The role of NOTCH2 gene in human malignant glial brain tumours

Background: Glioblastoma multiforme (GBM), astrocytoma (A) and oligodendroglioma (OG) are the neoplasms of the glial lineage in the Central Nervous System (CNS). Among them, GBM occurs at the highest frequency and shows the shortest patient median survival time of some 10 months as compared for instance to the survival time of OG of about 10 years. Genetically, OG differs from GBM by the frequent combination of loss of heterozygosity (LOH) on chromosomes 1p and 19q, which is associated with more favourable prognosis in OG patients. However, the clinical significance of LOH on 1p in other glioma subtypes remained unknown. Methods and Results: We identified a subgroup of GBM with LOH on centromeric chromosome 1p together with longer survival. The minimally lost area(s) in both GBM and OG converged at the NOTCH2 locus on 1p11 and positively correlated with prognosis in GBM as well as in OG patients. Comparison between gene expression of NOTCH2 and the genetic status at the NOTCH2 locus on chromosome 1p11 supported the hypothesis of a loss of function alteration of NOTCH2 in tumours. However, many GBMs do not display deletions at the NOTCH2 locus on 1p11 and do express the NOTCH2 gene. Abundant expression of components of canonical NOTCH signaling in these tumors and a positive correlation between NOTCH2 transcripts with the target gene HES-1 (P=0.0001) indicated that functional NOTCH signaling in glioma is mainly driven by NOTCH2. In addition, we defined TNC, the gene for the cell migration factor tenascin-C as a novel target gene for NOTCH signaling. We further showed that activation of NOTCH signaling was indeed promoting TNC-dependent glioma cell motility. Thus, together with the ability to increase proliferation, canonical Notch signaling turned out to be critical for glioma progression. We also found that non-canonical Notch signaling was associated with the maintenance of tumorigenic potential of the GBM cells in soft agar culture. In addition, Notch2 had a prosurvival effect on GBM cells by upregulating anti-apoptotic proteins Bcl-2 and Mcl-1, independently of the canonical pathway. Finally, defective degradation pathway of Notch receptors in GBM cells led to slow receptor turnover, thereby providing additional contribution to the oncogenic function of Notch2. Conclusion: This study identified aberrant multi-facetted oncogenic behaviours of Notch proteins, in particular of Notch2, in GBM. This provided a molecular basis for the higher aggressiveness of Notch2-positive GBM compared to Notch2-negative GBM or OG, and suggested Notch2 as a sensible target for new therapeutic approaches against GBM

Loss of NOTCH2 Positively Predicts Survival in Subgroups of Human Glial Brain Tumors

The structural complexity of chromosome 1p centromeric region has been an obstacle for fine mapping of tumor suppressor genes in this area. Loss of heterozygosity (LOH) on chromosome 1p is associated with the longer survival of oligodendroglioma (OD) patients. To test the clinical relevance of 1p loss in glioblastomas (GBM) patients and identifiy the underlying tumor suppressor locus, we constructed a somatic deletion map on chromosome 1p in 26 OG and 118 GBM. Deletion hotspots at 4 microsatellite markers located at 1p36.3, 1p36.1, 1p22 and 1p11 defined 10 distinct haplotypes that were related to patient survival. We found that loss of 1p centromeric marker D1S2696 within NOTCH2 intron 12 was associated with favorable prognosis in OD (P = 0.0007) as well as in GBM (P = 0.0175), while 19q loss, concomitant with 1p LOH in OD, had no influence on GBM survival (P = 0.918). Assessment of the intra-chromosomal ratio between NOTCH2 and its 1q21 pericentric duplication N2N (N2/N2N-test) allowed delineation of a consistent centromeric breakpoint in OD that also contained a minimally lost area in GBM. OD and GBM showed distinct deletion patterns that converged to the NOTCH2 gene in both glioma subtypes. Moreover, the N2/N2N-test disclosed homozygous deletions of NOTCH2 in primary OD. The N2/N2N test distinguished OD from GBM with a specificity of 100% and a sensitivity of 97%. Combined assessment of NOTCH2 genetic markers D1S2696 and N2/N2N predicted 24-month survival with an accuracy (0.925) that is equivalent to histological classification combined with the D1S2696 status (0.954) and higher than current genetic evaluation by 1p/19q LOH (0.762). Our data propose NOTCH2 as a powerful new molecular test to detect prognostically favorable gliomas

