fehlerhaften Kontrolle von Th2-Antwort bestätigt werden, wobei zusätzlich aufgedeckt wurde, dass bereits unbehandelte CK2βTreg-/- Mäuse Zeichen einer Entzündungsreaktion in der Lunge aufweisen. Bei der Suche nach den molekularen Ursachen der fehlerhaften Suppression Th2-vermittelter Immunantworten durch CK2β-defiziente Tregs konnten zwei mögliche Erklärungsansätze gefunden werden. Zum einen zeigen CK2β-defiziente Tregs eine verringerte Expression von Foxp3, was, in Analogie zu Ergebnissen der Gruppe von R. Flavell (Wang Y.Y. Nature. 445, 766-770 (2007)), zu einer Konversion von Tregs zu Th2 Zellen und damit zur Entstehung eines Th2-basierten, autoimmunen Phänotyps führt. Des Weiteren weisen CK2β-defiziente Tregs eine reduzierte Expression des Transkriptionsfaktors IRF4 auf, der in Tregs entscheidend für die Kontrolle Th2-basierter Immunreaktionen ist (Zheng Y. Nature. 19; 351-356 (2009)). Die dargelegten Ergebnisse identifizieren die CK2 damit als Kinase, die entscheidend an der Treg-vermittelten Suppression speziell Th2-basierter Immunantworten beteiligt ist. Demnach könnten pharmakologische CK2 Inhibitoren beispielsweise dazu eingesetzt werden, um die Treg-vermittelte Suppression im Rahmen chronischer Parasiten-Infektionen aufzuheben. Die in CK2βTreg-/- Mäusen beobachtete Prävalenz der Funktion der CK2 für Mukosa-assoziierte Organe stellt dabei einen zusätzlichen Vorteil dar, weil systemische Nebenwirkungen, die durch die Blockade der Treg-vermittelte Suppression entstehen, zumindest in nicht-Mukosa-assoziierten Geweben nicht zu erwarten sind.rn

Surfactant protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model

Background Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. Methods SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. Results SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. Conclusion These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

3CAM - A NOVEL TERNARY CONTENT ADDRESSABLE MEMORY USING THREE-VALUED LOGIC

Content Addressable Memory (CAM) is a special type of Complementary Metal-Oxide-Semiconductor (CMOS) storage element that allows for a parallel search operation on a memory stack in addition to the read and write operations yielded by a conventional SRAM storage array. In practice, it is often desirable to be able to store a “don’t care” state for faster searching operation. However, commercially available CAM chips are forced to accomplish this functionality by having to include two binary memory storage elements per CAM cell,which is a waste of precious area and power resources. This research presents a novel CAM circuit that achieves the “don’t care” functionality with a single ternary memory storage element. Using the recent development of multiple-voltage-threshold (MVT) CMOS transistors, the functionality of the proposed circuit is validated and characteristics for performance, power consumption, noise immunity, and silicon area are presented. This workpresents the following contributions to the field of CAM and ternary-valued logic:• We present a novel Simple Ternary Inverter (STI) transistor geometry scheme for achieving ternary-valued functionality in existing SOI-CMOS 0.18µm processes.• We present a novel Ternary Content Addressable Memory based on Three-Valued Logic (3CAM) as a single-storage-element CAM cell with “don’t care” functionality.• We explore the application of macro partitioning schemes to our proposed 3CAM array to observe the benefits and tradeoffs of architecture design in the context of power, delay, and area.

Differential gene and protein expression in abluminal sprouting and intraluminal splitting forms of angiogenesis