GSK3β Regulates Differentiation and Growth Arrest in Glioblastoma

Cancers are driven by a population of cells with the stem cell properties of self-renewal and unlimited growth. As a subpopulation within the tumor mass, these cells are believed to constitute a tumor cell reservoir. Pathways controlling the renewal of normal stem cells are deregulated in cancer. The polycomb group gene Bmi1, which is required for neural stem cell self-renewal and also controls anti-oxidant defense in neurons, is upregulated in several cancers, including medulloblastoma. We have found that Bmi1 is consistently and highly expressed in GBM. Downregulation of Bmi1 by shRNAs induced a differentiation phenotype and reduced expression of the stem cell markers Sox2 and Nestin. Interestingly, expression of glycogen synthase kinase 3 beta (GSK3β), which was found to be consistently expressed in primary GBM, also declined. This suggests a functional link between Bmi1 and GSK3β. Interference with GSK3β activity by siRNA, the specific inhibitor SB216763, or lithium chloride (LiCl) induced tumor cell differentiation. In addition, tumor cell apoptosis was enhanced, the formation of neurospheres was impaired, and clonogenicity reduced in a dose-dependent manner. GBM cell lines consist mainly of CD133-negative (CD133-) cells. Interestingly, ex vivo cells from primary tumor biopsies allowed the identification of a CD133- subpopulation of cells that express stem cell markers and are depleted by inactivation of GSK3β. Drugs that inhibit GSK3, including the psychiatric drug LiCl, may deplete the GBM stem cell reservoir independently of CD133 status

A surprising system : polymeric nanoreactors containing a mimic with dual-enzyme activity

Reactive oxygen species have been implicated in various diseases, but attempts to find efficient antioxidant treatments for such conditions have met with only limited success. Here, we have developed an antioxidant nanoreactor by encapsulating a dual-enzyme mimic of superoxide dismutase and catalase, in polymeric nanovesicles and examined how this nanoreactor combats oxidative stress. The mimic (CuIIENZm) is encapsulated inside poly-(2-methyloxazoline)–poly-(dimethylsiloxane)–poly(2-methyloxazoline) polymer vesicles that feature membranes permeable to superoxide, enabling the enzyme mimic to act in situ. We ensured that the size and shape of polymeric vesicles were not changed during the encapsulation procedure by analysis with light scattering and transmission electron microscopy, and that the structural geometry of CuIIENZm was preserved, as demonstrated by electron paramagnetic resonance and UV-vis spectroscopy. Due to its bi-functionality, CuIIENZm detoxified both superoxide radicals and related H2O2. The intracellular localization of the nanoreactor in THP-1 cells was established using confocal laser scanning microscopy and flow cytometry. No evident toxicity was found using MTS and LDH assays. As CuIIENZm remained active inside the vesicles therefore, these CuIIENZm-containing nanoreactors exhibited efficient antioxidant activity in THP-1 cells. Development of this simple, robust antioxidant nanoreactor represents a new direction in efficiently fighting oxidative stress

Interaction between lidocaine hydrochloride (with and without adrenaline) and various irrigants: A nuclear magnetic resonance analysis

Background: Interaction between local anesthetic solution, lidocaine hydrochloride (with and without adrenaline), and root canal irrigants such as sodium hypochlorite (NaOCl), ethylene diamine tetra-acetic acid (EDTA), and chlorhexidine (CHX) has not been studied earlier. Hence, the purpose of this in vitro study was to evaluate the chemical interaction between 2% lidocaine hydrochloride (with and without adrenaline) and commonly used root canal irrigants, NaOCl, EDTA, and CHX. Materials and Methods: Samples were divided into eight experimental groups: Group I-Lidocaine hydrochloride (with adrenaline)/3% NaOCl, Group II-Lidocaine hydrochloride (with adrenaline)/17% EDTA, Group III- Lidocaine hydrochloride (with adrenaline)/2% CHX, Group IV-Lidocaine hydrochloride (without adrenaline)/3% NaOCl, Group V-Lidocaine hydrochloride (without adrenaline)/17% EDTA, Group VI-Lidocaine hydrochloride (without adrenaline)/2% CHX, and two control groups: Group VII-Lidocaine hydrochloride (with adrenaline)/deionized water and Group VIII-Lidocaine hydrochloride (without adrenaline)/deionized water. The respective solutions of various groups were mixed in equal proportions (1 ml each) and observed for precipitate formation. Chemical composition of the formed precipitate was then analysed by nuclear magnetic resonance spectroscopy (NMR) and confirmed with diazotation test. Results: In groups I and IV, a white precipitate was observed in all the samples on mixing the respective solutions, which showed a color change to reddish brown after 15 minutes. This precipitate was then analysed by NMR spectroscopy and was observed to be 2,6-xylidine, a reported toxic compound. The experimental groups II, III, V, and VI and control groups VII and VIII showed no precipitate formation in any of the respective samples, until 2 hours. Conclusion: Interaction between lidocaine hydrochloride (with and without adrenaline) and NaOCl showed precipitate formation containing 2,6-xylidine, a toxic compound