In adult skeletal muscle, abluminal sprouting or longitudinal splitting of capillaries can be initiated separately by muscle overload and elevated microcirculation shear stress respectively. In the present study, gene and protein expression patterns associated with the different forms of angiogenesis were examined using a targeted gene array (Superarray), validated by quantitative RT (reverse transcription)-PCR and immunoblots. Sprouting angiogenesis induced large changes in expression levels in genes associated with extracellular matrix remodelling, such as MMP-2 (matrix metalloproteinase-2), TIMP (tissue inhibitor of metalloproteinases), SPARC (secreted protein, acidic and rich in cysteine) and thrombospondin. Changes in neuropilin, midkine and restin levels, which may underpin changes in endothelial morphology, were seen during splitting angiogenesis. Up-regulation of VEGF (vascular endothelial growth factor), Flk-1, angiopoietin-2 and PECAM-1 (platelet/endothelial cell adhesion molecule-1) was seen in both forms of angiogenesis, representing a common angiogenic response of endothelial cells. In conclusion, the present study demonstrates that general angiogenic signals from growth factors can be influenced by the local microenvironment resulting in differing forms of capillary growth to produce a co-ordinated expansion of the vascular bed.

Quantum dot / optical protein bio-nano hybrid system biosensing

The integration of novel nanomaterials with highly-functional biological molecules has advanced multiple fields including electronics, sensing, imaging, and energy harvesting. This work focuses on the creation of a new type of bio-nano hybrid substrate for military biosensing applications. Specifically it is shown that the nano-scale interactions of the optical protein bacteriorhodopsin and colloidal semiconductor quantum dots can be utilized as a generic sensing substrate. This work spans from the basic creation of the protein to its application in a novel biosensing system. The functionality of this sensor design originates from the unique interactions between the quantum dot and bacteriorhodopsin molecule when in nanoscale proximity. A direct energy transfer relationship has been established between coreshell quantum dots and the optical protein bacteriorhodopsin that substantially enhances the protein’s native photovoltaic capabilities. This energy transfer phenomena is largely distance dependent, in the sub-10nm realm, and is characterized experimentally at multiple separation distances. Experimental results on the energy transfer efficiency in this hybrid system correlate closely to theoretical predictions. Deposition of the hybrid system with nano-scale control has allowed for the utilization of this energy transfer phenomena as a modulation point for a functional biosensor prototype. This work reveals that quantum dots have the ability to activate the bacteriorhodopsin photocycle through both photonic and non-photonic energy transfer mechanisms. By altering the energy transferred to the bacteriorhodopsin molecule from the quantum dot, the electrical output of the protein can be modulated. A biosensing prototype was created in which the energy transfer relationship is altered upon target binding, demonstrating the applicability of a quantum dot/bacteriorhodopsin hybrid system for sensor applications. The electrical nature of this sensing substrate will allow for its efficient integration into a nanoelectronics array form, potentially leading to a small-low power sensing platform for remote toxin detection applications.

Bacteriorhodopsin protein hybrids for chemical and biological sensing

Bacteriorhodopsin (bR), an optoelectric protein found in Halobacterium salinarum, has the potential for use in protein hybrid sensing systems. Bacteriorhodopsin has no intrinsic sensing properties, however molecular and chemical tools permit production of bR protein hybrids with transducing and sensing properties. As a proof of concept, a maltose binding protein-bacteriorhodopsin ([MBP]-bR) hybrid was developed. It was proposed that the energy associated with target molecule binding, maltose, to the hybrid sensor protein would provide a means to directly modulate the electrical output from the MBP-bR bio-nanosensor platform. The bR protein hybrid is produced by linkage between bR (principal component of purified purple membrane [PM]) and MBP, which was produced by use of a plasmid expression vector system in Escherichia coli and purified utilizing an amylose affinity column. These proteins were chemically linked using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), which facilitates formation of an amide bond between a primary carboxylic acid and a primary amine. The presence of novel protein hybrids after chemical linkage was analyzed by SDSPAGE. Soluble proteins (MBP-only derivatives and unlinked MBP) were separated from insoluble proteins (PM derivatives and unlinked PM) using size exclusion chromatography. The putatively identified MBP-bR protein hybrid, in addition to unlinked bR, was collected. This sample was normalized for bR concentration to native PM and both were deposited onto indium tin oxide (ITO) coated glass slides by electrophoretic sedimentation. The photoresponse of both samples, activated using 100 Watt tungsten lamp at 10 cm distance, were equal at 175 mV. Testing of deposited PM with 1 mM sucrose or 1 mM maltose showed no change in the photoresponse of the xiv material, however addition of 1 mM maltose to the deposited MBP-bR linked hybrid material elicited a 57% decrease in photoresponse indicating a positive response for targeting of maltose. This chemically linked MBP-bR hybrid protein, with bacteriorhodopsin, as a photoresponsive transducing substrate, shows promise for creation of a universal sensing array by attachment of other pertinent sensing materials, in lieu of the maltose binding protein utilized. This strategy would allow significant reduction in sensor size, while increasing responsiveness and sensitivity at nano and picomolar levels.