CBSE-2014 [2 nd and 3 rd April 2014] Challenges in Biochemical Engineering and Biotechnology for Sustainable Environment Comparative Study on Candida Sp. for the Production of Glycerol

Abstract : Candidmagnoliae and Candida glycerinogenes are the yeast isolated from the natural environment used for the production of glycerol. In this work potential of glycerol production by both candida species was compared by the fermentation of glucose by optimizing certain parameters.C.glycerimogenesconverts up to 64.5% (w/w) of the available glucose into glycerol and it was 55% in C. magnolia. C. glycerinogenesyields 9% of higher concentration of glycerol than C. magnoliae. In C. magnoliaethe optimum temperature of 30 o C and a pH of 5 yields highest glycerol production whereas for C.glycerinogenesa temperature of 32 o C and a pH of 5. The parameters which found to influence the glycerol production like phosphate ,glucose were studied

Forensic video solution using facial feature‐based synoptic Video Footage Record

Person specific identification is an important problem in computer vision. However, forensic video analysis is the tool in surveillance applications, such as a specific person Video Footage Record can be used to help personalised monitoring. This study proposes a solution to identify the specific person very quickly through offline which will be valuable to analyse the incident/crime earlier. The main idea of this study is to reduce the enormous volume of video data by using an object‐based video synopsis. After that, Viola–Jones face detection, deformable part based models are used to detect the face attributes. Subsequently, histogram of oriented gradients and oriented centre symmetric local binary pattern features are extracted. Support vector machine classifier is used to classify the weak and strong features. These strong features are used to recognise the person. The algorithm works well even in complicated situations such as expression changes, pose, illumination variations and even if the face is partially as well as fully occluded in few frames. The advantage of synoptic video helps to recognise the person who is not occluded in some other frames. Experimental results on benchmark and real time datasets demonstrate the effectiveness of the proposed algorithm

Deltex-1 Activates Mitotic Signaling and Proliferation and Increases the Clonogenic and Invasive Potential of U373 and LN18 <em>Glioblastoma</em> Cells and Correlates with Patient Survival

<div><p><i>Glioblastoma</i> (GBM) is a highly malignant primary tumor of the central nervous system originating in glial cells. GBM results in more years of life lost than any other cancer type. Low levels of Notch receptor expression correlates with prolonged survival in various high grade gliomas independent of other markers. Different downstream pathways of Notch receptors have been identified. We tested if the Notch/Deltex pathway, which is distinct from the canonical, CSL-mediated pathway, has a role in GBM. We show that the alternative or non-canonical Notch pathway functioning through Deltex1 (DTX1) mediates key features of glioblastoma cell aggressiveness. For example, DTX1 activates the RTK/PI3K/PKB and the MAPK/ERK mitotic pathways and induces anti-apoptotic Mcl-1. The clonogenic and growth potential of established glioma cells correlated with DTX1 levels. Microarray gene expression analysis further identified a DTX1-specific, MAML1-independent transcriptional program - including <i>microRNA-21</i>- which is functionally linked to the changes in tumor cell aggressiveness. Over-expression of DTX1 increased cell migration and invasion correlating to ERK activation, miR-21 levels and endogenous Notch levels. In contrast to high and intermediate expressors, patients with low <i>DTX1</i> levels have a more favorable prognosis. The alternative Notch pathway via DTX1 appears to be an oncogenic factor in glioblastoma and these findings offer new potential therapeutic targets. </p> </div

Proliferation and mitotic signaling pathways are modified by DTX1 in established glioma cell lines.

<p>(A) Western blot analysis of signaling cascade proteins. Blots for total EGFR (t-EGFR), phosphorylated Akt/PKB (p-Akt/PKB), total Akt/PKB (t-Akt/PKB), phosphorylated Erk (p-Erk), total Erk (t-Erk), Mcl-1 and β-actin (actin) are shown. (B) Proliferation analysis by BrdU incorporation assay. Relative average values of 5 individual experiments are shown per genotype. (C) Proliferation analysis by cell counting. Equal numbers of cells were seeded, grown for 3d under standard conditions and counted afterwards. (D) Apoptosis in U373 cells after treatment with 100 µM temozolomide (TMZ) or DMSO control. Relative values of sub-G₁ cells measured by PI staining are shown normalized to control cells treated with vector control. Averages of at least three independent experiments are shown. Values are normalized to controls. Error bars: ±SEM. * p<0.05, ** p<0.01, *** p<0.001.</p