The PDZ protein MPP2 interacts with c-Src in epithelial cells

c-Src is a non-receptor tyrosine kinase involved in regulating cell proliferation, cell migration and cell invasion and is tightly controlled by reversible phosphorylation on regulatory sites and through protein-protein interactions. The interaction of c-Src with PDZ proteins was recently identified as novel mechanism to restrict c-Src function. The objective of this study was to identify and characterise PDZ proteins that interact with c-Src to control its activity. By PDZ domain array screen, we identified the interaction of c-Src with the PDZ protein Membrane Protein Palmitoylated 2 (MPP2), a member of the Membrane-Associated Guanylate Kinase (MAGUK) family, to which also the Discs large (Dlg) tumour suppressor protein belongs. The function of MPP2 has not been established and the functional significance of the MPP2 c-Src interaction is not known. We found that in non-transformed breast epithelial MCF-10A cells, endogenous MPP2 associated with the cytoskeleton in filamentous structures, which partially co-localised with microtubules and c-Src. MPP2 and c-Src interacted in cells, where c-Src kinase activity promoted increased interaction of c-Src with MPP2. We furthermore found that MPP2 was able to negatively regulate c-Src kinase activity in cells, suggesting that the functional significance of the MPP2-c-Src interaction is to restrict Src activity. Consequently, the c-Src-dependent disorganisation of the cortical actin cytoskeleton of epithelial cells expressing c-Src was suppressed by MPP2. In conclusion we demonstrate here that MPP2 interacts with c-Src in cells to control c-Src activity and morphological function.

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Glioma-specific alternative RNA splicing: A role for polypyrimidine tract binding protein-1

Alternative RNA splicing plays an integral role in cell fate determination and function, especially in the cells of the brain. Errors in RNA processing contribute to diseases such as cancer, where it leads to the production of oncogenic proteins or the loss of tumor suppressors. In silica mining suggests that hundreds of splice isoforms are misexpressed in the glial cell-derived glioma. However, there is little experimental evidence of the prevalence and contribution of these changes and whether they contribute to the formation and progression of this devastating malignancy. To determine the frequency of these aberrant events, global profiling of alternative RNA splice patterns in glioma and nontumor brain was conducted using an exon array. Most splicing changes were less than 5-fold in magnitude and 14 cassette exon events were validated, including 7 previously published events. To determine the possible causes of missplicing, the differential expression levels of splicing factors in these two tissues were also analyzed. Six RNA splicing factors had greater than 2-fold changes in expression. The highest differentially expressed factor was polypyrimidine tract binding protein-1 (PTB). Evaluation by immunohistochemistry determined that this factor was elevated in both early and late stages of glioma. Glial cell-specific PTB expression in the adult brain led me to examine the role of PTB in gliomagenesis. Downregulation of PTB slowed glioma cell proliferation and migration and enhanced cell adhesion to fibronectin and vitronectin. To determine whether PTB was affecting these processes through splicing, genome-wide exon expression levels were correlated with PTB levels. Surprisingly, previously reported PTB target transcripts were insensitive to changes in PTB levels in both patient samples and PTB-depleted glioma cells. Only one validated glioma-specific splice target, RTN4/Nogo, had a significant PTB-mediated splicing change. Downregulation of PTB enhanced inclusion of its alternative exon 3, which encodes an auxiliary domain within a neurite inhibitor protein. Overexpression of this splice isoform in glioma cells slowed proliferation in a manner similar to that observed in PTB knockdown cells. In summary, aberrant expression of splicing factors such as PTB in glioma may elicit changes in splicing patterns that enhance tumorigenesis. ^